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Ca<sup>2+ and calmodulin have been found to have a role in initiating the proliferative cycle of cells as well as in the G1/S, G2/M and the metaphase-anaphase transitions see (see Means et al., 1999; Lu and Means 1993). Calmodulin, and CaMKII were also found to be essential for the initiation of centrosome duplication (see Matsumoto and Maller, 2002). These findings suggest that Ca2+ and that the calmodulin,-CaMKII cascade are part of the signal required for centrosome duplication and subsequent cell division. Centrosomes have been found essential for cell division (see Section K, below) Considering the key role of proteins in gene expression, it is of special interest to examine whether a new protein appears during the cell cycle. The degradation of proteins involved in the cell cycle is no less important as addressed in the various sections. below. A. The Cyclins Newly synthesized protein can be detected by introducing a radioactive amino acid such as [35S] methionine. The proteins synthesized can be separated by molecular weight in polyacrylamide gels using SDS-gel electrophoresis (see Chapter 1). The gel acts as a sieve and the smaller polypeptides will migrate more rapidly than larger molecules. The denatured polypeptides are covered with negatively charged detergent so that the migration will be in the direction of the positive electrode. The position of the newly synthesized peptides can be seen using autoradiography (see Chapter 1) and will appear on a photographic film as a dark band. In such an experiment, the entire cell population has to be synchronized. For eggs of the sea urchin or clam, this synchronization can be readily obtained by introducing sperms which will fertilize the eggs. In sea urchin eggs a protein was found to closely anticipate each division. This protein was promptly named cyclin. The results of an experiment are displayed in Fig. 4 (Evans et al., 1983). This figure represents results obtained from a suspension of eggs after fertilization (the 0 time on the abscissa) in a medium containing [35S] methionine. The cyclin is shown by the circles and line A, whereas the triangles and line B represent another protein. The amount of each protein was estimated from the density of the bands (right hand ordinate) after autoradiography of the gel using SDS-PAGE (see Chapter 1). The percentage of cells which are dividing, also known as the cleavage index (open squares, the dashed line and left hand ordinate), identifies the time at which the cells divide. The results not only show that cyclin is synthesized before each cell division, but it is degraded at each mitosis. In contrast, the protein represented by line B is independent of the cell cycle.
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Fig. 4 Correlation of the level of cyclin with the cell division cycle in sea urchin eggs. The dashed lines and squares indicate the cleavage index (see text). The circles and full line (A) represent the intensity of the bands corresponding to cyclin and the triangles and dotted line (B) correspond to the intensity of another protein, which exhibits no cyclicity (from Evans et al., 1983). Reproduced by permission.
The results of Fig. 4 can be interpreted in two different ways. The degradation of cyclin following the initiation of cell division could occur while its synthetic rate remained constant. Alternatively, both the synthesis and the degradation could occur cyclically, with the synthesis obviously preceding the degradation. These two separate hypotheses can be tested. The synthetic ability of the cells can be followed by pulse label experiments. With this design, the eggs are exposed to the radioactive amino acid for a short time, after which the radioactivity is diluted by adding an excess of unlabelled amino acid. When pulse-label is introduced at different stages of the cycle, the results provide an estimate of the synthetic capacity of the system during each of these pulse periods. These experiments show that in sea urchin eggs the rate of cyclin synthesis is not greatly changed with the cell cycle, implicating degradation as the major regulatory factor. This is not true in somatic cells, where cyclin synthesis is generally under transcriptional control. In fertilized sea urchin, the mRNA for cyclin is a maternal contribution, so no new mRNA is immediately needed. The mRNA is in an inactive state until it is activated by fertilization. The maternal mRNA continues to be used in the early cell divisions. In mammalian somatic cells and S. cerevisiae, the concentration of cyclin mRNA is cycle-dependent (see below, e.g., see Fig.5).
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Several cyclins are now known and some of these are listed in Table 1 (Lew and Reed, 1992). Their classification is based on their amino acid sequence. Yeast cyclins Many of the studies of cyclins were carried out with yeast. In S. cerevisiae, three proteins with some analogies to the known cyclins were identified. These proteins were found to be needed for Start. Elimination of one or two of the genes controlling these cyclins (CLN1, CLN2 and CLN3) by insertional mutations (that is, mutations that incorporate a piece of foreign DNA and eliminate the wild type gene, e.g., see Chapter 1) is deleterious and delays Start. However, elimination of all three produces large amorphous cells that cannot proceed past G1. These findings indicate that the three genes that code for cyclins have a similar function, and this function is crucial for the process of cell division. Do the yeast cyclins exhibit the same periodicity as those discussed for the sea urchin eggs? In such a study the cells must be dividing synchronously. In S. cerevisiae the cells can all be stopped at G1 in the presence of mating pheromone. When the pheromone is removed, the cells begin dividing synchronously. As we have seen before, proteins can be labelled by maintaining the cells in a medium containing radioactive amino acid. Each individual protein can be recognized and isolated using a specific antibody, which precipitates the antigen by crosslinking several molecules together (this procedure is called immunoprecipitation, see Chapter 1). Immunoprecipitation using CLN2antibody, showed that CLN2 protein appears cyclically, peaking at the G1-S transition. Similar experiments were carried out with other cyclins. Table 1 Cyclins in Yeast and Mammalian Cells. Reproduced from Lew and Reed, 1992. Reproduced by permission.
Cyclin CLN1 CLN2 CLN3 HCS26 CLB5 CLB3 CLB4 CLB1 CLB2
Species
Cell Cycle Function
Class
S. cerevisiae
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CLN CLN CLN B B B B B
G1 (START) G1 (START) G1 (START) G1 (START)? G1 (START)? G1-S and G2M? G1-S and G2M? G2-M G2-M
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CDC13 cig1 mcs2 puc1
S. pombe
B B
Cyclin A Cyclin B1 Cyclin B2 Cyclin C CyclinD1 Cyclin E
H. sapiens
A B B C D E
G1-S and G2M G2-M G2-M ? ? G1 or G1-S
CYL1 CYL2 CYL3
M. musculus
D D D
? ? ?
CLN
G2-M G1 or G1-S M? ?
Is the control of cyclins transcriptional? Transcriptional control would produce each cyclin mRNA at a different time during the cycle. Transcripts can be recognized by hybridization to cDNA probes (which are complementary to the mRNA) attached to nitrocellulose sheets (Northern blot, see Chapter 1). The results show that the amount of mRNA transcribed does indeed reflect the events of the cycle for CLN1 and CLN2. Careful comparison of the mRNAs shows that they slightly precede the synthesis of the cyclins. In contrast to these findings, the level of CLN3-RNA remains unchanged. For CLN1 and CLN2 these results indicate that the control is transcriptional. Furthermore, both the proteins and the mRNAs are degraded following the phase of the cycle that they trigger. While CLN1, CLN2 and CLN3 act at the G1-S transition, the B-class cyclins have different roles. CLB1 accumulates during the S and G2 phases and CLB5 at G1. Other cyclins are thought to play a role in the earlier stages. A diagrammatic summary of the synthesis and degradation of the various cyclin-mRNAs in yeast is shown in Fig. 5 (Lew and Reed, 1992).
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Fig. 5 Periodicity of cyclins during the cell cycle in Saccharomyces cerevisiae. Cyclins are grouped according to suggested times of action during the cell cycle and mRNA periodicity. As indicated by *, CLN3 is the exception to the rule in that its mRNA levels remain constant throughout the cell cycle. Reproduced from Trends in Cell Biology, vol.2, Lew, D.J. and Reed, S.I., A proliferation of cyclins, pp.77-80, copyright ©1992, with permission from Elsevier Science.
Mammalian cyclins As already mentioned, the major control of mitosis in mammalian cells is at the G1-S interface. The early studies with mammalian cells, however, only identified G2-M cyclins. As summarized in Fig. 5, several cyclins active at the G1-S interface have been identified in yeast. Why not use this knowledge to study mammalian cyclins? Generally, proteins that have a universal role are highly conserved from organism to organism. Why not search for cDNAs of mammalian cells that restore function in yeast strains lacking CLN1, CLN2 and CLN3 (Lew et al., 1991; Xiong et al., 1991; Koff et al., 1991)? The experiment was carried out by first inactivating the CLN genes of a S. cerevisiae strain by insertional mutation (mutations that replace the wild type gene with foreign DNA, see Chapter 1). A human cDNA library in yeast expression vectors would then be used to attempt to restore function. A cDNA library is synthesized in vitro from the mRNA present in cells, therefore the cDNA is complementary to the mRNA (c stands for complementary) and contains the sequence information needed for proteins produced by the cell (see Chapter 1). The rationale for this experiment is simple. However, the details of the design must be more complex. Deletion of three G1-S CLN genes in yeast is lethal: the cells are unable to divide. How can you get cells to reproduce without these genes to provide cells that can be used for the experiment? Furthermore, how can you select for the cells which have incorporated the mammalian cDNA? The production of a yeast strain from the transformation would be extremely rare and would be confused with possible spontaneous
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revertants. The task of bypassing these two problems would be difficult, if not impossible, without reaching into the bag of tricks of the molecular biologist. Expression of the CLN genes was put under the control of the experimenter by first fusing the GAL1 promoter region to the CLN2 coding region and then, using a vector, this DNA combination was incorporated into the yeast lacking the CLN genes. The transfected cells were able to grow in a galactose medium. However, they were CLN-less and unable to divide when placed into a glucose medium. The expression of the CLN provided by the vector could be turned on (when the cells have to be grown) or off (when they are to be tested for the CLN ability provided by the mammalian cDNA). Selection of cells containing components of the human cDNA library was obtained by combining the DNA with a yeast expression vector containing the URA2 gene. This gene provides an enzyme which allows the cells to produce their own uracil. Then the transformed cells could be selected in a medium lacking uracil and containing glucose. In a glucose medium they will be able to grow only if they contain the mammalian equivalent of the CLN gene and the URA2 gene. The experimental design used by Lew et al. (1991) is represented in Fig. 6. Fig. 6 Experimental design to identify mammalian CLN genes by replacing the missing genes in CLN- yeast.
A. How to grow cells missing the CLN yeast genes. (1) A vector containing the galactose promoter attached to CLN2 DNA transfects CLN- cells. (2) In a galactose-containing medium the CLN2 is expressed to produce CLN2-mRNA, allowing the cells to divide.
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B. Rescue of CLN- cells by mammalian cDNA. (1) Production of cDNA library from mammalian cell's mRNA. (2) Production of ura+cDNA (3) transfection of CLN- with ura+-cDNA; these cells will be viable if the cDNA contained the CLN+ mammalian gene, (4) transfection with DNA containing ura+ but no CLN+ : these cells will not grow because of the absence of CLN.
Three novel mammalian cyclin genes (C,D and E) were recognized in this way. Cyclin A was recognized in similar experiments; it reaches a maximum near the G2-M transition. In mammalian cells, the function of cyclin A has been examined by micro-injecting antisense cyclin A plasmids in rat fibroblasts in the G1 phase (Girard et al., 1991). The production of RNA that is antisense (that is, complementary) in relation to the mRNA of the target gene, in effect inactivates its translation supposedly by binding to the mRNA (see Chapter 1). The cells are then unable to replicate DNA. However, injecting cyclin A reestablished cell function. These experiments support a role of cyclin A either in the G1-S transition or in the S phase. Cyclin E-mRNA reaches a maximum near the G1-S transition. The physiological role of cyclin E was also examined by overexpressing cyclin E in fibroblasts by transfection with the appropriate cDNA (Ohtsubo and Roberts, 1993). In these cells, the duration of G1 and cell size are decreased and the cells are less dependent on serum, suggesting http://www.albany.edu/~abio304/text/8part1.html (13 of 29) [3/5/2003 7:53:42 PM]
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a role of cyclin E in the G1-S transition. The D-cyclins (D1, D2 and D3) are probably the major factors responsible for cells to enter the S-phase. Expression of cyclin D1 speeds up the progression through the G1 phase (Quelle et al., 1993; Resnitzky et al., 1994). The microinjection of anti-cyclin D1 antibodies or antisense plasmids prevents cells from entering the S phase (Quelle et al., 1993, Baldin et al., 1993; Lukas et al., 1994). Mice lacking the cyclin D1 gene have severe defects (Fantl et al., 1995; Sicinski et al., 1995). In contrast, strains in which the gene coding for cyclin D1 has been replaced by human cyclin E genes, have a normal phenotype. These results suggest the cyclin E is a downstream target of cyclin D (Geng et al., 1999). Basically, D and E cyclins function in the early stages and are generally thought of as G1 cyclins (see Sherr, 1993). In contrast, cyclins A, B1 and B2 predominate in the S and M phases (see Norbury and Nurse, 1992). B. The Cyclin-dependent Kinases Early experiments were able to demonstrate the involvement of other factors in the control of cell division. The experiments were carried out by the fusion of HeLa cells in culture, at either G1 or G2 stages, to cells in the S-phase (Rao and Johnson, 1970). The fusion was induced by introducing a virus to the mixed cultures. The cells in G1 which fused to the S cells synthesized DNA earlier, as estimated by [3H]thymidine incorporation (Fig. 7). Furthermore, the time at which the incorporation occurred was accelerated if 2 S nuclei were fused with G1 cells (Fig. 8). The presence of G2 nuclei did not delay the onset of DNA synthesis when G2 and G1 nuclei were fused (Fig. 9). The factor that promotes DNA synthesis has been referred to as the S-phase promoting factor (SPF). An additional factor must be present to promote the M-phase. Fusion of G2 to G1 or S cells induces an earlier onset of mitosis as shown in Fig. 10 for G1 nuclei. The proportion of G1 nuclei undergoing mitosis, as indicated by the mitotic index (the ratio of dividing cells/ all cells) in the ordinate, occurs earlier when G2 nuclei are fused to G1 nuclei. The greater the G2 dosage, the earlier the onset of mitosis. These results speak for a second factor, the M-phase or maturation promoting factor (MPF). The term "maturation" refers to the induction of mitosis in amphibian oocytes.
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Fig. 7 Rate of induction of DNA synthesis determined from the labelling with [3H]- thymidine in G1 nuclei of G1/S fused cells. Reproduced with permission from Nature, Rao, P.N. and Johnson, R.T.,225:159-164, copyright ©1970 MacMillan Magazines Ltd.
Fig. 8 Dosage effect on the induction of DNA synthesis in G1 nuclei in trinucleate cells. Reproduced with permission from Nature, Rao, P.N. and Johnson, R.T.,225:159-164, copyright ©1970 MacMillan Magazines Ltd.
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Fig. 9 Rate of induction of DNA synthesis comparing G1/G2, to G1/G2 and parent G1. Reproduced with permission from Nature, Rao, P.N. and Johnson, R.T.,225:159-164, copyright 1970 MacMillan Magazines Ltd.
Fig. 10 Dosage effect of the G2 component on the rate of mitotic accumulation. Reproduced with permission from Nature, Rao,P.N. and Johnson,R.T.,225:159-164, copyright ©1970 MacMillan Magazines Ltd.
Amphibian oocytes are arrested at the first meiotic division and remain at this stage until fertilization. After fertilization meiosis is completed and the egg begins a series of mitotic divisions. Extracts collected during the cell cycles of fertilized eggs, which contained MPF, were found to activate cell division in the arrested oocytes. The MPF was found to peak before each mitosis. The activity was assayed by microinjecting extracts into unfertilized oocytes and observing the activation of cell division (Masui and Markert, 1971). The extracts were prepared from fertilized http://www.albany.edu/~abio304/text/8part1.html (16 of 29) [3/5/2003 7:53:42 PM]
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eggs at different stages of the cell cycle. The study of the extracts led to the extensive purification of the protein (Lohka et al., 1988), which was found to be a complex of a protein kinase and a cyclin. The kinase activity depends on the presence of a cyclin. The kinases that require binding to a cyclin are referred to as cyclin dependent protein kinases (CDKs). The CDKs are activated at the transition points and presumably phosphorylate mitosis-specific substrates that have a key role in either initiating events, such as transcription or inactivating inhibitors of cell division (see next section). A model outlining some of the events in yeast is presented in Fig. 11A (Wittenberg et al., 1990). The models discussed here (for example, Fig. 1112) incorporate the knowledge available at the time they were formulated. Many more elaborations (including changes in nomenclature) were introduced later. These are areas of active research and many more additions and modifications can be expected (e.g., consult Morgan, 1995 for a review of CDKs). In Fig. 11A, the CLN genes are periodically transcribed and a different cyclin (Cln) is produced depending on the stage of the cycle. The CLN-mRNA and the Clns are degraded. As shown for G1/S and G2/M, the formation of the complex of the active Cln (perhaps phosphorylated) with the protein kinase (p34 in Fig. 11A; also referred to as p34cdc2; CDC2 is the gene coding for p34cdc2, in budding yeast CDC28) activates the latter to phosphorylate a "mitotic" substrate. The substrate could correspond, for example, to transcription factors such as E2F. Protein phosphatases that hydrolyze the phosphate of the phosphorylated proteins under the control of cyclins also play a role in the cell cycle. Cdc25-phosphatase of the MPF (the p34cdc2-cyclin B complex) allows the translocation p34cdc2 into the nucleus and its activation of mitosis. The phosphatase CDC14 plays a key role in the departure from the M-phase (see below). In yeast, the data are consistent with the presence of a single CDK (p34 in Fig. 11A) activated at different parts of the cycle (mostly at the G1-S, G2-M transitions) by a different cyclin. In contrast, in animal cells there are different CDKs which are activated by different cyclins. This is shown in the diagram of Fig. 11B. A protein kinase homologous to the yeast p34cdc2 is also present in mammalian cells and as indicated in the figure, it is essential to initiate the M-phase (see Nurse, 1990). p34cdc2 is one of the components of MPF. In normal animal cells, the centrosome reproduces only once per cell cycle. Then the two centrosomes separate and nucleate the mitotic spindle at the two poles. By ensuring a bipolar mitotic spindle, the cell avoids the misdirection of chromosomes that could lead to genetic instability (see Hinchcliffe and Sluder, 2001). Centrosome duplication requires the presence of active Cdk2-cyclin E in Xenopus (Hinchcliffe et al., 1999; Lacey et al., 1999) and in somatic human cells (Matsumoto et al., 1999). A protein kinase present in centrosomes which mediates centrosome duplication, has been found in Caenorhabditis elegans. However, this kinase is not needed for cell cycle progression. The absence of the corresponding gene (zyg-1) produces a monopolar spindle and the failure of duplication of the centrosome (O'Connell et al., 2001). The protein kinase, ZYG-1, acts at least one cell cycle before to each spindle assembly. In embryos, paternal ZYG-1 regulates duplication during the first cell cycle, and maternal ZYG-1 regulates subsequent duplications. In Drosophila embryos (Vidwans et al., 1999), Cdc25-phosphatase initiates mitosis and is also needed for daughter centriole assembly. Cdc20 by activating the APC initiates cyclin degradation and initiates transition from metaphase to anaphase (see below) and apparently also has a role in the separation of mother and http://www.albany.edu/~abio304/text/8part1.html (17 of 29) [3/5/2003 7:53:42 PM]
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daughter centrioles. In addition, the stabilization of cyclins prevent exit from mitosis and the assembly of the daughter centrioles.
Fig. 11A A model of the regulation and role of cyclin proteins in yeast. Two transitions, G1/S and G2/M are represented. See text for details (from Wittenberg et al., 1990). Reproduced by permission.
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Fig. 11B A model summarizing the interactions between cyclins and the cyclin dependent kinases in multicellular animal cells. The R point is the restriction point. The diagram shows the stages of the cell cycle and the binding of the specified cyclins with the corresponding CDKs at each stage. cdc2 is kinase, p107 and E2F are proteins involved in transcription. Reproduced from Trends in Biochemical Science,vol.18, Pines, J., Cyclins and cyclin-dependant kinases:take your partners, pp.195-197, copyright ©1993, with permission from Elsevier Science.
Binding of cyclin only partially activates the CDKs (see Morgan, 1995). The CDKs are subject to regulation by phosphorylation. They are fully activated only when phosphorylated at a threonine residue (Thr 161). They can be inhibited not only by dephosphorylation at threonine 161 but also by phosphorylation at tyrosine sites near the amino terminal. In order to activate the appropriate genes, most CDK/cyclin complexes must be translocated into the nucleus. Therefore, the localization of the various cyclins and CDKs, whether cytoplasmic or nuclear, is likely to play a role in the regulation of the cell cycle (see Yang and Kornbluth, 1999). What mechanisms determine the localization of these components? Apparently, a sequence of 42 amino acids the amino acid terminal region of cyclin B, the so-called cytoplasmic retention sequence (CRS), is necessary for a cytoplasmic localization of that cyclin (see Pines and Hunter
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1994). However, the CRS sequence contains a nuclear export sequence (NES) (see Chapter 5) (Hagting et al., 1998; Toyoshima et al., 1998) suggesting that, in actuality, the cyclin is capable of cycling between nucleus and cytoplasm. The cytoplasmic localization of cyclin B1 during interphase is directed by an NES-dependent export. In addition, the use of leptomycin B, an inhibitor of nuclear export, was found to lead to an interphase nuclear accumulation (Toyoshima et al., 1998) suggesting that normally the localization is determined by a balance between import and export. Although they have no obvious NLS domains, the mechanisms for the import of these complexes are beginning to be elucidated (Moore et al., 1999). Permeabilized mammalian cells were exposed to fluorescently labelled complexes of CDK2/cyclin E and Cdc2/cyclin B1. The nuclear import apparatus imports the complexes by direct interaction with the complexes. Whatever sequence is required for import is contained in the cyclin units. In contrast, the nuclear import of Cyclin A requires its CDK (Maridor et al., 1993g1). Cyclins E and B1 are imported by different mechanisms. Cyclin binds to the α subunit of importin-α and β. In contrast, cyclin B1 binds to importin-α alone. Knowledge of what genes are activated during the various phases would go far in completing the story of the mitotic cycle. Little by little new information is being collected. However, new technology, namely DNA arrays and chromatin-immunoprecipitation, has permitted taking a giant leap in this area for the G1/S transition. This transition initiates the whole cycle and for this reason has attracted a good deal of attention. In yeast, as shown in Fig. 11A the cyclins CLN1, CLN2, CLB5 and CLB6 appear at G1/S transition (along with other proteins). Their arrival is driven transcriptionally (e.g., see Fig. 5) in late G1. In addition, the CLN/CDK combination activates a variety of needed components such as transcription factors which initiate a cascade of biochemical events needed for the transition. The transcribed genes have been divided into two groups depending on the sequences motifs in their promoters. The SCB motif has been found in the promoters of CLN1 and CLN2 and in other genes. A second set has another motif, the MCB element. Two separate transcription factors, SBF and MCF, respectively, act on the two promoters (see Koch and Nasmyth, 1994). The two factors are heterodimers of Swi4 and Swi6 (SBF) and Mdp1 and Swi6 (MBF) (see Koch et al, 1993; Andrews and Herskowitz, 1989). Swi4 and Mbp1 are the components which bind DNA. Swi6 probably has a regulatory function (Primig et al., 1992 ; Dirick et al., 1992). Which are the genes activated in the G1/S transition? These genes can be identified by finding the DNA binding sites of SBF and MBF by immunoprecipitation with antibodies to SBF and MCF of chromatin (after chemical crosslinking) and using microarrays of most of the yeast genome. 200 probable targets were identified (Iyer et al., 2001). SBF was found to be primarily involved in budding as well as membrane and wall biosynthesis. In contrast, MBF was found to be involved in DNA replication and repair. C. Inhibitors of Cell Division Just as cyclins play a role as positive effectors of the cell cycle, other proteins block the cell cycle. As already discussed, in mammals progression through the cell cycle depends on several kinds of CDKs. These are constrained by CDK-inhibitors (CKIs) (see Morgan, 1995; Scherr and Roberts, http://www.albany.edu/~abio304/text/8part1.html (20 of 29) [3/5/2003 7:53:42 PM]
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1995, 1999). In mammals, CKIs have been assigned to two different groups on the basis of their structure and target CDKs. The inhibitors of CDK4 (INK4) block the catalytic subunits of CDK4 and CDK6 specifically. The Cip/Kip family of inhibitors are much less specific and interact with cyclin D, E and A-dependent kinases by binding to subunits of both cyclins and CDKs. In addition, they act as positive regulators of cyclin D-dependent kinases. The Cip/Kip family of inhibitors include three different gene products (p21, p27 and p35) with a broad range of specificity so that they are able to inhibit all of the G1 cyclin-CDK complexes and to some extent cyclin B-CDK (see Lees 1995). Some repressors of cell division such as as p53 (see below) act by a mechanism involving CKIs. In addition to the functions described in this section, certain CKIs have a role in producing senescence phenotype in mammalian cells (McConnell et al., 1998). The inhibitors of the Cip/Kip family can also promote the function of the cyclin-CDK complexes. Cyclin D-CDK complexes are resistant to Cip/Kip inhibition. The inhibitors promote cyclin D-CDK assembly (LaBaer et al., 1997, Cheng et al., 1999). In addition, the Cip/Kip inhibitors target cdk4 and cyclin D1 to the nucleus, needed for cell cycle progression. This does not conflict with the role of the inhibitor to function as such. p21 at low concentrations favors the assembly of active kinase complexes. Inhibition takes place at higher concentrations. RB-protein is a repressor protein involved in regulation. These repressors have been called "pocket proteins" (see Weinberg, 1995). They include several family members in addition to RB-protein (p107 and p130). Phosphorylation of pRb in mid-G1 is catalyzed by G1-phase CDKs. p107 is phosphorylated in mid- and late-G1-phase. The phosphorylation removes the repression. This section examines the involvement of three proteins: the RB-protein, p53, and p27. RB-protein There are three members of the RB-family of proteins: RB, p107 and p130. The three proteins are coded by separate genes (e.g., see Cobrinik et al., 1996) and they have different functions (see below). The RB gene product is a nuclear phosphoprotein of about 110 kDa which is present in all cells (referred to as pRb, p110RB or RB-protein). The RB gene earned its initials from its role in producing retinoblastoma. Retinoblastoma is a childhood cancer of the retina. Retinoblastoma cells lack the expression of normal p110RB and correspond to a mutation in both alleles of the RB gene. However, RB gene defects are also responsible for other carcinomas. Replacement of the missing functional RB gene by transfection with a retrovirus containing the gene, suppresses the tumorigenic potential of the cells (e.g., Bookstein et al., 1990). Besides possessing a domain capable of binding DNA, the protein can bind to transcription factors of the E2F family required for the S-phase to take place. These observations suggest a role of p110RB in regulating cell division by controlling the activity of E2F (see Weinberg, 1995). A role of RB-protein can be examined using purified RB-protein or RB-antibody (Goodrich et al., 1991). These experiments were carried out using osteosarcoma cells which lack expression of the RB gene. The cell cycle of these cells can be synchronized using the drug nocodazole, which blocks http://www.albany.edu/~abio304/text/8part1.html (21 of 29) [3/5/2003 7:53:42 PM]
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reversibly the formation of microtubules. The drug blocks in the M-phase, which requires microtubules for the formation of the mitotic spindle. When nocodazole is washed away, the cell resumes dividing. The synthesis of the DNA can be followed from the incorporation of bromodeoxyuridine, for which there is an antibody. When fixed cells are exposed to the the antibody labelled with a fluorescent probe, the fluorescence serves as a measure of DNA synthesis. Microinjection of RB-protein in early G1 inhibited cell division and the effect was blocked by the coinjection of RB-antibody. However, RB had no effect in late G1 or G1-S transition: injection of the RB-protein or the RB antibody at these later stages had no effect. These observations support a role of RB-protein in blocking cell division. One might presume, therefore, that the concentration of RB-protein decreases when cell division is induced. However, this is not true. Unlike the cyclins, the amount of RB-protein remains constant throughout the cell cycle. How could RB-protein function as an inhibitor of cell division? As we saw for other cases, RB-protein might be present in active and inactive forms. In many cases, the phosphorylative state of the protein determines its biological activity. Why not examine the phosphorylative state of RB-protein during the various cell cycle stages? A study of the phosphorylative state of the RB-protein (Buchkovich et al., 1989) was carried out with human tumor HeLa cells. The cells were grown synchronously after they were isolated at G1. The HeLa cells' size and mass change as they progress through the mitotic cycle. Thus, cells in different stages can be separated by centrifugation, because their sedimentation velocity increases with each stage. The technique permits separating cells that are primarily in G1 and S. However, the cells in G2 and M remain together. In this experiment, aliquots of the cell fractions were incubated with [32P]-Pi. After incubation, the cells were disrupted and the RB-protein was immunoprecipitated with an antibody, run on a gel (SDS-PAGE), and the location of the proteins recorded with fluorography. In fluorography (analogous to scintillation counting), the gel contains a fluor. The fluor emits light when excited by the radioactive disintegrations and the light emission is recorded on photographic film. The RB-protein was found to be relatively unphosphorylated at G1, the stage at which the control of cell proliferation is thought to take place, and becomes highly phosphorylated in the S phase and the G2/M phases. These results suggest that the unphosphorylated RB-protein is the molecule blocking the initiation of cell division and that phosphorylation of this protein releases the the block. Naturally, this conclusion raises a series of questions. What is responsible for the phosphorylation of RB-protein? How does RB-protein inhibit the cell cycle? The phosphorylation of RB begins in late G1 and continues until the M-phase (e.g., Weinberg, 1995). RB was shown to be phosphorylated by CDKs in vitro (Taya et al., 1989). At least three different CDKs are involved in the phosphorylation of RB (see Taya, 1997). Cyclin E is thought to be one of the activators of a CDK responsible for the phosphorylation. However, the D-cyclins, the major factors responsible for cells to enter the S-phase are also involved. Inhibition of the cell cycle by RB-protein is complex, because this protein binds as many as seven nuclear proteins. At least one of these proteins is a transcription factor, E2F. E2F binds DNA and has been found in complexes containing RB-protein and cyclin A. This suggests that the formation of the complex blocks transcription. Present information supports a model in which cyclin D-dependent kinases initiate RBprotein phosphorylation at mid-G1 phase after which cyclin E-CDK2 are activated and further http://www.albany.edu/~abio304/text/8part1.html (22 of 29) [3/5/2003 7:53:42 PM]
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phosphorylate the RB-protein (e.g., Lundberg and Weinberg, 1998). RB is maintained in a hyperphosphorylated form by cyclin A and cyclin B-dependent kinases until cells exit mitosis and then the RB-protein is returned to its hypophosphorylated form by a phosphatase (Ludlow et al., 1993). The RB-protein hyperphosphorylation in late G1 frees it from its association of the E2F transcription factors which permit the transcription of genes needed for DNA synthesis (e.g., see Dyson, 1998; Nevins, 1998). Among the trascribed genes are those coding for cyclin E and A and thymidylate kinase, needed for the G1/S transition (e.g., Pagano et al., 1992; Ohtsubo et al., 1995). Fig. 12 (Hollingsworth et al., 1993) summarizes some of these features. The arrow at the bottom of the figure indicates the stage of the cell cycle. Upon activation by an extracellular signal (a) the cyclin binds a protein kinase (b), which is activated by the binding and phosphorylates RB-protein (c). The phosphorylated RB-protein no longer binds to E2F that remains attached to the DNA, (d). The E2F begins transcribing the mRNA of a specific gene (e), and the cell is now able to proceed from G1 to S. The genes containing E2F-binding sites are crucial during the G1-phase.
Fig. 12 A model for the interaction between E2F and RB-protein induced by the cyclins during the cell cycle. (a) G1 cyclins are synthesized in response to an environmental stimulus. (b) A complex between cyclins and cyclin dependent kinase (CDK) which activates the kinase. (c) phosphorylation of RB-protein (d) releases E2F for (e) transcription of appropriate mRNAS (e.g., corresponding to http://www.albany.edu/~abio304/text/8part1.html (23 of 29) [3/5/2003 7:53:42 PM]
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activators of key reactions of cell division. From Hollingworth et al., 1993, reproduced by permission.(Available in the BioMedNet library at:http://biomednet.com/cbiology/cel)
However, the picture currently emerging is somewhat more complex than that outlined so far in this section. The key phases of cell division are regulated by the interaction of five proteins of the family of E2F transcription factors, composed of six members (E2F1-E2F6) (see Dyson, 1998) and the three proteins of the RB-family. The function of the proteins of the RB-family differs. In certain settings p107 and p130 perform growth-regulatory functions that are not fulfilled by pRB. ( e.g., Cobrinik et al., 1996). Indeed, pRB and p107/p130 have different roles in their interaction with E2F (see Hurford et al., 1997). RB-protein has other effects not involving CDKs but still blocking cell growth. RB specifically blocks the activation of the promoter of polymerase I, by binding the upstream binding factor (UBF) needed for the activation of transcription of the ribosomal DNA genes by polymerase I (see Cavanaugh et al., 1995). Similarly, RNA polymerase II (e.g., Weintraub et al., 1995) and III (White et al., 1996) are also repressed by RB. Therefore, RB-protein is likely to have a general effect in restraining protein synthesis, by blocking production of ribosomal and other small RNAs involved in translation, explaining further its anti-tumor effect. It is worth noting that RB can also act by activating transcription (see Section H, below). This is the case for the glucocorticoid-system that is not involved in proliferation, but in differentiation, so that this effect is not contrary to the usual role of RB. RB activates the glucocorticoid-receptor mediated transcription by binding to hBmr (Singh et al., 1995). hBmr is a gene that codes for a protein involved in regulating the activity of DNAbinding transcription factors. Recent data indicate that RB protein has an active role in repression, by forming part of an assembly that remodels chromatin. Unlike the process silencing heterochromatin which affects large chromatin sectors, RB only blocks the genes that are needed to enter the S-phase (see Brehm and Kouzarides, 1999). Repression of RB protein depends on a complex constituted by E2F, RB-protein, the histone deactylase HDAC1 and the hSWI/SNF nucleosome remodeling complex, thereby inhibiting the transcription of genes for cyclins E and A (Zhang et al., 2000) (see Chapter 2). E2F and HDAC1 do not interact directly but require the presence of the RB-protein order to bind. HDAC1 in the presence of RB represses the cyclin E promoter. The HDAC1-RB combination catalyzes the removal of acetyl groups from core histones (Brehm et al., 1998; Magnaghi-Jaulin et al., 1998; Luo et al., 1998). Removal of the charged groups supposedly produces a tighter association between DNA and nucleosomes and blocks the access of the transcription factors (see Chapters 2 and 3 and Wolffe, 1997). Luo et al. (1998) were able to show changes in acetylation at a Gal4-dependent promoter after binding a chimera of Gal4-RB. Normally, Gal4 is resposible for activating the genes needed to metabolize galactose in yeast. An antibody specific for acetylated histone H3 was found to precipitate promoter DNA only when the Gal4 DNA-binding domain was present and not when the Gal4-RB fusion protein was expressed, indicating that RB deacetylated the histone. This technique of chromatin immunoprecipitation is referred to as CHIP (see Chapter 1). In addition, RB has been found to act through a mechanism similar to that producing heterochromatin http://www.albany.edu/~abio304/text/8part1.html (24 of 29) [3/5/2003 7:53:42 PM]
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(see Chapter 2). RB binds to the complex of SUV39H1 and HP1 and represses the cyclin E promoter. RB is necessary to direct methylation of histone H3, and for binding of HP1 to the cyclin E promoter. Therefore, the SUV39H1-HP1 complex is involved in the repression of euchromatic genes by RB (Nielsen et al., 2001). However, in contrast to the silencing of heterochromatin, the events leading to repression of the cyclins is limited and may involve as little as one nucleosome. The action of RB is not always through the recruitment of histone deacetylase and this protein can act directly on transcription factors. Experiments with trichostastin, an inhibitor of histone deacetylase, show that it blocks RB-repression in the cases in which RB is recruited to the promoter. However, in the cases where RB acts by inhibiting neighboring transcription factors, the drug has no effect, as for example in the case of p107, a member of the RB-family. In epithelial cells, transforming growth factor β (TGFβ) is one of the regulators of the G1 to S transition (see Massague, 1996) by several mechanisms including the accumulation of CDKIs (e.g., see Sherr and Roberts, 1995). Cell-to-cell contact also acts by multiple mechanisms, including the increase of CDKIs (e.g., St. Croix et al., 1998). The CDK inhibitors prevent the phosphorylation of RB, thereby blocking cell division. Apparently both the contact inhibition and the effect of TGFβ are both mediated by a repression brought about by the RB-E2F complex (Zhang et al., 1999). p53 The gene coding for p53 is highly conserved and, like RB, functions as an inhibitor of cell proliferation (Milner, 1984). The mutations of this gene are responsible for in vitro transformation of cells. Furthermore, the loss of function in the two alleles for p53 is implicated in the production of 50 to 55% of human tumors (see Levine 1997), such as colorectal carcinoma and human lung carcinoma. The regulation of p53 has been recently reviewed (see Giaccia and Kastan, 1998) Activation of p53 can occur in response to a number of cellular stresses such as DNA damage, hypoxia and nucleotide deprivation. By blocking the division of cells and activating programmmed cell death (apoptosis) (see Chapter 2), p53 avoids genome instability that may produce multiple genetic alterations that result in tumor formation (see Hanahan and Weinberg, 2000). In the absence of activation, p53 is present mostly at a low level and in an inactive state which activates transcription inefficiently. When p53 is activated in response to DNA damage, the p53 level rapidly increases and is more effective in binding DNA and activating transcription. Apparently, the increase in activity is in part a reponse to phosphorylation, de-phosphorylation and acetylation events on the p53 polypeptide (see Agarwal et al., 1998; Lakin and Jackson, 1999). Several genes activated by signals involved in cell proliferation, checkpoint-arrest, DNA repair and apoptosis depend on p53 for their transcription (e.g., Di Leonardo et al., 1994; see Lakin and Jackson, 1999).
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How does p53 affect cell division? p53 protein has been found to have a transcriptional activation domain at the amino terminal and it binds to specific DNA sequences. In addition, some p53 mutants which fail to block cell proliferation have lost the ability to bind. These observations suggest that p53 could activate genes capable of inhibiting cell growth or could repress genes needed for cell growth or cell cycle progression (see also below). Both possible modes of action have been shown. p53 binds to, and activates, the growth arrest DNA damage (GADD) gene, a growth suppressor gene. Both the binding site in p53 (Kastan et al., 1992) and the response element in the GADD gene have been identified. Furthermore, a high level of p53 represses a large number of promoter regions of genes regulating the cell cycle (e.g., Ginsberg et al., 1991). The activation, synthesis and degradation of p53 are inextricably interwined. The level of p53 is a balance between its synthesis and its degradation. In undamaged dividing cells, p53 has a short halflife. In addition, p53 is largely inactive. However, after radiation damage, p53 is more stable, becomes activated and accumulates in the cell, followed by the expression of genes under its control. The instability of p53 depends on Mdm2. The oncoprotein Mdm2, binds to the amino terminal of p53 and targets it for degradation by the ubiquitination pathway, where it functions as an E3 ubiquitin-protein ligase ( Honda and Yasuda, 1999). The stability of p53 is regulated negatively by a feedback loop where Mdm2 decreases stability and the Mdm2 gene is activated in turn by p53. After treatment of cells with ionizing radiation, the action of Mdm2 is blocked by its phosphorylation in an ATM-dependent manner (e.g., Khosravi et al.,1999). ATM is discussed in more detail below. In a number of cellular responses to ionizing irradiation, the protein kinase, ATM, exhibits enhanced activity in all phases of the cell cycle (e.g., see Pandita, et al. 2000). Responding to DNA damage, Msm2 is phosphorylated by the protein kinases ATM and Chk2 (Khosravi et al.,1999). Since p53 is also activated by ATM, this protein kinase may promote p53 activity and stability by mediating the phosphorylation of both p53 and Msm2. What protein kinase or kinases are responsible for the phosphorylation of p53? A DNA-dependent protein kinase (DNA-PK) that requires the presence of DNA breaks for its activity (e.g., Morozov et al., 1994), has been suspected as the trigger for the action of p53 in response to DNA damage. However, this is not likely, since cells lacking DNA-PK show no defect in the p53 block of the cell cycle (Huang et al., 1996). CHK2 (the homolog of the checkpoint kinase Cds1 of Schizosaccharomyces pombe, and RAD53/SPK1 of Saccharomyces cerevisiae, also called hCds1), phosphorylates tetrameric p53. The phosphorylation at Ser20 of p53 stabilizes it ( Hirao et al., 2000; Chehab et al., 2000; Shieh et al., 2000). The human homolog of the S. pombe checkpoint kinase, Chk1 (hCHK1) also phosphorylates p53 in vitro at Ser20 (Shieh et al., 2000). p53 also exerts its effect through CKIs; in mammals the proteins p16, p21 and p27 (e.g., Harper et al., 1993). Tumor cells lacking p21 (Waldman et al., 1996) continue synthesizing DNA without mitosis, arguing for the absence of a checkpoint between the S and the M phase (possibly at the
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G2/M interface). p53 acts as a transcription factor for the p21/WAF1 promoter (see Levine, 1997). The p53 protein acts on the gene p21/WAF1, in conjunction with another protein encoded by another tumor suppression gene, ING1 (Garkavtsev et al., 1998). Neither protein alone can inhibit growth by themselves. p33INGI and p53 actually form a complex detected by immunoprecipitation (see Chapter 1). Some of the p53 actions require the gene for interferon regulatory factor, IRF1 (Tanaka et al, 1996), in addition to p33INGI. Whether the interaction actually involves a binding is not known at this time. The proliferation of eukaryotic cells requires maintenance of telomere length and function (see Chapter 2). Telomere dysfunction produces the activation of p53 leading to growth arrest (e.g., Chin et al., 1999) or apoptosis (Karlseder et al., 1999). Telomere loss occurs with cell division in telomerase deficient mice (Chin et al., 1999). It is overcome by a deficiency in p53 that leads to carcinogenesis. Two genes related to the p53 gene have also been found: p73 and p63 (p63 is also called p40, p51, KET, or p73L) (see Chen, 1999). These genes are expressed only in certain tissues. Each one of the corresponding proteins has several isoforms produced by alternative splicing of their mRNA (see Kaelin, 1999). The function of these additional molecules differ from that of p53. p73 is not induced by DNA damage and is not targeted for inactivation by viral oncoproteins. So far neither p73 nor p63 has been found to be mutated in human cancers. p73 is involved in apoptosis resulting from DNA damage (e.g., Gong et al., 1999) (see Section IIIJ). Both p73 and p63 have important developmental roles still under scrutiny. Mutations of p63 produce defects in limb and skin development in mice (Yang et al., 1999; Mills et al., 1999) and cause the ectrodactyly, ectodermal dysplasia and cleft lip (EEC) syndrome in humans (Celli et al., 1999). p73 mutations produce neurological and inflammatory defects (Yang et al., 2000). The involvement of RB-protein and p53 in the control of cell proliferation is now well established, although much has to be learned about the underlying mechanism. The involvement of other as yet unknown suppressor genes is suspected. Many investigators are now examining DNA lesions in tumor cells in the hope of unmasking other inhibitory proteins. Proteins coded by the INK4a gene The gene INK4a has a central role in proliferation and tumorigenesis. Mutations of this gene have been implicated in a variety of cancers (see Kamb, 1995). The gene is capable of producing two different proteins by alternative splicing of the corresponding RNA (e.g., Quelle et al., 1995): p16INK4a and p19ARF (corresponding to p14ARF in human cells). p16INK4a is a CKI that inhibits the association between CDK4/6 and D cyclins, blocking the phosphorylation of RB and thereby the exit from G1 (Serrano et al., 1993). p16INK4a is also necessary for the apoptosis that follows the loss of contact in nontransformed epithelial cells (Plath et al., 2000). Mice lacking p19ARF develop http://www.albany.edu/~abio304/text/8part1.html (27 of 29) [3/5/2003 7:53:42 PM]
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cancer (Serrano et al., 1996). This protein is involved in the arrest at G1 and G2 (Quelle et al., 1995), an effect that requires the presence of p53 (Kamijo et al., 1997). The mechanism is not known, but MDM2, which is involved in p53 degradation, has been implicated (e.g., Pomeranz et al., 1998). p19ARF appears to block the degradation (see Larsen et al., 1996; Zhang et al., 1998). The human supressor gene p14ARF (p19ARF in mice) is activated by E2F-1 (Bates et al., 1998). Therefore, the inactivation of RB (that frees E2Fs) can supress cell proliferation by this alternative mechanism, providing one more line of defence against unregulated cell growth. p27 p27 is a CKI specific for CDK2. p27 normally acts at the G1 stage. The absence of the p27-gene in mice, leads not only to increased cell proliferation in virtually every organ but also increased body size (e.g., Nakayama et al., 1996). The structure of CDK2, in its various states, has been fully elucidated. The structure of the cyclinCDK2-p27 complex has been recently reported (Russo et al., 1996) and accounts for the inhibition in molecular terms by binding cyclin, disrupting the structure of CDK2 and occupying the ATP binding site of the kinase. Eventually, p27 is phosphorylated before becoming a substrate for the ubiquitin/proteasome machinery (e.g., Sheaff et al., 1997; Vlach et al., 1997). A yeast two-hybrid system (see Chapter 1) identified a protein interacting with p27Kip1, a mouse protein coded by the Jab1 gene (p38) (Tomada et al., 1999). Increased levels of p38 caused increased breakdown of p27Kip1. The binding of the two proteins directed the translocation of p27Kip1 from the nucleus to the cytoplasm. Mutants lacking Jab1 remained in the nucleus and were not degraded. Proteasome inhibitors blocked the transfer and the degradation, possibly because the proteasomes may control the degradation of a factor blocking protein export form the nucleus. The involvement of translocation from the nucleus in the control of proteins factors regulating the cell cycle may have general applicability. Tumor viruses Some DNA tumor viruses act through their binding of RB proteins. Tumor DNA viruses can disrupt the normal controls of cell division and induce uncontrolled proliferation when they transform cells. Much can be learned about normal cell division by studies of how oncoproteins exert their effect. Several nuclear oncoproteins associated with tumor DNA-viruses have been found to interact with RB-protein. The regions of the oncoproteins interacting with RB-protein overlap with those needed for transformation. These results suggest that the protein generated from the DNA-virus acts by binding and inactivating the non-phosphorylated version of RB-protein in a manner analogous to the RB mutations leading to carcinogenesis.
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D. Initiation of DNA Replication In eukaryotes, the capacity to initiate DNA replication at G1 rests on the ordered assembly of multiprotein complexes at origins of DNA replication. Once these components are assembled DNA synthesis can proceed under the action of CDKs and the Cdc7 family of protein kinases. CDKs also have a role in the prevention of the formation of new initiation complexes (see Kelly and Brown, 2000). In Saccharomyces cerevisiae, the chromosomal origins of replication, the autonomously replicating sequences (ARS) are modular, composed of short sequences distributed in a region of 100 to 200 base pairs (bp). One of these sequences, the A element, contains 11 bp AT-rich, ARS consensus sequences (ACSs). The ACS is a component of the binding site for the origin recognition complex (ORC). The ORC of budding yeast has been purified. It functions in recruiting replication factors to the origins of DNA replication (see Dutta and Bell, 1997). Cdc6 in Saccharomyces cerevisiae (Cdc18 in S.pombe) has a central role in the initiation of DNA replication. High level of expression of Cdc18 initiates multiple rounds of DNA replication in the absence of mitosis (e.g., Muzi Falconi et al., 1996) Six related proteins, the minochromosome maintenance proteins (MCM), also have a role in DNA replication. All six form a complex apparently involved in DNA unwinding (see Ishimi, 1997). Cdc45 (Saccharomyces cerevisiae) and analogs in other organisms are essential of DNA replication and associate with chromatin periodically. After assembly, the initiation complexes are activated by the action of the CDKs and Cdc7-dbf4 kinase. Apparently, Cdc6/18 as well as the MCM proteins and various ORC subunits are phosphorylated by the kinases.
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E. The Inactivation of the Cell Cycle Regulators The timely removal of inhibitory proteins, the appearance of the cyclins and their activation of the appropriate kinases are responsible for the cell cycle. As with other biological regulators, removal of inhibitors, cyclins and kinases are as important as as their appearance. The inactivation of the CDKs involves two interacting systems: the degradation of the cyclins and in yeast the activity of the kinase inhibitor Sic1 which binds to the CDKs and inhibits their activity (see Morgan, 1997; King et al., 1996). The degradation of the cyclins takes place through ubiquitination (Chapter 15, Section 2B) and the proteolytic machinery of the 26S proteasome. Two distinct entry pathways open the proteolytic pathway. One of these is involved in the G1-S transition and uses an E2 known as CDC34 and involve the SCF complex (see below). The other underlies the onset of anaphase and the termination of mitosis and involves a complex of E3, the anaphase promoting complex (APC/C) or cyclosome. The vertebrate APC is a multicomponent complex (see Morgan, 1999). The APC of yeast has 12 subunits and most are homologous to the vertebrate variety. Its activity follows the cell cycle. First we will discuss the reactions involved in the proteolytic pathway. This will then be followed by a discussion of the regulation of the system. The ubiquitination pathway The details of the involvement of protein degradation in the cell cycle have recently been reviewed (King et al., 1996; Pagano, 1997; Townsley and Ruderman, 1998; Koepp et al., 1999). Cyclin degradation occurs via the ubiquitination pathway described in Chapter 15 (Section 2B). Ubiquitination involves three enzyme catalyzed processes. In an activating reaction, E1 uses ATP to form a thiolester between itself and ubiquitin. The activated ubiquitin is then transferred to E2. Some E2s catalyze the formation of an isopeptide bond between ubiquitin and the ε-amino group of lysine in the substrate molecule. The degradation of other proteins require a second protein, E3. Long polyubiquitin chains must be formed to direct proteins to the proteasomes. Polyubiquitination may require only E2, although both E2 and E3 may be involved in forming more than one kind of ubiquitin-ubiquitin bond (Kalderon, 1996). An additional factor, E4 (UFD2 in yeast) (Koegl et al., 1999), was found necessary for polyubiquitination. Novel E2s and E3s involved in the ubiquitination of cyclin A and B and other mitotic proteins have been identified in yeast and in cell free systems, generally derived from clam or Xenopus eggs (Irniger et al., http://www.albany.edu/~abio304/text/8part2.html (1 of 19) [3/5/2003 7:53:53 PM]
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1995; Hershko et al., 1994; King et al., 1995; Sudakin et al., 1995; Tugendreich et al., 1995). The cyclins preceding the M-phase are targeted for degradation by phosphorylation and ubiquitination by phosphoprotein-ubiquitin ligase E3 complexes and the SCF ubiquitin ligase system (the complex is named after its components one of which is an F-box containing protein) (see Skowyra et al., 1997). The SCF complex, responsible for the specificity of the degradation (see Craig and Tyers, 1999) is composed of the subunits Skp1, Rbx1, Cdc53 (in yeast) or Cull (mammals) and any one of a large number of different F-box proteins. Specific F-box proteins determine the specificity by recruiting a particular set of substrates for ubiquitination to the core complex composed of Skp1, Rbx1, Cdc53 and the E2 enzyme Cdc34. F-box proteins have a common F-box motif which links F-box proteins to Skp1 and the core complex and in addition a motif that binds to one of the various proteins to be degraded. Most SCF substrates are degraded in the early part of the cell cycle except for the mitotic regulator Wee1 which is degraded later. Wee1-like kinases inhibit CDK function by tyrosine phosphorylation and preventing entry into mitosis. In contrast to the G1-S components, mitotic B-type cyclins and some S-phase cyclins such as cyclin A (see Table 1 and Fig. 11B) are ligated to ubiquitin by the APC/C (the cyclosome) (Sudakin et al., 1995). The APC/C favors the entry into anaphase and the termination of mitosis. Mutations of some of the subunits of APC/C block the ubiquitination and degradation of mitotic cyclins (Zachariae and Nasmyth, 1996). They also arrest cells in metaphase. The APC/C also mediates the degradation of proteins responsible for the assembly and disassembly of the mitotic spindle. The proteolysis involving APC/C starts at anaphase continues during G1 and is stopped by cyclin-dependent kinases. The sections below will discuss in more detail the G1-S transition and the metaphase-anaphase transition separately. A large number of deubiquitination enzymes work in the opposite direction and play a significant regulatory role which is still to be fully evaluated. As an indication of their importance, present estimates suggest that more genes code for the deubiquitinating enzymes than for E2. In addition, in budding yeast, interference with the deubiquitinating enzyme activity disrupts the physiology of the cells (e.g., Zhu et al., 1996). Degradation requires the presence of specific amino acid sequences. For the G1 and S cyclins, A and B, this sequence is the destruction box of nine amino acids. The yeast G1 cyclins CLN2 and CLN3 contain a sequence with a similar mission, the PEST-sequence (see Deshaies, 1995). The destruction occurs after the cyclin containing the PEST-sequence is phosphorylated. Cdc28-protein kinase activity is required for phosphorylation (Lanker et al., 1996). Cdc34 kinase is required for cyclin destruction (Salama et al., 1994, Deshaies et al., 1995). However, it should be recognized that ubiquitination need not lead to degradation in all cases. For example, an entirely different mechanism is involved when the transcription factor Met4p is ubiquitinated. A gene or genes regulated by Met4p are thought to delay the G1-S transition (e.g., Patton et al. 2000). This transition requires inhibition by SCFMet30. Met30 is the substrate recognition F-box http://www.albany.edu/~abio304/text/8part2.html (2 of 19) [3/5/2003 7:53:53 PM]
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protein subunit of SCFMet30 which specifically targets the transcription factor, Met4p for ubiquitination. However, although the transcriptional activity of Met4p is blocked by ubiquitination, Met4p is not degraded (Kaiser et al., 2000). Apparently, ubiquinated Met4p associates with target promoters which become unable to initiate transcription. Met4p appears to control its own activity by regulating the amount of assembled SCFMet30 ubiquitin ligase (Rouillon et al., 2000). The ubiquitination of a ribosomal protein (L28) also has an entirely different role. In this case, the ubiquitins are attached to Lys63 of the ribosomal protein, an association that does not affect its stability. In Saccharomyces cerevisiae, the level of phosphorylation of L28 was found to vary with the stages of the cell cycle (highest during the S-phase, lowest during G1 or Go). Mutants at the Lys63 site were found o be defective in ribosomal function. The ubiquitination is likely to increase the association between the ribosomes and the nascent mRNAs and facilitate protein synthesis (Spence et al., 2000). M-G1 and the G1-S transitions In somatic cells and yeast the cycle, a prolonged G1 is required to allow enough time for growth or differentiation. Cdk inactivation in late mitosis activates Cdh1-APC so that cyclin continue being maintained to a minimum level throughout G1. At least in yeast, the Cdc20-APC dependent destruction of Clb2 was found to be necessary for mitotic exit (Wäsch and Cross, 2002). In mammalian cells, the CDK inhibitor p27 (discussed above), functions in the control of proliferation (see Sherr and Roberts, 1999). The entry into cell division requires its degradation which follows its phosphorylation by cyclin-CDK complexes (the SCF complex, see above). An F-box protein (see Craig and Tyers, 1999), SKP2 is the F-box protein of the ubiquitin-protein ligase that specifically recognizes phosphorylated p27 (Carrano et al., 1999) so that it is degraded by proteasomes. In budding yeast, a single CDK, Cdc28 (also referred to as Cdc2 , p34 or p34cdc2), combines with various cyclins (for the G1-S transition, CLN1, 2 and 3). The S-phase involves the cyclins CLB5 and CLB6, and the M-phase, CLB1 and CLB4. In higher eukaryotes, the equivalent cyclins are D and E during G1, cyclins E and A during S-phase and A and B during mitosis (see Table 1 and Fig. 11A and B). The gene coding for the E2, CDC34, is required for G1-S transition in budding yeast and it encodes a ubiquitinating conjugation step needed before initiation of the S-phase (Goebl et al., 1988). CDC34 is involved in the degradation of CLN2 and CLN3. In addition, it permits progression by mediating the degradation of the S-phase CDK inhibitor Sic1 (see Schwob et al., 1994) (see next section). The various substrates of CDC34 are degraded differentially during the cell cycle. The G1 cyclins rapidly turnover throughout the cycle in yeast (Willems, 1996) and so does Cyclin E in animal cells (Clurman et al., 1996). However, Sic1 is stable during G1 but is degraded upon entering the S-phase (Schwob et al., 1994; Donovan et al., 1994). The regulation of the stability of the substrates depends on the substrate specific phosphorylation which is required for CDC34-dependent ubiquitination. CLN2 and CLN3 are phosphorylated by Cdc28 (Deshaies et al., 1995; Yaglom et al., 1995).
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The stability of Sic1 is controlled by a similar mechanism. CDK activity is needed for the multiubiquitination of Sic1. An important function of the G1 cyclins is to phosphorylate Sic1 to allow for the ubiquitination mediated by CDC34 (Tyers, 1996). The degradation of the cyclins of G1 are needed before G2 cyclins can be effective. In yeast, the proteolysis of the G1 cyclins CLN1 and CLN2 requires the presence of the G2 cyclins CLB1, CLB2, CLB3 and CLB4, thereby providing a mechanism for coupling the two processes (Blondel and Mann, 1996). The metaphase-anaphase transition Anaphase is initiated by an increase in APC activity probably by its phosphorylation by CDKs and an increase in the level of an activator of APC, CDC20. In turn APC degrades cyclins and anaphase inhibitors. Proteolysis of cyclins involving the APC complex and inhibition of the CDKs by Sic1 interact for the exit from mitosis. In addition the protein phosphatase CDC14 plays an important role in these interaction. The APC recognizes a destruction box with a minimum sequence of RXXLXXIXXN (see Yamano et al., 1998). in Saccharomyces cerevisiae, APC is activated by two proteins, CDC20 and Hct/Cdh1 (see Morgan 1999, for the names in other organisms) (e.g., see Fang et al., 1998a,b). These two activators are likely to confer different substrate specificity and are activated by separate mechanisms. Initially, the APC is inactive because its activator, Cdh1, is inhibited by its phosphorylation mediated by cyclin B/CDK1. The CDC14 phosphatase is responsible for the dephosphorylation of Cdh1 (e.g., Zachariae et al., 1998; Jaspersen et al., 1999). As mentioned above, inhibition of the CDKs by Sic1 is important for terminating the M-phase. The regulation of the events involve an interplay between the kinase inhibitor and the phosphatase CDC14. The level of Sic1 depends on the balance between its production and its degradation. Sic1 synthesis depends on the transcription of its mRNA. On the other hand, Sic1 is targeted for degradation by CDK1 phosphorylation (Skowyra et al., 1997; Feldman et al., 1997). The role of the phosphatase CDC14 is complex. CDC14 increases Sic1. This effect depends on the dephosphorylation and consequent activation of the transcription factor Swi5. This activation permits an increase in transcription of the Sic1 mRNA. In addition, CDC14 interferes with the degradation of Sic1 by removing the phosphate (thereby making it less susceptible to degradation) and by decreasing the CDK1 activity (Visintin et al., 1998). The two together, the accumulation of Sic1 and the activation of APC by activators, bring an end to mitosis. As we saw, the presence and availability of the phosphatase CDC14, is of key importance in the regulation of mitosis by releasing cdh1 from its inactive state to activate ADC and by controlling the level of the CDK inhibitor Sic1. How is CDC14 regulated? The localization of CDC14 in the cell has shed more light on the mechanism controlling the arrest of mitotic cycle. Immunofluorescence studies http://www.albany.edu/~abio304/text/8part2.html (4 of 19) [3/5/2003 7:53:54 PM]
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(Visintin et al., 1999: Shou et al., 1999) found CDC14 in the nucleolus during G1 and the S phase. At the beginning of anaphase, CDC14 spreads throughout the nucleus and, to a limited extent, the cytoplasm. Therefore the activation of CDC14 may correspond to its release from the nucleolus. A nucleolar protein Cfi1 (Tem1 in yeast) (which might be a phosphatase) binds to CDC14, suggesting that it might be the anchor which holds it to the nucleolus. In agreement with this notion, cfi1 deletion mutants are unable to retain CDC14 in the nucleolus (Visintin et al., 1999, Shou et al., 1999) and the cells have difficulty in entering S phase. In Saccharomyces cerevisiae, the complex anchoring CDC14 to the nucleolus has been called RENT (Shou et al., 1999). The sequestration in nucleolus may be a general regulatory mechanism. The tumor suppressor protein p19Arf activates p53 by sequestering its inhibitor Mdm2 in the nucleolus (see Weber et al., 1999). F. Polo-like and Other Kinases We have seen that cyclins and CDKs have a central role in cell division. Other protein kinases have been shown to be produced and degraded at specific transition points in the cell cycle, e.g., NIMA protein kinase (a kinase coded by the nimA gene) has its highest concentration at the end of G2 and the M-phase and has a role in the regulation of the cell cycle. The polo-like kinases (plks), like the CDKs are also members of the serine/threonine kinase family. They play a complex and often distinct role in the progression throughout mitosis (see Glover et al., 1996; 1998), including centrosome maturation, bipolar spindle formation, activation of APC and cytokinesis (i.e., the actual separation into two cells) (see Field et al., 1999). The plks may function in conjuction with the CDK system. plk can help maintain the mitotic state by phosphorylating the Cdc25 phosphatase that activates p34cdc2 (Kumagai and Dunphy, 1996). MPF (the p34cdc2-cyclin B complex) allows the translocation p34cdc2 into the nucleus and its activation of mitosis. The nuclear translocation depends on the phosphorylation of the cyclin (e.g., Hagting et al, 1998). A protein kinase from Xenopus M-phase extract phosphorylates a serine residue in the middle of the nuclear export sequence of the cyclin. Apparently the kinase corresponds to a Polo-like kinase (homologous to plk-1). Antibodies to plk-1 block the kinase activity and an antiphosphate-serine147 antibody binds cyclin B1 only during the G2/M phase. Constitutive Plk1 stimulates the entry of cyclin B1 to the nucleus during phophase (Toyoshima-Morimoto et al., 2001). plks can also act separately from CDKs in maintaining functional centrosomes and a mitotic spindle. The polo1 mutation in Drosophila have a disorganized centrosome and an abnormal microtubular organization (Sunkel and Glover, 1988). The loss of polo-like kinases results in monopolar mitotic spindles in other cases as well. For example, the microinjection of Plk1-antibodies into human cells, blocks the cells in mitosis with monopolar spindles and unseparated centrosomes (Lane and Nigg, 1996). Immunological cytochemistry (see Chapter 1) indicates that Plk1 is associated with the polar region of the spindle from prophase to metaphase (Golsteyn et al., 1995). The Plk1 of S. cerevisiae behaves differently from the other systems studied. The cells can form a spindle. Nevertheless, cell division is stopped when the chromosomes have separated and the spindle is elongated (Hartwell et al., 1973).
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The polo kinase Cdc5 is needed for cytokinesis. HeLa cells transfected with either wild-type or mutant plk exhibit a failure of cytokinesis independently from its kinase activity ( Mundt et al., 1997). The kinase probably favors APC activation and the cytokinetic pathway (Song and Lee, 2001). G. Cohesion of the Chromatids For the M-phase to take place in an orderly manner, the sister chromatids have to be held tightly together. Failure to maintain cohesion between each pair, produces serious malfunctions. Congenital aneupleudy is one of the causes of Down's syndrome and chromosome-segregation defects uncover recessive mutations which can produce tumors. When the sister kinetochores are attached to the spindle, forces are generated which would separate the chromatids (Losada et al., 1998) if it were not held firmly together. Cohesion is responsible for the alignement during metaphase, needed for the proper distribution of the chromosomes. Generally, separation is initiated at anaphase, when the elimination of the cohesiveness triggers the separation of the sister chromatids (see Nasmyth, 1999). In Saccharomyces cervisiae, a complex of at least four subunits (Scc1, Scc3, Smc1 and Smc3), cohesin, is responsible for the cohesion (Michaelis et al., 1997; Guacci et al., 1997, Toth et al., 1999). Smc1 and Smc3 are members of the SMC family, which are putative ATPases with coiled-coil domains. Two other proteins are needed to establish cohesion (Toth et al., 1999; Uhlmann and Nasmyth, 1998; Skibbens et al., 1999). The separation of the sister chromatids occurs after the disappearance of Scc1 and Scc3. Both the dissociation of Scc1 from the chromosomes and the separation of the sister chromatids depend on a separin protein (Esp1) (Ciosk et al., 1998). Esp1 dissociates Scc1 from the chromosomes by stimulating its proteolysis (Uhlmann et al., 1999) thereby initiating the separation of the sister chromatids. Before this, Esp1 is held inactive by binding to the inhibitor Pds1 (Ciosk et al., 1998) until the inhibitor is ubiquitinated and then degraded at the metaphase-anaphase transition by the anaphase-promoting factor (APC) (Cohen-Fix et al., 1996). The activation of APC depends on CDC20 (see above). H. Anchorage Requirement and the Cell Cycle The dependence of cell growth on cell anchorage has been the subject of studies for many years. Benecke et al. (1978) showed that, in culture, absence of substratum inhibited mRNA and protein synthesis. This inhibition decreased with increasing degrees of cell transformation (Wittlesberger et al., 1981). The block in normal cells appeared to be in the G1-phase. The cyclins that play a role in the G1 phase are indicated in Fig. 11B. In addition, cyclin inhibitors (CKI) are also involved and the various factors interact in a complex manner (see Assoian, 1997). Cyclin D1 is
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the primary cyclin for several cell types. ECM and mitogens are jointly required to induce cyclin D1 expression. In the absence of substratum, quiescent cells in culture did not produce cyclin D1 mRNA even in the presence of mitogens. In addition, the production of cyclin D1 using preexisting mRNA is also blocked in the absence of a substratum. Cyclin E and CDK2, active in late G1, are also growth factor dependent and require attachement to the substratum. However, the induction is presumed to occur by decreasing the levels of CKIs. The induction of cyclin A, active in the S-phase, is also strongly dependent on cell anchorage. In this case, however, the mitogenic effect is through a mechanism in which RB or p107 are phosphorylated. These effects of anchorage are profound because the lack of these enzyme activities precludes phosphorylation of pRb and p107 and the subsequent activation of E2F-dependent transcription. The effects of anchorage are mediated in some way by the cytoskeleton. Adhesion and dependence on the cytoskeleton appear to go hand in hand (Böhmer et al., 1996). For example, cytochalasin D, that disrupts the cytoskeleton, blocks the progress through G1. However, as is the case for anchorage, cytochalasin is totally without effect between the Go and the S-phase. Anchorage and the cytoskeleton are needed for the phosphorylation of Rb-protein. The ECM and mitogenic growth factors can also act synergistically. Cell adhesion to substratum and the aggregation of integrins in focal contacts, activate several signalling molecules (see Bottazzi et al., 1997). Many of these signalling molecules are also activated by mitogenic growth factors. The extent of signaling and progression through the cell cycle is determined by the interaction between receptor tyrosine kinases and integrins. The molecular connections between these two pathways is still unknown. I. Licensing Factors In proliferating eukaryotic cells, DNA replication is confined to the S phase. In this process each sequence in the chromosomes is replicated only once. Consequently, DNA replication does not occur again until the segregation of the chromosomes in mitosis. The mechanisms for preventing additional replications during the mitotic cycle could be positive or negative. That is, a factor could induce the replication or, alternatively, the control could be through an inhibitor that prevents replication. The inactivation of the inhibitor would then initiate replication. The induction of DNA synthesis and mitosis by the S-phase promoting factor (SPF) and the maturation promoting factor (MPF), argues for a positive control, which has been referred to as a replication licensing factor (RLF). Xenopus egg extracts support the initiation and synthesis of the entire DNA complement, which replicates only once. Rupture of the nuclear envelope, however, allows cells arrested in G2 to re-replicate with fresh extract (Blow and Laskey, 1988). This finding argues that the RLF licenses only in the absence of a functioning nuclear envelope during the M-phase. Such a mechanism would clearly allow only one replication per cycle. SPF induces initiation of the "licensed" origins and terminates the license. The action of RLF and SLF is sequential. RLF cannot cross the nuclear envelope and it can license the DNA synthesis only during http://www.albany.edu/~abio304/text/8part2.html (7 of 19) [3/5/2003 7:53:54 PM]
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mitosis. However, the SPF can initiate DNA replication on licensed DNA even in an intact nucleus. However, the sequential nature of the process is guaranteed by the fact the both activities occur at specific points in the cells cycle. RLF is abruptly activated after the metaphase-anaphase transition and is degraded at interphase (Blow, 1993). In contrast the SPF activity can be detected only in interphase (Blow and Nurse, 1990). The RLF activity is found in two components, both needed for licensing, RLF-M and RLFB (Chong et al., 1995; Tada et al, 1999). RLF-M is a complex of six members of the MCM/P1 family (see Tye, 1999) such as MCM4 discussed below, originally identified in yeast. Inactivation of the MCM proteins in mammalian cells by microinjection with antibodies, blocks DNA replication in the S-phase (Kimura et al., 1994). All six assemble at the replication origin during the late stages of mitosis and G1 and form a pre-replicative complex (PreRC) (see Tye, 1999). Proteins in this family are: (1) localized in the nucleus in G1 and late mitosis in yeast (e.g., Hennessy et al., 1990), (2) present in a broad variety of eukaryotes ranging from plants to mammals (Chong et al., 1996, Kearsey et al., 1996), (3) required for the replication of DNA in higher eukaryotes (e.g., Todorov et al., 1994, Treisman et al., 1995), and (4) reassociated with chromatin only after permeabilization or breakdown of the nuclear envelope (e.g., Chong et al, 1995, Kubota et al., 1995). Only two contain nuclear localization sequences (NLSs) so that it is likely that the complex moves to the nucleus after being assembled. Two other proteins are involved: the originrecognition complex (ORC) and Cdc6. The presence of CDKs acts in the opposite direction by blocking initiation. One of the mechanisms that insures that DNA replication occurs only once per cell cycle rests in the spatially separation of key components such as MCM4 and template DNA. In yeast cells, MCM4 is exported from the nucleus when no longer needed. This process avoids re-replication. MCM is present in the nucleus in late mitosis and the G1 phase and cytoplasmic during the S-phase, the following G2 phase and early mitosis (Hennessy et al., 1990). The movements of MCM can be followed using MCM4 fused to green fluorescent protein (GFP) (see Chapter 1) (Labib et al., 1999). The MCM-GTP hybrid was found in the nucleus only in the absence of CDK activity. The inhibition by CDKs is probably indirect, the result of the activation of an inhibitor which requires the presence of CDKs (Mahbubani et al., 1997). When the CDKs are inactivated in the G2-phase, the MCM-GFP returns to the nucleus. In mammalian cells, entry of the MCM proteins into the nucleus does not determine their binding to the chromatin. The binding requires a rupture of the nuclear membrane, suggesting the presence of another factor involved in the licensing, a loading factor, which cannot pass through the nuclear envelope (Madine et al., 1995). In Saccharomyces cerevisiae, other experiments indicate that the cyclin B-Cdk complexes prevent replication during S, G2 and M phases - probably by blocking the transition of replication origins to a prereplicative state (Dahmann et al., 1995). Experiments carried out with cell free extracts of Xenopus eggs, show that CDK2-cyclin E and A kinases negatively regulate DNA synthesis. Once the chromatin is assembled, CDK2 kinase is accumulated in the chromatin 100 fold. CDK2-cyclin E did not block the http://www.albany.edu/~abio304/text/8part2.html (8 of 19) [3/5/2003 7:53:54 PM]
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association of the ORC complex with sperm chromatin, but prevented MCM3 from associating (Hua et al., 1997). A role of CDK2 in limiting the replication of DNA has been shown in rat fibroblasts, where inhibitors of cdc2 activity produce multiple rounds of replication in the absence of mitosis (Usui et al., 1991). J. Sequential Processes Controlled by Checkpoints As discussed above, the cell cycle seems to be regulated to a large extent by interactions between cyclins, CDKs, inhibitory proteins and their degradation. These proteins are instrumental in activating and deactivating specific sequential steps. A sequential order could also be imposed by the nature of the reactions. For example, reactive sites that permit the addition of the next component may be created by the previously synthesized protein. This process would introduce a well-defined sequential assembly pathway. Such endogenous sequential processes undoubtedly play a role in cell division. For example, aster formation requires the presence of functioning microtubule organizing centers. However, there may be special mechanisms to produce a sequential pattern. A temporal inhibition may take place so that a particular step is not activated until the preceding step is completed. In effect, these controls act as checkpoints and represent a block in some of the essential steps in the mitotic progression. The precise mechanism of these controls is not entirely clear at this time. They are of obvious importance in maintaining function and avoiding abnormalities that could lead to serious problems such as malignancy. Completion of DNA replication and DNA damage After one round of duplication, the re-initiation of DNA replication has to be blocked to maintain a single replication per cell cycle. CDKs have been implicated in blocking re-initiation in eukaryotes (see Kelly and Brown, 2000) by preventing the assembly of preinitiation complexes at the origins (see above). High CDK activity in the G2/M prevents the association of MCM proteins with the origin of duplication. In addition the CDKs phosphorylate Cdc6. In Saccharomyces cerevisiae, B-type CDKs block re-initiation via three inhibitory pathways: nuclear exclusion of MCM 2-7 complex, phosphorylation of the origin recognition complex (ORC) and inhibition of Cdc6 activity ( Nguyen et al., 2001). Checkpoints responsive to DNA damage stop the cell cycle either at G1 (G1-DNA-damage checkpoint) or just before mitosis (G2-DNA-damage checkpoint). The loss of checkpoints involved in blocking cell division because of DNA damage have been implicated in human cancers and genetic instability (e.g., see Hartwell and Kastan, 1994; Paulovich et al., 1997). Cell fusion experiments (see section IIIB, above) show that when cells in the S phase and G2 phase are fused, the G2 nucleus is delayed in entering mitosis until the DNA duplication of the S-phase cell is completed (Rao and Johnson, 1970). These experiments highlight how checkpoints work. Similarly, interference of DNA synthesis, either by the introduction of specific inhibitors or mutational inactivation of enzymes involved in DNA synthesis, prevents mitosis.
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How do incomplete DNA replication or damage prevent the completing of mitosis? The mechanisms and targets of these controls are still unclear. However, several patterns are beginning to emerge. Entry into mitosis requires dephosphorylation by the Cdc25-phosphatase of the MPF (the p34cdc2-cyclin B complex) which allows the translocation p34cdc2 into the nucleus and its activation of mitosis. Many of the checkpoints have been found to converge on this mechanism. p34cdc2 (also referred to as Cdc2 and in budding yeast, Cdc28) is the protein kinase involved in initiating several phases of the cell cycle in Saccharomyces cervisiae and some steps in many other systems, including mammals (see Fig. 11A and 11B). At interphase, the complex is maintained inactive by kinases which phosphorylate p34cdc2 (in Schizosaccharomyces pombe, wee1 kinase). The cell cycle starts only after p34cdc2 is dephosphorylated by the phosphatase Cdc25. Conversely, a block in the cell cycle at G2 persists as long as p34cdc2 remains phosphorylated (see Elledge, 1996). Phosphorylation of p34cdc2 prevents the activation of MPF (Nurse, 1990). The binding of Cdc25-phosphatase to a 14.3.3 protein blocks its action so that p34cdc2 remains phosphorylated. DNA damage leads to the degradation of Cdc25 (Mailand et al., 2000) blocking the progression. The 14-3-3 protein family (see Aitken, 1996) (so named because of their two-dimensional migration pattern on DEAE-cellulose chromatography and starch gel electrophoresis) has seven known members in humans. Homologues have been found in plants, insects, amphibians and in the nematode Caenorhabditis elegans as well as in fission and budding yeast. The homologues in budding yeast are Rad24 and Rad25. The 14-3-3 proteins bind to phosphoserine of certain proteins such as Cdc25-phosphatase (e.g., Muslin et al., 1996). In fission yeast, the components of this checkpoint were identified by means of a genetic screen based on the sensitivity to radiation. Without a functional check point responding to DNA damage, mutant cells begin mitosis despite the presence of damaged DNA. Among other proteins, Chk1 (a serinethreonine kinase) and Rad24, were identified as involved in the block. Chk1 and Rad24 act through the Cdc25-phosphatase. In both fission yeast and human cells, damaged DNA activates the protein kinase, Chk1. In turn, Chk1 phosphorylates the Cdc25-phosphatase creating a phosphoserine binding site for a 14.3.3 protein (e.g., see Peng et al., 1997). However, Cdc25-phosphatase is active whether bound to the 14.3.3 protein or not. Why then is the phosphorylated Cdc25-phosphatase able to block mitosis? The study of Lopez-Girona et al. (1999) provides the answer to this paradox. They found the Cdc25-phosphatase is mostly cytoplasmic and, in contrast, its substrate, cyclin B/p34cdc2 , is mostly nuclear. The localization of the Cdc25-phosphatase acts as a regulator of cell division. When the DNA is damaged, the phosphorylated Cdc25-phosphatase leaves the nucleus combined with the protein Rad24. Thereby Cdc25-phosphatase becomes separated spatially from its substrate, the cyclin B/p34cdc2 kinase, which is nuclear, and the kinase remains phosphorylated and inactive. Therefore, the initiation of mitosis is blocked. In contrast, in the absence of DNA damage, the cell cycle is initiated when some Cdc25-phosphatase molecules are translocated to the nucleus. Presumably, the cell cycle is triggered when the phosphatase, Cdc25, overwhelms the effect of the kinase, wee1. Two related protein kinases, either together or separately, appear to coordinate the signals following DNA damage: ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia and Rad3 related) (see http://www.albany.edu/~abio304/text/8part2.html (10 of 19) [3/5/2003 7:53:54 PM]
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Shiloh, 2001). The two are serine/threonine protein kinases of the phosphatidylinositol 3-kinase (PI3K) family. ATM functions mostly in response to DNA strand breaks and ATR in several different checkpoint responses. Although they follows similar pathways, their functions are quite different as shown by mutations. In humans, ATM mutation produces ataxia telangiectasia, a condition with multiple defects and progressive neurodegeneration. Mutation in the ATR gene in mice, which leads to early embryonic death, produces extensive chromosomal fragmentation. In mammals, ATM, ATR and p53 are gene products that play a role in maintaining the integrity of the genome by controlling checkpoints (see Morgan and Kastan, 1997; Shiloh, 2001; Walworth, 2001). Although ATM and ATR were both found to act via the phosphorylation of p53, supposedly ATM responds to ionizing radiations, whereas ATR responds to UV radiation. ATR may also act independently. ATR can act by activating the Chk1 kinase by phosphorylation (see Liu et al., 2000). The p53 protein is an important player in the G1 DNA-damage check point (Kastan et al., 1991). However, p53 also makes a contribution to the G2-DNA damage checkpoint. p53, as discussed above, acts as an inhibitor of cell division. ATM is also involved in other cellular processes such as S phase and G2-M phase arrest and in radiosensitivity. The kinases of the ATM family are needed for the checkpoint arrest following DNA damage or incomplete replication. Disruption of the ATM gene is responsible for causing a disease with cerebellar dysfunction, chromosomal instability and pre-disposition for cancer (Xu and Baltimore, 1996; Xu et al., 1996; Xu et al., 1999). ATM is part of a complex that contains BRCA1 (coded by the breast cancer gene 1, Brca1). ATM, Brca1 and Brca2 are tumor suppressor genes (see Kinzler and Voglestein, 1997). BRCA1 is required to maintain genetic stability by regulating the duplication of the centrosomes and provide a G2-M checkpoint (Xu et al., 1999). Therefore ATM has a dual role, one in the cell cycle and the other in cell-cycle arrest when DNA is defective (Xu and Baltimore, 1996). ATM phosphorylates BRCA1 after γ radiation (Cortez et al., 1999), although BRCA1 is also controlled independently of ATM (Scully et al., 1997). Presumably Brca1 and Brca2 are involved in the recombination repair after DNA damage (see Moynahan et al., 1999). Brca1 has also been implicated in transcription (e.g., Anderson et al., 1998; Kleiman and Manly, 1999). p53 is also be phosphorylated by ATM or by an ATM homologue (e.g., Canman et al., 1998; Lakin et al., 1999; Tibbets et al., 1999). Mutations of the Brca1 gene are associated with breast and ovarian cancers (e.g., Miki et al., 1994). These mutations account for 45% of families with high incidence of breast cancer and for 80-90% of families with both breast and ovarian cancer. BRCA1 has several motifs characteristic of transcription factors and has been shown to function as a transcription factor (e.g., Monteiro et al., 1996): its carboxy-terminal region, fused to GAL4 DNA binding domain has been shown to activate transcription of GAL4 (see Chapter 1). BRCA1 has also been found to be present in a complex related to SWI/SNF, a complex with chromatin remodeling activity (see Chapter 2 and 3). The binding is to the BRG1, the ATPase subunit indispensable for its action in chromatin. BRCA1 mutants were unable to act as coactivators of transcription which also requires p53 (Bochar et al., 2000).
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In the G1 arrest, the cyclin-dependent kinase inhibitor p21WAF1/clp1/SA11 (e.g, Brugarolas et al., 1995) mediates the effect. p21 is transcriptionally regulated by p53 (Xiong et al., 1993). p53 also activates the transcription of other proteins such as 14-3-3σ (Hermeking et al., 1997) in response to DNA damage and blocks the cell cycle at G2 (Hermeking et al., 1997). In the presence of p53, Cdc25-phosphatase cannot activate p34cdc2 because the phosphatase remains in a phosphorylated form, bound to 14-3-3 proteins. As we saw above, the phosphorylated phosphatase is tranferred to the cytoplasm and cannot act on the kinase. Damage apoptosis (see White, 1996), whether induced by malignancy (e.g., see Hartwell and Kastan, 1994), radiation damage (e.g., Lowe et al., 1993), hypoxia (Graeber et al., 1996) or infection by certain viruses, genrally involves p53. ATM has also been implicated in triggering apoptosis via the phosphorylation of p73 (See Shaul,2000). p53 exerts its effect through several CKIs (such as p21). The inhibition is reversible if the defect is minor or of short duration. However, in the absence of a recovery the cell proceeds to apoptosis (see Chapter 2). Apoptosis is a systematic process of programmed cellular death. It takes place in well-defined steps in individual cells, including cytoplasmic condensation and formation of cytoplasmic protuberances, chromatin condensation and DNA fragmentation (see Wyllie et al., 1980). In addition to a role in cell-cycle arrested cells, apoptosis plays a role in organogenesis, tissue homeostasis and in the functioning of the immune system. The role of 14-3-3σ in p53-controlled checkpoints was evaluated by producing knockout somatic cells (see Chapter 1) lacking the 14-3-3 protein. After DNA damage the cells were arrested at G2 but could not maintain the arrest and the cells died upon entering mitosis. The failure to block mitosis was caused by the absence of sequestration of cyclin B1 and p34cdc2 (Chan et al., 1999b) by 14-3-3σ. Normally, the p34cdc2-cyclin B1 complex is shuttled between nucleus and cytoplasm (see Yang, J. and Kornbluth, S., 1999) All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners, Trends Cell Biol. 9:207-210. Yang and Kornbluth, 1999). In the knockout mutants, the absence of 14-3-3σ allows the p34cdc2/cyclin B1 to accumulate in the nucleus and eventually bypass the block. The results of other studies, some with Aspergillus nidulas have shown other aspects of DNA damage checkpoints. In addition to the phosphorylation of p34cdc2 , the phosphorylation of a component (Cdh1 also called Hct1) of the anaphase promoting complex (APC) (Ye et al., 1996; Zachariae et al., 1996; Peters et al., 1996) is also involved. Phosphorylation of Hct1 block its incorporation into APC, eliminating the degradation of cyclins. Another check point, at least in A. nidulans, is the regulation of the mitosis-promoting NIMA protein kinase (Osmani and Ye, 1995, 1996). NIMA appears to be activated by p34cdc2cyclin B (Ye et al., 1995). The presence of both protein kinases is necesary for the G2-M transition. Mutations of CHK2 have been shown to be responsible for the predisposition to cancer in individuals with Li-Fraumeni syndrome ( Bell et al., 1999). This gene codes for checkpoint kinase CHK2 (Cds1 in
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Schizosaccharomyces pombe or Rad53 in Saccharomyces cerevisiae). Studies of the CHK2-deficient mice indicate that this kinase acts upstream from p53. The mice had a defective CHK2 gene produced by gene targeting (see Chapter 1). These cells, have several defective checkpoint after irradiation ( Hirao et al., 2000). The CHK2-deficient cells were embryonic mice stem cells lacking arrest in G1 (but having an arrest in G2) and apoptosis or thymocytes. The CHK2 absence failed to maintain G2 arrest in the stem cells and the deficient thymocytes were defective in DNA-damage apoptosis. The deficient cells were also defective in p53 stabilization (see discussion of p53 stability, above) and p53 dependent transcripts (such as that of p21). Normal function was reestablished by re-introducing the CHK2 gene. CHK2 stabilizes p53 which has a central role in G1 arrest. Therefore, the absence of CHK2 produces an inability to enter apoptosis (which depends on p53) and the G2 damage checkpoint (not requiring p53). Two genes related to the p53 gene (see Section IIIC) have also been found: p73 and p63 (p63 is also called p40, p51, KET, or p73L). The two genes are expressed only in certain tissues. Each one of the proteins has several isoforms produced by alternative splicing (see Kaelin, 1999). The p73 protein is involved in responses due to DNA damage. p73 has been found to be a target of non-receptor tyrosine kinase-cAbl following DNA damage (Gong et al., 1999). DNA damage activates c-Abl kinase activity (Kharbanda et al., 1995). Both cAbl and p73 act together in producing apoptosis (Agami et al., 1999; Yuan et al., 1999). In addition to the checkpoints dependent on completion of functional DNA duplication, other checkpoints are under study. These include the block to the separation of chromosomes when the mitotic spindle is damaged or chromosomes fail to attach properly to the spindle, the anaphase to metaphase transition which requires: the completion of anaphase, the inhibition of cell division until they have reached a critical size, the prevention of a new round of DNA replication until completion of the M phase, and the requirement for DNA synthesis before the centrosome can duplicate. The centrosomes are required for the G1-S transition (Hinchcliffe et al., 2001) and the completion of cytokinesis ( Piel et al., 2001). These finding suggest that there may be checkpoints linked to centrosome function. When other DNA replication and damage checkpoints fail, another mechanism causes mitotic spindle defects and chromosome-segregation failures and can be considered an additional check-point (see Sibon et al, 2000). This check point results in inactivation of the centrosome with dissociation of the γ-tubulin ring complex. Checkpoints of the mitotic apparatus The capture of kinetochores by microtubules during mitosis is a random process (e.g., see Nicklas, 1997). The orderly distribution of the chromosomes requires that mitosis be delayed until all chromosomes are properly aligned at the spindle equator. The checkpoint responsible for this control is sensitive even to a single unattached kinetochore (Rieder et al., 1995). The mechanism by which an unattached kinetochore inhibits entry into anaphase is unknown. However, it appears to respond to the attachment of kinetochore to microtubules (Rieder et al., 1994) and the level of tension exerted on the kinetochore (Li and Nicklas, http://www.albany.edu/~abio304/text/8part2.html (13 of 19) [3/5/2003 7:53:54 PM]
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1995). Immunofluorescence microscopy (see Chapter 1) using antibodies sensitive to phosphoproteins has demonstrated that kinetochore proteins are phosphorylated when the chromosomes are unattached and become dephosphorylated after the chromosomes attach to the spindle (Gorbsky and Ricketts, 1993; Nicklas et al., 1995). Misattached chromosomes remain phosphorylated. In order to identify kintechore phosphoproteins, this technique requires the extraction of soluble phosphoproteins from the cell sections and the inhibition of endogenous phosphatases which remove the phosphates from the phosphoproteins. Micromanipulation accompanied by immunofluorescence with the phosphoprotein antibodies demonstrates that tension obtained by micromanipulation causes kinetochore protein dephosphorylation, while relaxation causes rephosphorylation (Li and Nicklas, 1997). In view of these findings, it would not be surprising to find that protein kinases are associated with this checkpoint. What is the mechanism of chromosome separation and what are the events underlying the checkpoint that block it? After DNA replications the sister chromatids are held together by cohesin complexes (see Nasmyth, 2001). The separation of the two sister chromatids to opposite spindle poles depends on the intervention of separins which mediate the release of cohesins from the chromatids. However, the activity of separins is blocked by a class of proteins known as securins (PTTG in vertebrates) . The separation of the chromosomes during the metaphase-to-anaphase transitions requires the degradation of the securins initiated by a subunit of APC (CDC20), an ubiquitin ligase. CDC20 is one of the target of the checkpoint. When the degradation is blocked, the chromosome cannot separate. We have already seen that APC is involved in the progression of cell division to allow the initiation of cell division (see Section IIIC). How is CDC20 blocked? The protein Mad2 at unattached kinetochores binds and inhibits the activity of APC (see Shah and Cleveland, 2000). Other proteins are also present in unattached kinetochores such as Bub3, Mad1 and the mitotic kinases Bub1, MAP kinase and BubR1 (the mammalian Mad3) and may play a role in the checkpoint. A complex which has been called the mitotic checkpoint complex (MCC) (Sudakin et al., 2001) is composed of the hBubR1, hBub3, CDC20, and Mad2 checkpoint proteins in near equal stoichiometry. MCC inhibits APC/C. However, MCC is not assembled at kinetochores and is also present and active in interphase cells. Only APC/C isolated from mitotic cells was sensitive to inhibition by MCC suggesting the presence of an activator. The centromeres of misaligned chromosomes inactivate the APC by sequestering the protein CDC20, essential for APC function by forming a complex with MAD and BUB proteins. Without an active APC the progression is arrested APC is also required for spindle disassembly and cytokinesis (see Hardwick, 1998). The mitotic cyclins (A and B in higher eukaryotic and CLB in budding yeast) function as activators of CDK1 (CDC28 in budding yeast), required for spindle formation and pre-anaphase processes (e.g., see King et al., 1994). They are degraded via APC ubiquitination. The metaphase allignement of the chromosomes as well as the separation of the chromosomes require movement, indicating an involvement of motors. However, the motor proteins have been found to be needed for the spindle checkpoint itself. Three microtubule motors and some binding proteins have been found in kinetochores. CENP-E is a kinesin-like motor (plus-end directed) active in metaphase allignment
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and checkpoint activity (e.g., Yao et al., 2000; Abrieu et al., 2000). CENT-E binds to spindle microtubules as well as kinetochore-associated checkpoint kinase BUBR1. CENP-E is involved in delivery of components from kinetochores to poles. Depletion of this motor leads to checkpoint activation (Yao et al., 2000). Cytoplasmic dynein is the only minus-directed motor that might drive the poleward movement of the sister chromatids after anaphase (see Banks and Heald, 2001). The contribution of dynein at the knetochore for chromosome movement in prometaphase and anaphase was demonstrated by preventing the expression of AW10 or ROD, proteins needed for localizing dynein to the kinetochores. (Savoian et al., 2000). Dynein inhibitors were found to disrupt the alignment of kinetochores on the metaphase spindle equator and also chromatid-to-pole movements during anaphase A (Sharp et al., 2000). Zw10 and Rod not present in yeast, are also required for the spindle checkpoint in metazoan cells (e.g., Chan et al., 2000). Therefore, dynein may be the target of spindle-checkpoint regulators. K. The Centrosomes Centrosomes are present in most animal cells, but they are missing in higher plants, some meiotic cells, eggs and certain embryos. When present, centrosomes have been shown to be the major MT organizing centers (MTOCs) and are thought to have a role in spindle assembly. The assembly of MTs at MTOCs is also discussed in Chapter 23. Centrosomes play an important role in most cells (see Doxsey, 2001; Rieder et al., 2001). Their absence in some cells suggests that unrecognized components may fill a similar role. Many important questions about centrosomes are still at least in part unresolved including their precise function, the mechanism of centrosome duplication and assembly, as well as the regulation and mechanism of the centrosomal microtubule nucleation activity (see Andersen, 1999). In non-dividing cells, the centrosomes are usually located in approximately the center of the cell near the nucleus. They are composed of two centrioles and pericentriolar material. Centrioles are cylindrical in shape and composed of nine sets of triplet microtubules. The centrioles are at right angle from each other. The centrioles contain several specific proteins such as centrin, cenexin and tektin. The α-β- tubulin subunits of the centrioles are modified, in one case by polyglutamylation (see Andersen, 1999; Bobinnec et al., 1998). Centrioles are involved in the assembly of other centrosome components. Most kinds of cells have a non-motile cilium formed from the oldest "mother" centriole ( Roth et al., 1988). Basal bodies of cilia and flagella, responsible for their formation, are similar to centrioles. Normally, microtubular nucleation occurs mainly at centrosomes. The MTs are anchored to the centrosome through their minus ends, whereas the plus ends are toward the cytoplasm. The microtubules formed inside the cell have 13 protofilaments (see Evans et al. 1985) (the number is variable when formed in vitro). The precision of the number of protofilaments in vivo, suggests the formation of MTs on a template. This template has been found to be the γ-tubulin ring complex (γTuRCs) (Stearns and Kirschner, 1994; Zheng et al., 1995). Small and large complexes are present in most cells with only small
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complexes in budding yeast. Supposedly, the γ-tubulins are organized in a ring which can interact either longitudinally (the template model) or laterally with αβ-tubulins (the protofilament model) (see Erickson and Stoffler, 1996; Erickson, 2000). Anchoring and nucleation are considered two different processes. The apical region of epithelial cells (Mogensen et al., 1997, 2000) has anchoring domains but the molecules involved in nucleation such as γtubulin or perincentrin are lacking. Pericentrin is involved in the recruitment of γTuRCs to centrosomes in vertebrate cells (Dictenberg et al., 1998). Only two proteins have been isolated at this time in the anchoring sites: ninein and centriolin. In recent years, the role of centrosomes has had to be reevaluated. They have been found not to be essential for spindle formation in mammalian cells (Hinchcliffe et al., 2001; Khodjakov et al, 2000) in experiments in which centrosomes were removed either microsurgically or by laser ablation. Nevertheless, when present they have a dominant function in MT assembly (Heald et al., 1997). Rieder et al. (2001) suggest that their role may be more important for whole organisms since stable cell lines can be established in the absence of centrosomes ( Debec et al., 1995). However, in Drosophila a mutant lacking centrosomin (a core component of centrosomes in Drosophila) can develop into an adult fly (VaizelOhayon and Schejter, 1999; Megraw et al., 2001), despite the absence of centrosomes, a defective formation of astral microtubules and cytoskeletal defects during embryonic development. In mammals, centrosomes are required for formation of astral MTs and for positioning the mitotic spindle (Khodjakov and Rieder, 2001), although apparently alternative mechanisms exist (e.g., Megraw et al. 2001). Centrosomes have been found to have a role in the checkpoints of the cell cycle. In the absence of centrosomes, somatic cells are arrested in G1 (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001). In Drosophila embryos, mutations in the DNA-replication checkpoint produce an inactivation of centrosomes in mitosis ( Sibon et al., 2000) preventing chromosome segregation. In addition to their role in checkpoints, centrosomes are thought to anchor regulators of cellular functions such as cAMP-dependent protein kinase A (PKA) via A-kinase anchoring proteins (AKAPs) (see Diviani and Scott, 2001; Feliciello et al., 2001). The replication of the centrosomes takes place during the G1-S phase transition and is completed before the cells enter mitosis, a process requiring cdk2 (see (Hinchcliffe and Sluder, 2001). After replication each daughter cell contains one old and one new centriole. In humans and mice, the centrins of somatic cells are centrin 2 and 3. In HeLa cells, RNA interference (RNAi) (see Chapter 1) blocks the synthesis of centrin-2, a calcium binding protein of centrioles, and the centrioles are unable to duplicate during the cell cycle (Salisbury et al., 2002) . In the absence of centrin-2 synthesis the pair of centrioles separate, and bipolar spindles have only one centriole at each spindle pole. The cells can undergo division, however, without centrioles they do not separate by cytokinesis in subsequent cell cycles, they are multinucleated and die. http://www.albany.edu/~abio304/text/8part2.html (16 of 19) [3/5/2003 7:53:54 PM]
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Although there are abundant observations of the details of the duplication of centrosomes (see Doxsey, 2001), the molecular mechanisms are still unresolved. The duplication of centrosomes was found to depend on the Ca2+-CAM-CAMKII system (see above) L. Regulation of Translation We have emphasized how cell division depends on transcriptional events and the degradation of both mRNAs and certain proteins. However, in order to be effective in promoting cell division, mitogens must also increase the activity of the translational machinery. Accordingly, the production of rRNA and 5S RNA is decreased during quiescent periods and increased in response to growth factors (Johnson et al., 1974). The control might involve the RB gene that, in addition to its control of cell division, also inhibits rRNA and 5S RNA production (Cavanaugh et al., 1995; White et al., 1996). In addition to the recognized role of the RB gene, the ncl-1 gene of the nematode Caenorhabditis elegans controls rRNA and 5S RNA synthesis by acting as a repressor of the transcription of ribosomal and 5S RNA (Frank and Roth, 1998). The expression or repression of this gene is, therefore, involved in the regulation. Part of the action of mitogens is to trigger signals that converge on the 70 kDa S6 kinase (p70S6K) (see Chou and Blenis, 1995). When the ribosomal subunit S6 is phosphorylated by p70S6K, there is an increased translation of mRNA (Jefferies et al., 1997). In particular, the translation of mRNAs involved in cell-cycle progression is increased. Inactivation of p70S6K with injection of the appropriate antibodies (Lane et al., 1993) into cells, or by treatment with rapamycin, causes G1 cell arrest in many cell types (Chou and Blenis, 1995). The activation of p70S6K is sequential by phosphorylation at three specific sites to produce a fully active kinase (Pullen and Thomas, 1997). 3-phosphoinositide-dependent protein kinase 1 (PDK1) is thought to be responsible for these phosphorylations (Alessi et al., 1997; Pullen et al., 1998). The activation of p70S6K is triggered by mitogens apparently acting through phosphatidylinositol 3kinase, protein kinase B and C and phospholipase C (see Chou and Blenis, 1995). IV. SUMMARY The cell cycle proceeds in steps controlled by mechanisms that have been highly conserved. In this process, genes are activated in a specific order. In mammalian cells, some of the initial steps of G1 depend on the presence of growth factors. The regulation of cell division involves an interplay between the signals that block cell division and those that activate the steps of the cell cycle. The activating signals, the cyclins, are synthesized and then degraded at the steps they control. The cyclins act by forming a complex with other proteins, and this complex has protein kinase activity. The phosphorylation can either activate or inhibit transcription factors. Further control is provided by the need to complete a step (e.g., DNA synthesis) before the next step is initiated (e.g., mitosis). http://www.albany.edu/~abio304/text/8part2.html (17 of 19) [3/5/2003 7:53:54 PM]
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SUGGESTED READING Brehm, A. and Kouzarides, T. (1999) Retinoblastoma protein meets chromatin, Trends Biochem. Sci. 24:142-145. (Medline) Dyson, N. (1998) The regulation of E2F by pRB-family proteins, Genes Dev. 12:2245-2262. (Medline) Elledge, S.J. (1996) Cell cycle checkpoints: preventing an identity crisis, Science 274:1664-1672. (Medline) Heichman, K.A. and Roberts, J.M. (1994) Rules to replicate by, Cell 79:557-562. (Medline) Hunter, T. and Pines, J. (1994) Cyclins and cancer II: cyclin D and CDK inhibitors come of age, Cell 79:573-582. (Medline) Johnston, L.H. (1992) Cell cycle control of gene expression in yeast, Trends in Cell Biol. 2: 353-357. King, R.W., Jackson, P.K. and Kirschner, M.W. (1994) Mitosis in transition, Cell 79:563-571. (Medline) Lees, M. (1995) Cyclin dependent kinase regulation, Current Opin. Cell Biol. 7:773-780. Morgan, D.O. (1995) Principles of CDK regulation, Nature 374:131-134. (Medline) Murray, A. and Hunt, T. (1993) The Cell Cycle. An Introduction. Freeman and Co., New York, Chapters 5-8. Nasmyth, K. (1999) Separating sister chromatids, Trends Biochem. Sci. 24:98-104. (Medline) Nicklas, R.B. (1997) How cells get the right chromosomes, Science 275:632-637. (Medline) Nurse, P. (1994) Ordering S phase and M phase in the cell cycle, Cell 79:547-550. (Medline) Pagano, M. (1997) Cell cycle regulation by the ubiquitin pathway, FASEB J. 11:1067-1075. (Medline) Prives, C. (1993) Doing the right thing: feedback control and p53, Curr. Opin. Cell Biol. 5:214-218. (Medline) Romanowski, P. and Madine, M.A. (1996) Mechanisms restricting DNA replication to once per cell cycle. MCMs pre-replicative complexes and kinases, Trends in Cell Biol. 6:184-188. http://www.albany.edu/~abio304/text/8part2.html (18 of 19) [3/5/2003 7:53:54 PM]
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Scherr, C.J., (1994) G1 phase progression: cyclin on clue, Cell 79:551-555. REFERENCES Search the textbook
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Chapter 8: References
ieder Back to Chapter 8
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Baldin, V., Lukas, J., Marcorte, M.J., Pagano, M. and Draetta, G. (1993) Cyclin D1 is a nuclear protein required for cell cycle progression in G1, Genes Dev. 7:812-821. (Medline) Banks, J.D. and Heald, R. (2001) Chromosome movement: dynein-out at the kinetochore, Curr. Biol. 11:R128-131. (MedLine) Bates, S., Phillips, A.C., Clark, P.A., Stott, F., Peters, G. Ludwig, R.I. and Vousden, K.H. (1998) p14ARF links the tumour supressors RB and p53, Nature 395:124-125. (Medline) Bell, D.W., Varley, J.M., Szydlo, T.E., Kang, D.H., Wahrer, D.C., Shannon, K.E., Lubratovich, M., Verselis, S.J., Isselbacher, K.J., Fraumeni, J.F., Birch, J.M., Li, F.P., Garber, J.E. and Haber, D.A. (1999) Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome, Science 286:2528-2531. (MedLine) Benecke, B.-J., Ben-Ze'ev, A. and Penman, S. (1978) The control of mRNA production, translation and turnover in suspended and reattached anchorage depedent fibroblasts, Cell 14:931-939. (Medline) Blondel, M. and Mann, C. (1996) G2 cyclins are required for the degradation of G1 cyclins in yeast, Nature 384:279-282. (Medline) Blow, J.J. (1993) Preventing re-replication of DNA in a single cell cycle: evidence for a Replication Licensing Factor, J. Cell Biol. 122:993-1002. (Medline) Blow, J.J. and Laskey, R.A. (1988) A role of the nuclear envelope in controlling DNA replication within the cell cycle, Nature 332:546-548. (Medline) Blow, J.J. and Nurse, P. (1990) A cdc2-like protein involve din the initiation of DNA replication in Xenopus egg extracts, Cell 62:855-862. (Medline) Bobinnec, Y., Moudjou, M., Fouquet, J.P., Desbruyeres, E., Edde, B. and Bornens, M. (1998) Glutamylation of centriole and cytoplasmic tubulin in proliferating non-neuronal cells, Cell Motil. Cytoskeleton 39:223-232. (MedLine) Bochar, D.A., Wang, L., Beniya, H., Kinev, A., Xue, Y., Lane, W.S., Wang, W., Kashanchi F. and Shiekhattar R. (2000) BRCA1 is associated with a human SWI/SNF-related complex: linking chromatin remodeling to breast cancer, Cell 102:257-265. (MedLine) Böhmer, R.-M., Sharf, E. and Assoian, R.K. (1996) Cytoskeletal integrity is required throughout the mitogen stimulation phase of cell cycle and mediates the anchorage-dependent expression of cyclin D1, Mol. Biol. Cell 7:101-111. (Medline)
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(1999)Centrosome amplification and a defective G2-M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells., Mol. Cell 3:389-395. (Medline) Yaglom, J., Linskens, M.H., Sadis, S., Rubin, D.M., Futcher, B. and Finley, D. (1995) p34Cdc28mediated control of Cln3 cyclin degradation, Mol. Cell Biol. 15:731-741. (Medline) Yamano, H., Tsurumi, C., Gannon, J. and Hunt, T. (1998) The role of the destruction box and its neighbouring lysine residues in cyclin B for anaphase ubiquitin-dependent proteolysis in fission yeast: defining the D-box receptor, EMBO J. 17:5670-5678. (Medline) Yang, J. and Kornbluth, S. (1999) All aboard the cyclin train: subcellular trafficking of cyclins and their CDK partners, Trends Cell Biol. 9:207-210. (Medline) Yang, A., Schweitzer, R., Sun, D., Kaghad, M., Walker, N., Bronson, R.T., Tabin, C., Sharpe, A., Caput, D., Crum, C. and McKeon, F. (1999) p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development, Nature 398:714-718. (MedLine) Yang, A., Walker, N., Bronson, R., Kaghad, M., Oosterwegel, M., Bonnin, J., Vagner, C., Bonnet, H., Dikkes, P., Sharpe, A, McKeon, F. and Caput, D. (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours, Nature 404:99-103. (MedLine) Yao, X., Abrieu, A., Zheng, Y., Sullivan, K.F. and, Cleveland, D.W. (2000) CENP-E forms a link between attachment of spindle microtubules to kinetochores and the mitotic checkpoint, Nature Cell Biol. 2:484-491. (MedLine) Ye, X.S., Xu, G., Pu, R.T., Fincher, R.R., McGuire, S.L., Osmani, A.H. and Osmani, S.A. (1995) The NIMA protein kinase is hyperphosphorylated and activated downstream of p34cdc2/cyclin B: coordination of two mitosis promoting kinases, EMBO J. 14:986-994. (Medline) Ye, X.S., Fincher, R.R., Tang, A., O'Donnell, K. and Osmani, S.A. (1996) Two S-phase checkpoint system, one involving the function of both BIME and Tyr15 phosphorylation of p34cdc2, inhibit NIMA and prevent premature mitosis, EMBO J. 15:3599-3610. (Medline) Yuan Z.-M., Shioya, H., Ishiko, T., Sun, X., Gu, J., Huang, Y.Y., Lu, H., Kharbanda, S., Weichselbaum, R. and Kufe, D. (1999) p73 is regulated by tyrosine kinase c-Abl in the apoptotic response to DNA damage, Nature 399:814-817. (Medline) Zachariae, W. and Nasmyth, K. (1996) TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast, Mol. Biol. Cell 7:791-801. (Medline)
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Zachariae, W., Shin, T.H., Galova, M., Obermaier, B. and Nasmyth, K. (1996) Identification of subunits of the anaphase-promoting complex of Saccharomyces cerevisiae, Science 274:1201-1204. (Medline) Zachariae, W., Schwab, M., Nasmyth, K. and Seufert, W. (1998) Control of cyclin ubiquitination by CDKregulated binding of Hct1 to the anaphase promoting complex, Science 282:1721-1724. (Medline) Zhang, Y., Xiong, Y. and Yarbrough, W.G. (1998) ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways, Cell 92:725-734. (Medline) Zhang, H.S., Postigo, A.A. and Dean, D.C. (1999) Active transcriptional repression by Rb-ELF complex mediates G1 arrest triggered by p16INK4a, TGFβ, and contact inhibition, Cell 97:53-61. (Medline) Zhang, H.S., Gavin, M., Dahiya, A., Postigo, A.A., Ma, D., Luo, R.X, Harbour, J.W. and Dean, D.C. (2000) Exit from G1 and S phase of the cell cycle is regulated by repressor complexes containing HDACRb-hSWI/SNF and Rb-hSWI/SNF, Cell 101:79-89. (MedLine) Zheng, Y., Wong, M.L., Alberts, B. and Mitchison, T. (1995) Nucleation of microtubule assembly by a γtubulin-containing ring complex, Nature378:578-583. (MedLine) Zhu, Y., Carroll, M., Papa, F.R., Hochstrasser, M. and D'Andrea, A.D. (1996) DUB-1, a deubiquitinating enzyme with growth-suppressing activity, Proc. Natl. Acad. Sci. USA 93:3275-3279. (Medline)
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9. Endocytosis
9. Endocytosis I. Receptor-mediated Endocytosis II. The LDL Receptor Family A. The LDL Receptors B. Other Receptors of the LDLR Family III. Other Receptors A. Involvement in Endocytosis B. Fate of Ligand and Receptor C. Hydrolysis, Recycling, and Sorting IV. Coated Pits A. Clathrin and Adaptor Complexes B. Structure of Clathrin and Assembly C. Sorting of Proteins D. Budding and Disassembly V. Caveolae, Rafts and Other Membrane Patches VI. Other Forms of Endocytosis VII. Endosomes and Lysosomes: Interactions VIII. Transcytosis IX. Involvement of the Cytoskeleton Suggested Readings Web Resources References Back to List of Chapters No discussion of cell structure can do justice to its dynamics. Cell components are in continuous motion and change. Even when cells appear to be in a steady state, membrane-enclosed vesicles are continuously formed at the cell surface, material is taken up and processed, new molecules are produced and components are broken down. The present chapter is concerned with the events of endocytosis. In endocytosis, cells ingest extracellular materials by trapping them in invaginations of the cell membrane, which then pinch off to form membrane-lined intracellular vesicles. Many of these events result in the trafficking of vesicles between the surface and the cell's interior and these topics will be treated in this chapter. Some of pertinent molecular details are presented in Chapter 11: they are common to other forms of intracellular transport. The trafficking between the site of synthesis and other parts of the cell will be discussed in Chapters 10 and 11. Since most of the trafficking involves a variety of intracellular vesicles and compartments, this account also concerns membranes.
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9. Endocytosis
Endocytosis includes a very broad spectrum of cellular activities including pinocytosis (the uptake of liquids) and phagocytosis (the uptake of large particles). There are several distinct endocytotic mechanisms (see Mukherjee et al., 1997; Lamaze and Schmid, 1995a). The discussion of this chapter includes the involvement of coated pits, (Sections IV) caveolae (Section V) and still other forms of endocytosis (Section VI). At this time, the relative importance of the various forms of endocytosis is not clear. There is evidence that clathrin mediated endocytosis is the major mechanism of fluid uptake in some cells (e.g., baby hamster kidney cells, BHKC; Griffiths et al., 1989). However, mechanisms not involving clathrin are thought to be significant in fluid uptake in other cells (e.g., Tran et al., 1987, Hewlett et al., 1994; see Sandvig and van Deurs, 1994). For example, fluid-phase endocytosis continues despite pharmacological blocks of clathrin mediated endocytosis (see Sandvig and Van Deurs, 1994) In protozoans endocytosis has a role in feeding. Macrophages, specialized phagocytic cells of multicellular organisms, use endocytosis to remove foreign particulate material. This is then followed by digestion in the lysosomal system. However, endocytosis is not limited to specialized cells. All mammalian cells are thought to be capable of endocytotic uptake which typically proceeds at a prodigious rate. In mouse fibroblasts the amount of surface membrane taken up by endocytosis has been estimated to be 50% per hour (Pearse, 1975). Endocytosis has several recognized roles, some already discussed. It permits internalization of receptors, nutrient uptake, antigen presentation, pathogen internalization and the maintenance of plasma membrane surface (see Riezman et al., 1997; Marsh and McMahon, 1999). Endocytosis generally requires receptors. Receptors are integral membrane proteins that specifically bind a ligand with high affinity. The receptors involved in the endocytosis of growth factors and polypeptide hormones are the same ones that initiate the cascade of events which are responsible for the effect of the ligands (Chapters 6 and 7). Receptors and channels are taken up by endocytosis only after monoubiquitination (see below. Ubiquitination, especially in relation to degradation of proteins is also discussed in Chapter 15). Monoubiquitination marks a protein at the cell surface for endocytosis in both Saccharomyces cervisiae and in mammals (see Hicke, 2001). The monoubiquitination of proteins of the endocytotic machinery also has a role in endocytosis. A short amino acid motif, the ubiquitin-interacting motif (UIM) at the carboxy termini of proteins is required as a signal to monoubiquitinate a protein and is also required for the recognition of the protein by the endocytotic machinery (Polo et al., 2002; Raiborg et al., 2002; Shih et al., 2002). See below for a discussion of ubiquitination. In receptor-mediated endocytosis, after the specific binding of ligands to the surface receptors, other proteins and components present in the medium are taken up without any selectivity as part of the pinching-off process of endocytosis. The purpose of receptor-mediated endocytosis of metazoans is not always clear. In some cases http://www.albany.edu/~abio304/text/chapter_9.html (2 of 41) [3/5/2003 7:54:40 PM]
9. Endocytosis
endocytosis provides biologically important compounds to the interior of the cell. The low density lipoprotein (LDL) receptor system functions in the processing of cholesterol by the cell. Transferrin receptors function in iron metabolism and are responsible for the uptake of transferrin that carries iron. The physiological role of the receptors at the cell surface is the best understood. Their importance is shown by the failure of certain drugs, such as alkylamines or the antibiotic bacitracin, to block the mitogenic activity of epidermal growth factor (EGF), while greatly interfering with the movement of receptors to coated pits needed for endocytosis (Maxfield et al., 1979). However, the functioning of receptors after endocytosis is well documented (e.g., Sorkin et al., 1993; Zhang et al., 2000). In addition. in the case of the nerve growth factor (NGF), the receptors at the two different cellular locations appear to have different functions. Survival responses are activated by the receptors at the cell surface, whereas responses leading to differentiation were found to depend on their presence in endosomes ( Zhang et al., 2000). Some regulative factors have been shown to exert their effect in the cell interior. Supposedly, endocytosis allows them to reach their target. The uptake of nerve growth factor (NGF) is likely to have a role in transporting the NGF to intracellular targets. There are indications that part of the activity of insulin and other factors may be exerted directly at intracellular sites such as the cell nucleus. Microinjection of insulin into Xenopus oocytes increases RNA and protein synthesis, indicating an intracellular receptor site (Miller, 1988). Furthermore, insulin (Harada et al., 1992) and insulin-like growth factor I (Peralta Soler et al., 1990) are translocated into the nucleus in intact cells and insulin-like growth factor-binding protein type 3 (IGFBP-3) has a nuclear localization (NLS)-like sequence (Radulescu, 1994). In addition, protein tyrosine kinases, which have a role in the signal transduction pathway involving receptors (Chapter 7) have been found in the nucleus (Wang et al., 1994). Endocytosis and the physiological roles of peptide hormones are unavoidably linked because the two functions involve binding to the same receptors. In these cases the role of the endocytotic uptake of receptor and ligand is likely to be the regulation of the surface concentration of receptor required to provide its physiological response (see Katzmann et al., 2002). In fact, the removal of receptors from the cell surface by endocytosis after they are activated by binding to their ligand is part of the mechanism by which cells return to the unstimulated condition. The receptors can either be recycled or degraded by lysosomes (see below) or by the proteasome system (see Chapter 15). In addition, with changing conditions, channels and transporters can be regulated by a similar endocytotic pathway. This mechanism is physiologically significant as indicated by hypertension brought about by the failure of internalization of epithelial Na+ channels (Snyder et al., 1995) (Liddle’s syndrome). Furthermore, the inability to internalize the epidermal growth factor (EGF) leads to transformation (Wells et al., 1990; Vieira et al., 1996). The various organelles involved in receptor mediated endocytosis have been defined by their kinetic relationships (see Mellman, 1996; Gruenberg, 2001). The uptake of material is typically initiated by formation of coated vesicles from coated pits, although other alternatives are emerging from more recent studies. After internalization, the chlathrin coated vesicles loose their coating, a process driven by a 70 kDa ATPase (Braell et al., 1984; Rothman and Schmid, 1986). The early endosomes may have a variety of shapes from tubular to granular. Some have are vesicles about 250-400 nm surrounded by tubules (50http://www.albany.edu/~abio304/text/chapter_9.html (3 of 41) [3/5/2003 7:54:40 PM]
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60 nm in diameter) ( Gruenberg et al., 1989). The vesicles possess numerous invaginations which may have detached from the limiting membranes. Initially early endosomes have a high level of raft sphingomyelin and cholesterol as well as the raft-associated proteins caveolin-1 and flotillin-1. Their contents include cargo, recycling receptors and their bound ligands,and down regulated receptors. The late endosomes are multivesicular in appearance and have been referred to as multivesicular bodies (MVBs). The MVBs might be formed from invaginations of the endosomal membrane (e.g., see Hirsch et al., 1968). In yeast, the uptake of receptors culminating with their degradation in the vacuole (which corresponds to the lysosomes in mammals) involves as many as 50 genes. (see Katzmann et al., 2002). Phosphoinositides, phosphorylated derivatives of phosphatidylinositol (PI), have been implicated in endocytosis and the MVB pathway. They have a role in the invagination involved in the formation of endosomes by recruiting the necessary protein components.One of the lipids, lysobisphosphatidic acid (LBPA) is present in the lumenal membranes of the MVBs (Kobayashi et al., 1998) and is thought to have a role in lumenal vesicle formation and the distribution of cholesterol The early endosomes constitute the first step of sorting within the endosome system. In these endosomes, receptors and ligands are separated rapidly by acid pH. A vacuolar H+-ATPase is responsible for the acidity (see Al Awqati, 1986). Subsequently, some of the receptors are recycled while others are to be degraded by the lysosomes system. The sorting in the early endosomes is likely to take place by a mechanism in which the recycled components are segregated in the tubular elements (possibly forming recycling endosomes) whereas the cargo to be degraded remains in the central vesicle (see below). No protein motifs have been found for recycling to the cell surface. Selection to the degradation pathways (see Mukherjee et al., 1997; Gruenberg, 2001) may require signal mediated sorting. Specialized lipid domains, the so-called rafts capable of interacting with certain proteins (e.g., GPI anchored proteins) may contribute to sorting (e.g., Mayor et al., 1998, Mukherjee and Maxfield, 2000) (see Chapter 4 and below). The transit through the recycling endosomes may be as long as 5 to 10 min (Schmid et al., 1988; Daro et al., 1996) A certain proportion of the recycling vesicles are thought to be transferred directly from the early endosomes to the plasma membrane where the vesicles fuse to the plasma membrane by exocytosis. In the nervous sytem, endocytosis plays a very important role in replacing the vesicles discharged during synaptic conduction (see Chapter 22) A recycling route involves recycling endosomes (also called the perinuclear recycling vesicles) whose cargo contains only molecules to be recycled. The structures are distinct tubules 50-70 nm in thickness (Yamashiro et al., 1984; Gagescu et al., 2000) which frequently accumulate close to the microtubuleorganizing center by a mechanism involving microtubules (Yamashiro et al., 1984). These structures may have originated from the tubules of the early endosomes. Ligands and receptors destined for degradation follow a separate route. They are transferred to late endosomes and lysosomes in spheres (500 nm in diameter), in a process requiring functioning microtubules ( Gruenberg et al., 1989). The endosomal carrier vesicles/multivesicular bodies (ECV/MVB) derived from early endosomes form the late endosomes. Late endosomes, have the http://www.albany.edu/~abio304/text/chapter_9.html (4 of 41) [3/5/2003 7:54:40 PM]
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morphological characteristics of multivesicular bodies, can fuse with other late endosomes and are able to fuse to lysosomes. The latter initiates degradation of the endosomal contents. The passage from early endosomes to the degradation directed endosomes has been proposed to take place by a maturation process rather than by a transfer from compartment to compartment, since probe molecules are not diluted during the transition (Dunn and Maxfield 1992). The compartments and mechanisms involved in the traffic of polarized epithelial cells is more complex. These cells have distinct apical and basolateral surfaces (see Chapter 11). The biosynthetic pathway must sort out the components in the TGN (see Rindler et al., 1984). Although in some cases the endosomes and the recycling compartments may be involve as well (e.g., Futter et al., 1995). Endocytosis takes place in each of these surfaces (see Mukherjee et al., 1997). Furthermore, in recycling or transcytosis the targeting must be to one of the two surfaces. Reflecting this organization, there are two separate sets of early endosomes: The apical early endosomes (AEE) and the basolateral early endosomes (BEE). However, the late endosomes and lysosomes are compartments shared by both systems. AEE contents can be recycled, some transcytose and others are delivered to the late endosomes and lysosomes. However, these two are the main recipients of basolateral contents. There is a recycling compartment common to both systems and BEE can recycle components to the basolateral surface or the apical surface. The presence of a common recycling compartment indicates the presence of some sorting mechanism in this compartment. Some of these exchanges depend on actin, others on microtubules and some on both these components (see Apodaca, 2001). The progression of endocytosis from coated pits to endosomes can be followed in some cells by synchronizing the endocytotic events. The giant reticulospinal axon of the lamprey have proven very informative (see Brodin et al., 2000; Higgins and McMahon, 2002). When these cells are depleted of Ca2+ endocytosis is arrested. Reintroduction of Ca2+ reinitiates endocytosis. The changes can then be followed at various times with conventional transmission electron microscopy so that the time course of endocytotic events from coated pits to invaginated pits and pits with narrow necks can be followed. The use of mutants and blocking the effect of a protein by microinjection of either an antibody or a domain of the protein into cells have implicated various components in individual steps of endocytosis. The peptide domain blocks the effect of the native protein by competing with it. This approach can be illustrated by the study of a temperature sensitive mutation affecting the protein dynamin (shibire , ts-1) in Drosophila. This mutation arrests the process at the stage of in which coated pits have developed narrow necks and a collar ( Koenig and Ikeda, 1989), implicating dynamin in the pinching off of the vesicles. Similarly, mutations in mice implicated synaptojanin in the uncoating of vesicles. I. RECEPTOR-MEDIATED ENDOCYTOSIS The process that has been studied in most detail is the clathrin-mediated endocytosis where receptors, ligands and extracellular fluid are captured by 100-150 nm diameter coated pits (see below). Caveolae (Section V) have been found to be involved in receptor mediated endocytosis, transcytosis and the transport of certain blood macromolecules (see Schnitzer, 1997) as well as viruses, toxins and http://www.albany.edu/~abio304/text/chapter_9.html (5 of 41) [3/5/2003 7:54:40 PM]
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conformational altered proteins. Caveolae are also thought to contain signaling molecules (Liu et al., 1997). However, the present discussion will first be restricted to the better known system of coated pits and vesicles containing clathrin. The receptors involved in the endocytotic uptake of ligands are transmembrane glycoproteins with the carbohydrate moiety attached to the amino terminal region. In principle, the arrangement is similar to that of other intrinsic proteins discussed in Chapter 4. All have residues such as phosphate, palmitate, or oligosaccharides that have been added posttranslationally. In receptor-mediated endocytosis, the uptake of ligand occurs within minutes of binding to the receptor. In some cases, the receptors are preferentially located in coated pits, in others the receptors migrate to the coated pits after binding the ligand. The coated pits are indentations in the membrane in which the inner surface is lined with a fuzzy coat (see Fig. 1, Anderson, et al., 1977a). Most generally, the proteins taken up by endocytosis are in coated vesicles which lose their coat when they form endosomes (see Section IV). In many cases, the proteins are eventually digested by the lysosomal enzymes. Lysosomes are vesicles that contain a full array of hydrolytic enzymes capable of digesting materials taken up by endocytosis. The ways in which the endosomal contents and the lysosomal enzymes interact is presently under discussion (see Section VII, below). The low density lipoprotein (LDL) receptor system which functions in the transport of cholesterol from the plasma into cells, was one of the first to be examined in detail and will be the first subject of our discussion. II. THE LDL RECEPTOR FAMILY A. The LDL Receptors In mammals, cholesterol is transported in the bloodstream in spherical LDL particles, 22 nm in diameter, which originate in the liver. The particles are complexes containing a core of 1500 cholesterol molecules esterified to long-chain fatty acids which are covered by phospholipids, cholesterol, and protein (Goldstein and Brown, 1977). The LDL receptors (LDLRs), lipoprotein molecules with a molecular mass of 164 kDa (Schneider et al., 1982), are synthesized in the endoplasmic reticulum when cholesterol is required. The LDL particles first bind to the LDLRs. Then they are internalized at the cell surface and eventually the receptor-LDL particle complexes are delivered to the lysosomes. In these organelles the protein component of the LDL particles is hydrolyzed and cholesterol is regenerated from the cholesteryl esters. Cholesterol in the cytoplasm inhibits the synthesis of the receptor molecules and thereby prevents an excessive accumulation. Apparently, the LDL receptors are recycled: they return to the surface where they cluster again in the coated pits (Anderson et al., 1977b, Goldstein et al., 1976).
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What is the location of the LDLRs and what are the details of the LDL uptake? To answer these questions LDL was labelled with ferritin. Ferritin has a high iron content and therefore is visible with the electron microscope. In contrast to other systems, the LDL receptors are present in coated pits before the addition of LDL. This has been demonstrated by the binding of the ferritin-labelled LDL to cells that have been fixed and presumably cannot migrate. Between 50 and 80% of the receptors are clustered in 2% of the cell surface and have been shown to be present in coated pits by electron microscopy (Anderson et al., 1977a, Orci et al., 1978). The location of coated pits coincides with the position of the underlying cytoplasmic stress fibers (Anderson et al., 1978) which have been shown in other studies to contain actin (see Chapter 23. In fact, clathrin, the major protein of coated vesicles, can bind actin. Since actin fibers are associated with movement, it is possible that they have a role in the internalization of vesicles. Accumulation in the coated pits and binding of the ligand are two separate functions of the LDLRs since they are affected by different mutations. This conclusion is based on observations of patients with familial hypercholesterolemia (FH), a disease characterized by very high levels of cholesterol in the blood. One kind of FH can be traced to a mutation in which the receptors are unable to bind LDL; another kind involves a mutation in which the receptors are unable to be incorporated into coated pits. These observations suggest that the receptors have two separate sites: the site for binding LDL that faces the medium and the site that interacts with the coated pits which is presumably cytoplasmic (see discussion in section IVC). Some of the receptor molecules are likely to have other specialized sites responsible for their sorting out into various cellular compartments. The sequence of events underlying the binding of LDL and endocytosis is illustrated in Fig. 1 (Anderson et al., 1977a) which summarizes the electron microscopic observations with the LDL-ferritin marker. Cultured fibroblasts were first incubated at 4oC for 2 h and then, after extensive washing, were incubated for various lengths of time in the presence of ferritin-labeled LDL at 37oC. Fig. 1 A-C shows that after incubation for 1 min the various configurations of early endocytosis are already present in coated pits. Fig. 1D shows a coated vesicle that is beginning to lose its coat after a 2 min incubation, and Fig. 1E shows a vesicle or endosome without a coat, also after a 2 min incubation. Fig. 1 F-H correspond to configurations in the formation of secondary lysosomes, that is, lysosomes formed by fusion of endosomes and newly formed primary lysosomes. In Fig. 1 F and G the amount of ferritin per vesicle seems to be greater than in the earlier stages, possibly indicating a fusion of various vesicles; Fig. 1H corresponds to a fully formed lysosome containing ferritin.
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Fig. 1 Role of the coated endocytotic vesicle in the uptake of receptor-bound low-density lipoprotein in human fibroblasts. The bars represent 100 nm. See text. Reproduced with permission from Anderson, et al., (1977a), copyright © 1977 by Cell Press.
The general information derived from the electron microscopic studies has been confirmed biochemically in cultured fibroblasts after labelling LDL with the radioactive isotope [125I]. The actual endocytotic uptake was distinguished from binding by adding heparin to the cells. Heparin releases only surface LDL and does not have an effect on the LDL inside the cells. Therefore, the [125I]-labelled LDL that is released after heparin treatment can be assumed to be attached to the LDL receptor at the surface. Heparin, a glucosaminoglycan which acts as an anticoagulant, is produced by mast cells. The release of the LDL from the receptor is not the result of damage to the receptors because after incubation in heparin and its removal, the binding of LDL is the same as that of untreated cells. Fig. 2 (Goldstein, et al., 1976) shows that the surface-bound [125I]-labeled LDL is taken up very rapidly, most within 5 min. However, the heparin-insensitive uptake of LDL continues linearly with time at a lower rate, suggesting that it corresponds to rapid uptake into cytoplasmic vesicles. What is the fate of the LDL? Its later association with lysosomes, revealed by the electron microscopy, suggests a proteolytic breakdown. The proteolysis of LDL can be followed by measuring the radioactivity of trichloroacetic acid (TCA)-soluble cell extracts. Hydrolytic products of proteolysis, amino acids and low molecular weight peptides, are soluble in TCA. In contrast, protein is denatured by TCA and becomes insoluble. An increase in the TCA-soluble radioactivity of the extract serves as a measure of LDL hydrolysis. Fig. 3 (Goldstein et al., 1976) represents the degradation of the LDL at 37oC after an initial binding at 4oC. After the initial binding the labelled LDL is removed. The radioactivity of the bound protein disappears (curve 1) and most of it appears in a TCA soluble form (curve 2).
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Fig. 2 Relation between heparin-releasable and heparin-resistant 1251-labelled LDL binding at 37oC at early time points. Reproduced with permission from J.L. Goldstein, et al., Cell, 7:85-95. Copyright © 1976 by Cell Press.
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Fig. 3 Proteolytic degradation at 37oC of 125I-labeled LDL previously bound to normal fibroblasts at 4oC. Reproduced with permission from, J.L. Goldstein, et al., Cell, 7:85-95. Copyright © 1976 by Cell Press.
B. Other Receptors of the LDLR family LDLRs have been found to be part of a family of receptors. Nine have been recognized in mammals. They share common structural motifs and all have a role in the uptake of lipoproteins. However, many of them are involved in many other functions as revealed in knockout mice mutants or in human gene defects (see Nykjaer and Willnow, 2002, Gliemann, 1998). Members of the LDL receptor family have roles in neuronal migration, synaptic plasticity and vitamin metabolism. Their multiple functions may be the result of their ability to bind to a variety of ligands, their interaction with other proteins present at the cell surface (co-receptors), including seven-transmembrane-span receptors, glycosylphosphatidylinositol (GPI) anchored proteins and adhesion molecules, and in addition their interaction via their cytoplasmic tails with cytoplasmic adaptors . Two of these receptors are large (600 kDa): LDL-receptor-related protein (LRP) and megalin megalin (see Gliemann, 1998; Herz and Strickland, 2001). LRP is present in hepatocytes, macrophages, smooth muscle cells, and neurons. Megalin is present in various epithelia such as that of proximal kidney tubules and intestine. These two proteins bind numerous ligands at different combinations of sites. LRPs function in lipid metabolism, the regulation of proteinases and proteinase inhibitors, activation of lysosomal enzymes, cellular signal transduction and neurotransmission, and recognizes at least 30 different ligands. In addition, a variety of cytoplasmic proteins bind to the tail of LRP and some of these proteins initiate endocytosis. LRP binds to remanents of chylomicron, and the lipases directly involved in the forming lipoproteins from triglyceride-rich chylomicrons. Chylomicrons are particles which carry primarily dietary cholesterol from the intestine to the liver. Some ligands first bind to heparan sulfate proteoglycans before being picked up by LRP. LRPs are involved in the removal of proteinase and proteinase inhibitor complexes and are important regulators of extracellular proteolytic activity including matrix metalloproteinases. LRPs are involved in sphingolipid activator protein (SAP) uptake which is required for activation of cerebrosidases, sphingomyelinases, glucosidases, and hexosaminidases in the lysosomes. In neurons, LRPs have been implicated in NMDA receptor function. They interact with tissue plasminogen activator (tPA). tPA expression is thought to have a role in synaptic plasticity in the brain. In the kidney, magalin is involved in the uptake of low molecular plasma proteins that have been lost through the glomerulus such as plasma carriers that transport vitamins and ions (e.g. retinol binding proteins, vitamin A, vitamin D- binding proteins, tranferrin ). In megalin knockout mutants, vitamins are excreted. In some cases, megalin ligands bind to cubulin, a peripheral protein of 460 kDa which binds to ligands but is unable to be internalized by itself without attaching to megalin. III. OTHER RECEPTORS A. Involvement in Endocytosis http://www.albany.edu/~abio304/text/chapter_9.html (11 of 41) [3/5/2003 7:54:40 PM]
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Various proteins are taken up specifically by cells, as shown in Table 1 (Goldstein, et al., 1979); at the time of this compilation, 25 specific receptors involved in endocytosis had been recognized. Table 2 (Brown, et al., 1983) lists some of the cell surface receptors that have been purified and characterized. The receptors have been generally identified by their ability to bind the appropriate ligand. The LDL receptors were extracted from cell membrane preparations of bovine adrenal cortex. Adrenal cells are particularly rich in these receptors because they use cholesterol in the synthesis of steroid hormones. It has been estimated that there are 100,000 receptor molecules per adrenal cell. Binding of [125I]-labelled LDL was used as the assay of the receptor through the various steps of the fractionation procedures. Table 1 Systems for Receptor-mediated Endocytosis of Proteins Fate of internalized protein Cell Type Protein
Transport proteins LDL Yolk proteins (phosvitin, lipovitellin) Transcobalamin II Transferrin
Internalization via coated pits and coated vesicles
Fibroblasts, smooth Yes muscle cells, endothelial cells, Yes adrenocortical cells,lymphocytes Data not available Oocytes (chicken, mosquito) Yes Kidney cells, hepatocytes, fibroblasts Erythroblasts, reticuloblasts
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Degraded in lysosomes
Other
Yes; cholesterol retained by cells
---
No
---
Delivered to yolk granules
Yes, Iron retained by cells vitamin B12 retained by cells Data not available
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Protein hormones Epidermal growth factor
Fibroblasts, 3T3 cells Sympathetic ganglion cells
Nerve growth factor Insulin Chorionic gonadotropin β-Melanotropin
Hepatocytes, hepatoma cells, lymphocytes, adipocytes, 3T3 cells
Yes
Yes
---
Data not available
Data not available
Carried in vesicles retrograde up to the axon
Data not available
Yes Yes
Data not available
Data not available
Also delivered to Golgi apparatus and nuclei --Delivered to Golgi apparatus and melanosomes
Data not Leydig tumor cells, available ovarian luteal cells Melanoma cells
Other proteins Asialoglycoproteins Lysosomal enzymes
a2-Macroglobulin
Maternal immunoglobulins (lgG)
Hepatocytes Fibroblasts
Fibroblasts, macrophages, 3T3 cells
Data not available Data not available
Yes
---
No
Delivered to lysosomes and Golgi-associated structures; enzymes remain active for many days
Yes
Yes
---
No
Transferred intact in coated vesicles to basal surface of cells, where igG is discharged into fetal or neonatal circulation
Yes
Fetal yolk sac, neonatal intestinal epithelial cells
Source: From Goldstein et al. (1979). Reprinted by permission from Nature 279:679-685, copyright © 1979 Macmillan Magazines Ltd.
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Clathrin coated pits and vesicles have been shown to be involved in many cases of receptor-mediated endocytosis (see however, Section V and Section VI), although many of the details of the uptake and processing differ. We saw that the LDL receptors are located in the coated pits even before they bind LDL. Most or all receptors binding inert cargo such as LDL (i.e. not a signaling molecule) are internalized regardless of whether they are binding the ligand. Various receptors follow this pattern. However others, such as the EGF receptors, are distributed throughout the surface and cluster only after binding the ligand (Hagler, 1978). As we saw in Chapter 4, membrane components can diffuse twodimensionally inside the membrane. The rate of the diffusional movement of receptors toward the coated pits is sufficiently rapid to account for the clustering (Bretscher and Pease, 1984, Hopkins, 1985), so that no special mechanism has to be invoked to explain the migration. In contrast to receptors binding inert cargo, signaling receptors require ligand binding for uptake. What allows the receptor proteins to be taken up by endocytosis when bound to their ligand? Many of the details are still unknown. Some of these aspects will be discussed in the section below (Section IV). The presence of short motifs of four or five amino acids in the cytoplasmic domain of the receptors seems to play a role (Collawn et al., 1990, 1991, Vaux, 1992). In the absence of these motifs the receptors do not initiate endocytosis when bound to the ligand. In the case of EGFRs, ligand binding cause the receptor to autophosphorylate (see Schlessinger and Ulrich, 1992). The phosphorylation results in a conformational change that exposes the motifs in the cytoplasmic domain required for coated pit targeting (Cadena et al., 1994). However, downstream receptor signaling (Lamaze and Schmid, 1995b) and at least in the case of other receptors, recruitment of clathrin to form coated pits (Grimes et al., 1996) are required. Apparently the binding of EGF to the receptor activates a kinase (SRC kinase) that phosphorylates the clathrin heavy chain (Wilde et al., 1999). This phosphorylation triggers the formation of vesicles and movement of clathrin to the interior of the cell. Without SRC kinase activity EGF endocytosis is delayed. B. Fate of Ligand and Receptor Many of the receptors recycle. In contrast to LDL which is hydrolyzed, the LDL receptor is rapidly returned to the surface and has a half-life as long as 15 h (Brown et al., 1981). As might be expected, blocking protein synthesis with cycloheximide does not have an immediate effect on LDL endocytosis. In contrast to the recycling of the LDL receptor, the EGF receptor is degraded in most tissues (Schlessinger et al., 1978) although it is recycled in the liver (Dunn and Hubbard, 1984). Table 2 Cell Surface Receptors That Concentrate in Coated Pits Estimated molecular mass (kDa) Receptor
Source
Subunit
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Holoreceptor
Residues added posttranslationally
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LDL
Bovine adrenal cortex
160
160
human fibroblasts
O-linked oligosaccharide; sulfate on N-linked oligosaccharides
Transferrin
Human leukemia cells
90
180 (disulfide linked dimers)
palmitate; phopshate on tyrosine and serine
Epidermal growth
Human A-431 cells
170
170
phopshate on tyrosine and serine
Rat adipose tissue
90
phopshate on tyrosine and serine
Rat liver
125
350; 2 subunits of 90, 2 subunits of 125 (disufide linked)
factor Insulin
Human placenta Lysosomal enzyme Bovine liver
215
215
Rat chondrosarcoma Asialoglyco-
Chicken liver
26
26
proteins
rat liver
43,54,64
43, 54, 64
Fibroblast growth
Pituitary
15
15
phosphate on serine
factor From M.S. Brown et al., Cell 32:663-667, Copyright ©1983 by Cell Press, reproduced by permission.
As we saw for LDL, many of the proteins taken up by endocytosis are degraded. In contrast, yolk proteins are accumulate in yolk granules. NGF, which enters by endocytosis at the tip of the axon, accumulates in the cell body (Bradshaw, 1978). The fate of insulin differs from both of these cases: some of the insulin taken up by endocytosis is degraded and some remains intact inside the cell. Both receptor and ligand may recycle, as is the case for transferrin (Bleil and Bretscher, 1982), a protein that functions in the transport of iron in organisms. After uptake of transferrin by endocytosis, the iron is removed from the transferrin in the endosome; the apotransferrin (i.e. transferrin stripped of iron) remains attached to the receptor and both are returned to the cell surface (Geuze, 1984).
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Four possible pathways for the receptor-ligand complex are shown in Fig. 4 (Goldstein, et al., 1985).
Fig. 4 Four pathways of receptor-mediated endocytosis. The initial steps (clustering of receptors in coated pits, internalization of coated vesicles, and fusion of vesicles to form endosomes) are common to the four pathways. After entry into acidic endosomes, a receptor-ligand complex can follow any of the four pathways shown. From Goldstein, et al., 1985. Reproduced, with permission, from the Annual Review of Cell Biology, Volume 1, copyright ©1985 by Annual Reviews Inc.
C. Hydrolysis, Recycling, and Sorting What is the fate of ligands and receptors which are not recycled? As we saw for LDL, after endocytosis the coated vesicles shed their coat and presumably fuse to form smooth larger vesicles, called endosomes. These in turn either fuse with the lysosomes or transfer the LDL to the lysosomes by means of transport vesicles (see section VII, below). The digestion of receptor then takes place in the lysosomes as indicated in Fig. 4. As already indicated, in mammals the lysosomal degradation pathway is involved in the degradation of certain receptors such as growth hormone receptors (Strous et al., 1996). Treatment with NH4Cl disrupts
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lysosomal function and blocks the degradation of these receptors. Others, seem to be degraded by proteasomes. Proteasomes are huge (1,700 kDa!) multimolecular assemblies involved in the degradation of a variety of proteins (see Chapter 15). Proteasomes are implicated in the degradation of the plateletderived growth factor (PDGF) receptors since inhibitors of proteasome function interfere with their degradation (Mori et al., 1995; Jeffers et al., 1997). A proteasomal pathway is suspected in downregulating mammalian receptors by degrading their cytosolic domains (see Hicke, 1997). Until now, for the degradation of yeast integral plasma membrane proteins, only the lysosomal pathway has been implicated (e.g. Berkower et al., 1994, Kölling and Kollenberg, 1994) (see Section C, Chapter 15, and section on the degradation of integral proteins in Chapter 15). For example, normal internalization and degradation occurs in mutants lacking proteasome function (Galan et al., 1994). Ubiquitination (see also above) marks proteins for internalization and degradation by either the lysosomal system (e.g. Hicke and Riezman, 1996) or the proteasome (e.g. Strous et al., 1996). Ubiquitin (see Chapter 15) is a small protein (8.5 kDa) that has been found to tag proteins for proteolysis, although it is likely to have other functions as well. As little as one ubiquitin is necessary for internalization (Hicke and Rieezman, 1996) in yeast, whereas polyubiquitin with a minimum of four ubiquitin molecules is required for recognition by the proteasomes (Deveraux et al., 1994). Besides a role in internalization, ubiquitination is needed for sorting in the late endosomes and from the TGN to the lysosomes (vacuole in yeast) (see Rotin et al., 2000; Dupré et al., 2001). At least in Saccharomyces cerevisiae the downregulation of the membrane receptors and transporter proteins takes place by internalization signaled by monoubiquitination. The enzymes involved in ubiquitination, such as Cbl, an E3 or ubiquitin ligase recruited to phosphotyrosine motifs, may have some additional roles. Cbl ubiquitinates the epidermal growth factor receptor (EGF-R) at the cell surface. However, it remains bound throughout the endosomal pathway suggesting some other function ( de Melker et al., 2001; Levkowitz et al., 1998). The targeting of the monoubiquitinated transmembrane proteins such as the cell surface receptors to the multivesicular bodies/lysosomal vesicles (or vacuole in yeast) involves several Vps proteins (Vps2, Vps20, Vps24, and Snf7). The Vps are transferred from the cytoplasm to endosomal membranes where they oligomerize into protein complexes, ESCRTs. ( ESCRT-I ,II and III). ESCRT-III, a membrane associated complex. includes the AAA-type ATPase Vps4. The ESCRT complexes perform a cascade of events in which the cargoes are sorted out and delivered to the vacuole or lysosomes (Babst et al. 2002a and b). Ligands and receptors may have separate fates (Fig. 4). It is difficult to imagine how this can take place unless there is some special mechanism for separating them and conveying them to different compartments. During endocytosis, the interior of the endosome becomes acidic. The acidity of the endosome has a role in detaching the ligand from the receptor. The increase in acidity has been shown for the endocytotic uptake of α2-macroglobulin conjugated to fluorescein (Tycko and Maxfield, 1982). Fluorescein is a dye whose fluorescence varies with pH. After 20 min of endocytosis induced by the
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labeled macroglobulin, the fluorescence of the dye in the vesicle indicated a pH of approximately 5. Experiments with isolated vesicles show that the internal acidification results from an H+-pump powered by ATP hydrolysis (Galloway et al., 1983). Once separated, a special mechanism must be present to direct the two components to a different location. One of the systems that provides information on this process is that responsible for the uptake of asialoglycoprotein. The asialoglycoprotein receptor system has been studied with the electron microscope after labeling with separate antibodies for ligands, receptors, and clathrin (Geuze et al., 1983). Clathrin is the major coat component of coated pits (see Sections IV below). Asialoglycoproteins are abnormal plasma glycoproteins that have been stripped of the sialic acid residue that normally covers the terminal galactose. The antibodies used in this study are visualized and distinguished from one another by coupling to colloidal gold particles of different sizes. The results indicate that after separation, ligands and receptors are segregated in a vesicle (compartment of uncoupling receptor and ligand, CURL; probably corresponding to early endosomes) containing tubular extensions. The receptors attach to the membranes of the tubules, whereas the ligands remain in the lumen. Presumably, budding of the tubules produces smaller vesicles, which return the receptors to the surface. Fig. 5A (Geuze, et al., 1983) shows that both ligand (coupled to the 5-nm particle) and receptor (coupled to the 8-nm particle) are present in a vesicle close to the cell surface. Fig.5B and C show how the ligand remains in the lumen of the CURL, whereas the tubules favor the receptors. In (B) the receptor is labeled with the 8-nm gold particle and in (C) the labeling is reversed. The movement of receptor or receptor-ligand complex occurs through several compartments, with accurate sorting in each. As in the case of translocation into the nucleus, the targeting of newly synthesized proteins requires special domains (Chapter 5). The targeting of receptors may also have many functional domains for interaction not only with the ligand but also the various macromolecular species capable of redirecting it to a new target.
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Fig. 5 Simultaneous demonstration of receptor and ligand in CURL and in a multivesicular body. The bars correspond to 100 nm. (a) Vesicle just beneath plasma membrane at the sinus (S), with ligand (5-nm gold) associated with receptor (8-nm gold). (b) Coated pit with receptor (8-nm gold) and ligand (5-nm gold) at upper left. The slightly tangential view of early endosomes shows a heterogeneous distribution of receptor in the vesicular portion and abundant receptor in associated tubules. Simultaneous demonstration of receptor and ligand in CURL and in a multivesicular body. (c) Early endosomes profile shows peripheral ligand (5-nm gold) and heterogeneous labeling of receptor (8-nm gold). Intense receptor labeling is present over the tubules adjacent to the vesicular portion of early endosome. (d) Free ligand (5-nm gold) can be seen in the lumen of the vesicular portion of early endosome, which also shows scarce and heterogeneous receptor (8-nm gold) labeling. Receptor labeling is intense over the connecting tubules. (e) Early endosome profile in which the receptor (5-nm gold) is located predominantly at the pole, where a tubule with heavy labeling of receptor is connected. Most of the ligand (8-nm gold) is present free in the vesicle lumen. Reproduced with permission from Geuze, et al., (1983), copyright ©1983 by Cell Press.
IV. COATED PITS Coated pits and coated vesicles have been found in virtually all nucleated animal cells (see for a review Takei and Haucke, 2001). The coated pits have a fuzzy cytoplasmic coat which, at higher resolution, can be shown to correspond to periodically spaced fibers. Electronmicrographs of freeze-fractured and deep etched preparations of vesicles (Heuser et al., 1988) reveal a regular lattice composed of many clathrin molecules.
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A. Clathrin and Adaptor Complexes While coated pits have been found to be involved in the uptake of proteins from the medium, coated vesicles have been implicated not only in endocytosis but also in intracellular membrane transport. Four different coat proteins are recognized, including two clathrin proteins and COPI and COPII. The coat proteins of intracellular transport are discussed in Chapter 11. Adaptor proteins (APs) are components of the clathrin coats (see below). Clathrin associated with AP-2 and AP180 are involved in endocytosis, whereas clathrin associated with AP-1 and AP-3 are involved in the transport from the TGN to the lysosomes (see Chapter 10 and 11). AP-4 has been found to be associated with the TGN. In the case of AP-1 and AP-3, the guanosine triphosphatase ADP-ribosylation factor 1 and possibly other proteins are required instead of AP180 (see Dell’Angelica, 2001). A major role of clathrin in endocytosis is attested by the observation that the introduction of anticlathrin antibody into living cells results in inhibition of both receptor-mediated endocytosis and fluid endocytosis (Doxsey et al., 1987) but not the transport accompanying secretion. There are indications, however, that in some cases endocytosis (including receptor-mediated endocytosis) proceeds by mechanisms not involving clathrin (see below) Isolated coated vesicles are composed of the membrane components surrounded by a basket-like arrangement of protein, predominantly clathrin, a protein of 180 kDa. Clathrin itself is formed from three heavy and three light chains forming a so-called triskelion (see Fig. 6). The clathrin coat also contains the APs. APs have a role in the assembly and the attachment of clathrin to the plasma membrane (see Hirst and Robinson, 1998; Kirchhausen 1999; Kirchhausen, 2000). In addition, APs mediate the interaction with lipids. AP180 has been shown to aid in the assembly of clathrin in vitro and is needed for endocytosis and the maintenance of proper vesicular size in vivo (see McMahon, 1999; Ford et al., 2001). As indicated, the various populations of clathrin coated vesicles have distinct APs. In addition to the APs, β-arrestin and arrestin-3 may act as adaptors (e.g., see Goodman et al., 1997) for the internalization of receptors coupled to the heterotrimeric GTP-binding proteins (see Chapter 7). APs are dimers, and each monomer has four polypeptides called adaptins. Two of the adaptins are approximately 100 kDa in size, one of 50 kDa and another of 25 kDa. In vitro, in the presence of clathrin triskelia, the APs are attached to the terminal domain of the clathrin unit. After removal of clathrin from coated vesicles, the adaptins appear as blocks with the two carboxy-terminals of the larger subunits sticking out (Heuser and Keen, 1988). B. Structure of Clathrin and Assembly The structure of clathrin cages has been studied (see Smith and Pearse, 1999) with EM (Vigers et al., 1986; Smith et al., 1998) and fragments of clathrin have been the subject of crystallographic studies (ter Haar et al., 1998; Ybe et al., 1999). Vigers et al. examined tilt series of electron micrographs from unstained clathrin cages embedded in vitreous ice. The three-dimensional reconstructions of individual hexagonal barrels show details of the internal structure. Cryoelectron microscopy and single-particle http://www.albany.edu/~abio304/text/chapter_9.html (20 of 41) [3/5/2003 7:54:40 PM]
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reconstruction (Smith et al, 1998) shows details of the packing of entire clathrin molecules as they interact to form a cage with two polyhedral layers. A triskelion hub is at the vortex of the cage (see Fig. 6). Three legs extend in different directions from the hub. Midway along each leg there is a bend corresponding to the proximal and distal domains. The amino-terminal of each of each clathrin heavy chain is adjacent to the distal domain. The carboxy-terminal is at the hub of the triskelion at the so-called trimerization domain. Each leg contributes two adjacent faces of the cage structure. The distal legs are positioned alongside on the underside of the second edge below the next hub where they can interacts with the APs inside the cage. The crystal structure of a fragment of clathrin corresponding to the hub domain (see Fig. 6) has been elucidated (Ybe et al., 1999). It forms an elongated coil of α-helices. In addition, a 145-residue motif is repeated seven times along the filamentous leg. This motif is similar to that of other proteins involved in vacuolar protein sorting. The hub domain contains a light chain binding region and the portion mediating spontaneous clathrin heavy-chain polymerization. Light chains modulate polymerization negatively in vitro and may play a role in preventing the unphysiological assembly of cages in the cytoplasm (Ungewickell and Ungewickell, 1991). The structure of the amino-terminal portion (ter Haar et al., 1998) has also been studied by crystallography. It corresponds to a globular terminal domain forming a β sevenblade propeller joined to the leg by the linker domain which is in an α zig zag arrangement. The propeller domains form an inner polyhedral array in the coated vesicle that allows many binding sites for adaptor molecules (Smith et al., 1998). AP-2 form an inner shell of density in the EM map of the clathrin coat.
Fig. 6 Representation of the heavy (HC) and light chains (LC) of a clathrin trimer free in solution and within a clathrin lattice. The APs are located beneath the clathrin edges. From Kirchhuasen, 1993, reproduced by permission.
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Clathrin can be readily removed from coated vesicles which bind it noncovalently. Under special conditions, free clathrin spontaneously assembles into a network of hexagons and pentagons similar to the basket-like arrangement present in coated vesicles. In endocytosis, the clathrin coat is assembled in the cytoplasmic face of the plasma membrane, forming pits that pinch off to become vesicles. In cell cultures the production of clathrin coated vesicles takes about 1 minute (Marsh and Helenius, 1980; Gaidarov et al., 1999). However, at the synapse where speed is important the uptake can be much faster (De Camilli and Takei, 1996; Marks and McMahon, 1998). Several clathrin-binding proteins have been shown to recognize the cytoplasmic domains of transmembrane proteins which are either cargo or receptors for soluble cargo. For soluble cargo molecules, the first step of the transport using vesicles can be considered the binding of cargo molecules to a transmembrane receptor. When preexisting pits are not involved, the receptors are then concentrated by binding to coat proteins needed for the budding of the vesicles. Cargo concentration and formation of a coat are probably coupled. After budding, the vesicles are transported, targeted and fused to the acceptor compartment. The assembly of clathrin coat components is thought to first require a recruitment of the adaptor AP-2 and various accessory molecules molecules from the cytosol to the membrane (see Robinson, 1994; Cremona and De Camilli, 1997). The cytoplasmic tails of the receptors are thought to play a role in this recruitment (see discussion in Section IIIA). Subsequently, clathrin is thought to be bound to the AP complexes attached to the membrane. The β chains of AP-1 or AP-2 are thought to be sufficient for the interaction with clathrin and the formation of a coat. Among the needed accessory molecules required are the GTPase, dynamin, the amphysin dimer and synaptojanin 1. Synaptojanin, an inositol 5-phosphatase (e.g., McPherson et al., 1994), is a major presynaptic protein associated with endocytic coated intermediates (Ringstad et al., 1999). In neurons of synaptojanin 1 mutant mice, phosphatidyl inositol 4,5bisphosphate (PIP2) levels are increased and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings (Cremona et al., 1999). Other phosphoinositol metabolites are also is involved in endocytosis (e.g., see the role of endophilin, below). Dynamin (see below) oligomerizes into collar structures at the neck of the invaginated clathrin coated pits. A conformational change in dynamin was thought to produce the separation of the vesicle from its stalk, acting as a "pinchase" (Oh et al., 1998; Sweitzer and Hinshaw, 1998), however, other studies suggest an indirect regulatory role (see below). Another of these factors is Eps15, a protein associated with clathrin that binds to the α-adaptin subunit of AP-2 (e.g., Benmerah et al., 1998; Tebar et al., 1996). Another protein that binds to Eps15, one of the epsins, is also required for endocytosis of coated pit invagination of synaptic vesicles (Chen et al., 1998). The epsins constitute a family of proteins which contain many binding domains. The epsin aminoterminal homology (ENTH) domain is present at the amino terminal and is structurally related to the VHS domain. Next to the ENTH domain is the ubiquitin-interacting motif (UIM). At the carboxy terminal many epsins contain motifs that bind to clathrin, accessory components and adaptors such as AP2 (see http://www.albany.edu/~abio304/text/chapter_9.html (22 of 41) [3/5/2003 7:54:40 PM]
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Brett et al., 2002). Epsin binds to the EH-domain of proteins (see table 2 of Chapter 6) via two to four asparagine, proline, phenylalanine (NPF) motifs. The tripeptide Asp-Pro-Trp (DPW) binds to α-adaptin. and other motifs. The epsins are also thought to bind to specific cargoes (De Camilli et al., 2002; Shih et al., 2002) and might be considered adaptors. The UIM is required for ubiquitin binding and protein transport (Shih et al., 2002). Conjugation to ubiquitin has a role in marking a protein for endocytotic uptake and targeting (see above) Phosphoinositol derivatives are linked to endocytosis. AP-2 and AP180 as well as epsin bind to phosphorylated metabolites of inositol such as phosphatidyl inositol bisphosphate (PIP2) and phosphatidyl inositol trisphosphate (IP3). PIP3 binds to AP-2 when this complex is assembled into a coat structure (Gaidarov et al., 1996). The assembly of the various components is regulated by phosphorylation. Dephosphorylation promotes the assembly of the components. Phosphorylation generally inhibits the assembly (see Wilde and Brodsky, 1996; Slepnev et al., 1998). For example, phosphorylation of the tyrosine signal residues blocks the interaction (Boll et al., 1996; Ohno et al., 1996). Changes in AP-2 can also be regulatory. The interaction of AP-2 with clathrin increases the strength of the binding to tyrosine-containing signals (Rapoport, et al., 1997), thereby favoring the assembly of receptors to partially assembled coated pits. Furthermore, AP-2 can be phosphorylated in vivo (Wilde and Brodsky, 1996). 3'-phosphorylated phosphoinositides increase the binding of AP-2 and tyrosine signals (Rapoport et al., 1997). C. Sorting of Proteins Several clathrin-binding proteins have been shown to recognize the cytoplasmic domains of transmembrane proteins which are either cargo or receptors for soluble cargo. There are also indications that the LDL receptor is recognized directly by clathrin (Kibbey, 1998). All three AP-complexes have been shown to recognize the sorting signals for transmembrane proteins Yxxφ (x is any amino acid and φ a bulky hydrophobic amino acid, such as Leu, Ile, Phe, Val and Met) and the dileucine signals (see Kirchhausen, 1999). The tyrosine (Y) is present within a loop structure called a "tight turn" (Collawn et al., 1990; Eberle et al., 1991; Bansal and Gierasch, 1991). β-Arrestin and β-arrestin 2 bind to the cytoplasmic domain of ligand activated G-protein-coupled receptors and recruit them into the coated vesicles (see Kirchhausen, 1999). Other types of signal include a cluster of acid amino acids (Pond et al., 1995; Voorhees et al., 1995; Jones et al., 1995) and the di-lysine signal KKFF (Itin et al., 1995). The KKFF signal is present in the protein VIP36 that cycles between the plasma membrane and the Golgi, and the ER protein, ERGIC-53, that cycles in the same way when overexpressed. Ubiquitin added to lysine residues in plasma membrane proteins also serves as an internalization signal (Hicke and Riezman, 1996; Strous et al., 1996). Coat proteins may be involved in these processes as well. In addition, in the TGN pathway, GGAs (Golgi-localized, γ-adaptin ear homology and ADP-ribosylation-factor-binding proteins) recognize acid-cluster-dileucine motifs of sortilin (a multi-ligand receptor) and mannose-6-phosphate receptors (e.g., Nielsen et al., 2001; Puertollano et al., 2001). The µ2 subunit of AP-2 binds to the http://www.albany.edu/~abio304/text/chapter_9.html (23 of 41) [3/5/2003 7:54:40 PM]
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tyrosine-sorting motifs (Owen and Evans, 1998; Bonifacino and Dell’Angelica, 1999). Subsets of the signals are responsible for later targeting to lysosomes, lysosomal-endosomal compartments, the TGN or the basolateral membrane in polar cells (see Mellman, 1996; Marks et al., 1997). Supposedly, they are recognized by complexes similar to the APs. X-ray crystallography has also been used to examine the epidermal growth factor (EGF) receptor and the trans-Golgi protein TGN38, while attached to the signal recognition domain of the µ2 subunit of AP-2 (Owen and Evans, 1998). In the complex, the signal peptides acquire an extended conformation rather than the expected tight turn. The hydrophobic pockets of µ2 bind the tyrosine and leucine of the peptides. D. Budding and Disassembly The process of clathrin vesicle budding has been studied in vitro in systems consisting of broken cells, either mechanically disrupted or broken by freeze thawing. These systems have permitted examination of the biochemistry of pit and vesicle formation. Aside from its structural components, coated pit assembly requires ATP and cytosol (Smythe et al., 1989, Schmid and Smythe, 1991). Budding and vesicle formation involving clathrin differs significantly from that occurring in the TGN. The events of assembly and disassembly of clathrin coated structures during endocytosis can be outlined as shown in Fig. 7 (Schmid and Damke, 1995). An involvement of GTP (and therefore GTP-binding proteins such as dynamin) is shown in steps 3 and 4 of the diagram. An involvement of GTP-binding proteins Rho and Rac in receptor mediated endocytosis of tranferrin was demonstrated by the inhibition produced in HeLa cells transfected with a Rac or Rho mutants (Lamaze et al., 1996).
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Fig. 7 Summary of events during formation of coated pits and clathrin-coated vesicles. The open circles represent the GTP-binding molecule dynamin. The letter D indicates that it binds GDP, and T indicates that it binds GTP. Modified from Schmid and Damke, 1995. Reproduced by permission.
The mechanics of vesicle excision are still not clear. A cytoplasmic GTPase, dynamin, is obviously involved (see Schmid et al., 1998). Dynamin is a member of a subfamily of GTPases (see van der Bliek, 1999a), present as a homotetramer in its native state (Muhlberg et al., 1997). Mutants with a defective dynamin have a defect in endocytosis. In addition, dynamin is found at the neck of invaginating coated pits (see Schmid et al., 1998). Dynamin is discussed in relation to the formation of intracellular cargo vesicles in Chapter 11. This GTPase is a homotetramer of 100 kDa. Dynamin has several domains (see Muhlberg et al., 1997). Binding to these domains regulates the GTPase activity by regulating the stability or the assembly of the oligomer (e.g., Warnock et al., 1996). Dynamin binds to microtubules, acidic phospholipids and phosphatidyl inositol 4,5 bisphosphate (PI(4,5)P2) containing vesicles, oligomeric Src homology domain (SH)-containing proteins and the βγ subunits of hereromeric G-proteins. Dynamin can be found at the neck of tubular invaginations or buds in the process of pinching off vesicles. It may provide the force required for vesicle scission from tubules (Takei et al., 1998; Sweitzer and Hinshaw, 1988) as shown by the observation that purified recombinant dynamin binds to acidic lipid vesicles in a regular pattern to form helical tubes. The pinching off could result from constriction of the dynamin structures upon the addition of GTP (Oh et al., 1998; Sweitzer and Hinshaw, 1998), thereby producing vesicles. Alternatively, an elongation of the dynamin spiral (a spring-like conformational change) could cause the vesicle fission (Stowell et al., 1999). However, other observations suggest that the role is indirect. Dynamin in the presence of GTP, is not sufficient to produce vesicles in perforated cells (see Schmid, 1997), possibly because other components are also needed. The role of GTP hydrolysis may also be indirect (see van der Bliek, 1999b), where dynamin acts as a switch rather than a "pinchase". Sever et al. (1999) produced two dynamin mutants. One of these was defective in its GTPase effector domain (GED). The GED is needed to increase the GTPase activity when the complex forms rings around lipid structures. The other dynamin mutant interferes with the self assembly. However the two were found to accelerate tranferrin mediated endocytosis of perforated cultured cells in the presence of a cytosolic fraction. These findings have been interpreted to negate a direct role of dynamin in the constriction of tubules and formation of vesicles. However, it is difficult to find fault with the experiments using synthetic liposomes and purified recombinant dynamin (Sweitzer and Hinshaw, 1998). It is conceivable that some component of the cytosolic extract used by Sever et al. (1999) compensates for the defect in the mutant dynamin. Vertebrates have a minimum of three dynamin isoforms and more than 25 variants are produced in rats by alternative splicing (Cao et al., 1998). Dynamin-1 is neuron specific, dynamin-2 is expressed widely in various tissues but not in neurons and dynamin-3, expressed in testes and neurons, is also present in many other tissues. Experiments in which dynamin was attached to green fluorescent protein (see Chapter 1) http://www.albany.edu/~abio304/text/chapter_9.html (25 of 41) [3/5/2003 7:54:40 PM]
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(Cao et al., 1998) show that the various variants localize to different sites inside the cell. Some dynamins localized in clathrin coated vesicles and others on vesicles not coated with clathrin. Apparently two very short amino acid domains in the middle of the molecule control their targeting. Dynamin-2 has a role in vesicle formation in clathrin dependent and independent pathways. In vitro addition of peptide-specific anti-dynamin antibodies to the assay mixture inhibited exocytic and clathrin-coated vesicle formation from the TGN (Jones et al., 1998). The microinjection of antibodies to dynamin-2 into cultured hepatocyes (Henley et al., 1998) inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, invaginations resembling caveolae (see the next section, below) accumulated at the plasma membrane and caveolamediated endocytosis of labeled cholera toxin B was inhibited. A second protein, amphiphysin, has been found to be involved in the recruitment of dynamin. Amphiphysin binds to both AP2 and dynamin. Disruption of the interaction between dynamin and amphiphysin (e.g., by recombinant amphiphysin SH3 domains that bind to dynamin) blocks recruitment of dynamin to coated pits and blocks endocytosis (Wigge et al., 1997; Shupliakov et al., 1997). In nerve, the interaction of the two is negatively regulated by phosphorylation of serine residues of dynamin (Slepnev et al., 1998). A rapid endocytosis is of vital importance at synapses which require the recycling of vesicles in order to repackage synaptic vesicles (see Chapter 22). These vesicles would then be poised to carry out a new signal across the vesicle. One of the components required for endocytosis in nerve tissue is endophilin I. Depletion of endophilin I (Schmidt et al., 1999) in perforated cells blocks the formation of synaptic-like microvesicles. Similarly, the injection of antibodies to endophilin block clathrin-mediated endocytosis (Ringstad et al., 1999). Endophilin I is a lysophosphatic acid acyl transferase which catalyzes the formation of phosphatidic acid (two acyl chains) from lysophosphatic acid (one acyl chain) and arachidonoylCoA. Endophilin I is present in the cytoplasmic leaflet of the plasma membrane and may play a role in the formation of vesicles by altering the membrane curvature to facilitate invagination during endocytosis (see Scales and Scheller, 1999; Schmidt et al., 1999). Presumably this would be accomplished by increasing the surface area of the cytoplasmic leaflet (to become the external leaflet of the vesicle). See also Chapter 4 for a discussion of this question At synapses, during endocytosis, clathrin and AP-2 have been shown to interact with elements present in the newly incorporated membranes following exocytosis (Gad et al., 1998). These include synaptotagmin which binds to AP-2 and is present in synaptic vesicles (Zhang et al., 1994). Synaptotagmins are Ca2+ and phospholipid-binding proteins present in all cells and tissues in membranes from which clathrin-AP2coated vesicles are formed (e.g., Li et al., 1995; Sugita et al., 2001). They have been shown to serve as docking sites for AP2 at the plasma membrane (e.g., Haucke and de Camilli., 1999). Genetic experiments in mice, Drosophila and the nematode Caenorhabditis elegans, show that synaptotagmins are involved in both endo-and exocytosis (Littleton et al., 1994; Geppert et al., 1994; Jorgensen et al., 1995). In addition, to the interaction with synaptotagmin, we already saw that phosphoinositides bind to several proteins involved in endocytosis, e.g., the α subunit of AP-2 (Beck and Keen, 1991; Gaidarov and Keen 1999) http://www.albany.edu/~abio304/text/chapter_9.html (26 of 41) [3/5/2003 7:54:40 PM]
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suggesting an involvement of these lipids in the mechanics of endocytosis. No less important than the assembly of clathrin is its disassembly. The uncoating of the clathrin coated vesicles needed for subsequent processing proceeds by a process involving the chaperone-ATPase hsp70c (Ungewickell et al., 1995). Auxilin mediates the binding of hsp70 to the clathrin cage. Synaptojanin 1, a phosphatase degrades phosphatidylinositol (4,5)-bisphosphate, the latter being essential for assembly. Auxilins 1 and 2 recruit the chaperone Hsc70 and stimulate its ATPase activity to depolymerize clathrin (see Slepnev and de Camilli, 2000). Apart of the special case of synaptic vesicles, which are recycled, what is the fate of uncoated vesicles and what is their relationship to clathrin coated vesicles? The fates of cholera toxin (CT) and α2macroglobulin (α2m) added to fibroblasts in cell culture were followed simultaneously with the EM by labelling them with different size gold particles (7 nm and 15 nm in diameter respectively). Both ligands bind to specific cell surface receptors. The CT first bound to non-coated pits and then appeared in a network of tubules distinct from the Golgi apparatus. In contrast, the α2m bound to coated pits was taken up by coated vesicles and then appeared in the network of tubules. Despite being packaged in distinct vesicles, the two macromolecules were found in the same compartments beginning with their appearance in the tubular network. Eventually both were present in the multivesicular bodies (which probably correspond to lysosomes). The results are summarized in Fig. 8 (Tran et al., 1987). In this figure, the % of the gold particles are shown (the ordinate) in relation to time (the abscissa). New information has surfaced on mechanisms of endocytosis (see Kirchhausen,2000; Nichols and Lippincott-Schwartz, 2001). Studies taking advantage of mutants unable to form clathrin-vesicles have provided considerable evidence for clathrin-independent endocytosis (see Puri et al., 2001; Nichols et al., 2001; Lamaze et al., 2001). In addition, techniques that allow tagging proteins and lipids with fluorescent labels has introduced the possibility of visualizing the movement of these components (Pelkmans et al., 2001; Puri et al., 2001; Nichols et al., 2001). Many of the non-clathrin based processes have been found to involve caveolin or lipid rafts. (see Nichols et al., 2001). Their role can be defined by interfering with their formation. Cholesterol deprivation (which may also have other effects) does not interfere with clathrin-mediated endocytosis but blocks the caveolinlinked pathways (e.g., Puri et al., 2001; Nichols et al., 2001. Caveolae and lipid rafts (section V) are discussed below.
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Fig. 8 Relationship of CT-gold (7-15 nm) (upper) and α2m-gold (lower) with the various locations in fibroblasts. First incubation at 4oC for two hours and then without labelled ligand at 22 oC. Tran et al., 1987, reproduced by permission.
V. CAVEOLAE, RAFTS AND OTHER MEMBRANE PATCHES Many studies have centered on coated pits. However, there are other specialized areas at the surface of cells. Some of these have the appearance of flasks 50 to 100 nm in diameter. These structures have been called caveolae (Yamada, 1955), meaning "little caves". They are probably the same structures as those described by Palade and Bruns (1958) for endothelial cells, which they called plasmalemmal vesicles. Their importance in endocytosis and receptor mediated signaling is just beginning to be appreciated (e.g., see Parton, 1996; Anderson, 1998). They are likely to constitute a system parallel to that of the clathrin coated pits and vesicles. With most standard EM techniques, caveolae and vesicles derived from caveolae appear uncoated. However, high resolution EM and freeze-deep etch techniques have shown the caveolae have a distinctive cytoplasmic coat of delicate filaments arranged as striations (Peters et al., 1982, Izumi et al., 1988). The striations appear as ridges or strands 6 to 10 nm in width and are seen most clearly after treatment with the detergent saponin (which presumably removes other proteins). The striped structures were enhanced by treatment with subfragment-1 (S1) of myosin or the mushroom toxin phalloidin, suggesting an involvement of actin. Actin is discussed in relation to endocytosis in Section IX and in relation to cell movement in later chapters (e.g., in Chapter 23 and Chapter 24, Section IA and Section IVA). Phalloidin and S1 bind to F-actin and phalloidin prevents its depolymerization. The distinct protein present in the caveolar membranes is caveolin (e.g. see Schnitzer et al. 1995b). Whether all uncoated http://www.albany.edu/~abio304/text/chapter_9.html (28 of 41) [3/5/2003 7:54:40 PM]
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invaginations or vesicles can be considered part of the caveolar system is not clear. Caveolae have been isolated by a variety of methods, some in the absence of detergents (e.g., Smart et al., 1995b; Song et al., 1996). As important as caveolae are likely to be, mice lacking caveolae (because of disruption of caveolin-1) have been shown to survive, although defective in nitric oxide and calcium signaling in the cardiovascular system. These mice exhibit defects in endothelium-dependent relaxation, contractility, maintenance of myogenic tone and exhibit a thickening of alvaeolar septa in the lungs (Drab et al., 2001). However, all of these defects would be in harmony with the suspected role of caveolae in organizing signaling pathways in the cell (see below). Early studies suggested the presence of membrane patches rich in glycosphingolipids found to be detergent (such as Triton) insoluble. Structurally, these fractions were found to contain vesicles 50 to 100 nm in diameter, similar to caveolae. These domains were found to contain cholesterol, glycosphingolipids, glycosylphosphatidylinositol- (GPI)-linked proteins (see Chapter 4) and caveolin. Some studies challenge the idea that the supposed caveolar components are present in clusters. The results of these studies suggest that at least some of the components are distributed randomly throughout the plasma membrane. They attribute the earlier results on artifacts produced by the use of detergent or cross-linking by antibodies. However, a very strong case can be made for the clustering of GPI-anchored proteins in caveolae (see discussion in Chapter 4). Caveolin is a 21-24-kDa protein, the principal component of caveolar membranes. Unlike clathrin, the hydrophobicity plot of clathrin indicates a transmembrane sequence (Glenney, 1992), a conclusion supported by the accessibility of the protein in intact cells to chemicals from either the external or the cytoplasmic surface of the plasma membrane (see Chapter 4). The hydrophobic domain contains 33 amino acids and the carboxy- and amino-terminals are free in the cytoplasm. Recently, caveolin has been recognized as a family of proteins (see Parton, 1996) and the caveolin originally studied has been renamed caveolin-1. In mammals, there are three distinct caveolin genes coding for caveolin-1, -2 and -3. In addition, there are two different isoforms for caveolin 1, Cav-1α and Cav-1β produced by alternative initiation during translation. Caveolin-1 and 2 are most abundantly expressed in endothelial cells, smooth muscle cells, skeletal myoblasts, fibroblasts, and differentiated adipocytes (Schereret al. 1997). Caveolin3 is expressed mostly in muscle (Tang et al., 1996). Purified caveolin homo-oligomers have the capacity to self-associate into caveolae-like structures (Sargiacomo et al., 1995). They bind cholesterol with 1:1 stoichiometry (Murata et al., 1995) and insert into model lipid membranes only in the presence of cholesterol. In addition, caveolin-1 also binds fatty acid (Trigatti et al., 1999). Caveolin 1 and 2 form hetero-oligomeric complexes of high molecular weight (14-16 monomeric units per oligomer) and localize to caveolae as shown by immunoelectron microscopy (Scherer et al., 1997). Caveolin and cholesterol dynamics are clearly interrelated. The synthesis of caveolin depends on cholesterol concentration: caveolin mRNA levels in culture cells dropped to one-sixth of control levels http://www.albany.edu/~abio304/text/chapter_9.html (29 of 41) [3/5/2003 7:54:40 PM]
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after treatment an inhibitor of cholesterol synthesis, or a cholesterol sequestering drug (e.g., Hailstones et al., 1998). On the other hand, increases in caveolin increase cholesterol transport Cultured cells transport new cholesterol to caveolae with a half-time of approximately 10 min (Smart et al., 1996). The cholesterol then rapidly flows from caveolae to non-caveolar membranes. Cholesterol moved out of caveolae even when the supply of fresh cholesterol from the ER was interrupted. Some of the interactions are with the hydrophobic domain of caveolin, thought to traverse the membrane (Wary et al., 1996; Das et al., 1999). Another domain close to the insertion into the membrane (amino acids 80-100) binds to variety of signal transducing molecules (see Okamoto et al, 1998) including tyrosine kinase receptors, nitric oxide synthase and heterotrimeric G proteins. This domain, called the caveolin scaffolding domain was used as receptor to select caveolin-binding peptide ligands (Couet et al., 1997) from random sequences. Two similar caveolin-binding motifs were found [φXφXXXXφ and φXXXXφXXφ; φ can be any aromatic amino acid (Trp, Phy or Tyr)]. These motifs are present in most caveolae-associated proteins. The scaffolding domain is apparently not used in recruitment (Mineo et al. 1999) but is thought to have a role in modulating transduction (see Okamoto et al., 1998). The role of the various caveolin-1 domains in targeting was studied systematically by introducing mutations in caveolin-cDNA followed by transfection of cells in culture. (Machleidt et al., 2000). The amino acid domain between amino acid 66 and 70 was found to be required for exit from the ER. The domain between amino acids 71 and 80 was found to control the incorporation of caveolin-1 into detergent resistant regions of the Golgi (presumably rafts). The domains of amino acids 91 to 100 and 134 to 154 were needed for oligomerization and exit from the Golgi apparatus. The fate of the caveolin mutants or wild type were followed using immunofluorescence (see Chapter 1). Caveolae have a specific lipid composition, many lipid modified proteins, receptors and signaltransducing molecules. The lipid composition is high in glycosphingolipids, sphingomyelin and cholesterol. Proteins attached to either GPI or fatty acids (see Chapter 4) are enriched in caveolae. Mutations that remove the GPI-anchor (e.g., Ritter et al., 1995) or protein acylation (e.g., Robbins et al., 1995) redistribute the proteins to other environments. In the cell, caveolin is associated with cholesterol (e.g., Li et al., 1996) and cholesterol stabilizes caveolin oligomers (Monier et al., 1995) suggesting that the two must function together to provide a coat. The lipid core of caveolae forms in the transitional region of the Golgi (e.g., Lisanti et al., 1993). The proteins attached by GPI anchors and caveolin synthesized in the ER are incorporated into this lipid core (see Lisanti et al., 1993). Caveolae reach the cell surface via exocytotic vesicles (Dupree et al., 1993). Bidirectional ER-to-caveolae transport of cholesterol involves caveolin-1 (e.g., see Smart et al., 1994; 1996). Accordingly, caveolin-1 (then called VIP21) (see Glenney, 1992) was found in the Golgi, the plasma membrane and vesicular structures and appears to be involved in the machinery of vesicular transport (Kurzchalia et al., 1992). Tracer studies have implicated uncoated vesicles in transcytosis of LDL (see Simionescu, 1983), serum albumin and other molecules across capillary endothelial cells (Ghitescu et al., 1986; Vasile et al., 1983). http://www.albany.edu/~abio304/text/chapter_9.html (30 of 41) [3/5/2003 7:54:40 PM]
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Caveolae are also involved in the endocytotic uptake of macromolecules (Tran et al., 1987; Keller et al., 1992). Transcellular transport involves vesicles originating from caveolae (e.g., see Simionescu et al., 1975; Schnitzer et al., 1994, 1996) that may also form channels across cells (Simionescu et al., 1975). Caveolae are dynamic, actively interacting with the endocytic compartment including early-sorting endosomes (e.g., see Pol et al., 1999). A reconstituted cell-free system capable of forming vesicles from caveolae has been developed using endothelial plasma membranes (Schnitzer et al., 1996). Caveolae have a role in clustering glycosylphosphatidylinositol-linked receptors. Unclustered and clustered GPI-proteins are thought to be in dynamic equilibrium (van Meer et al., 1987). The unclustered proteins are highly mobile in the plane of the membrane (Zhang et al., 1991). The mobile fraction can bind to transmembrane proteins and be internalized. These results suggest that the GPI-anchored proteins spend part of the time in caveolae and part of the time free; the latter can interact with receptors so that they might be internalized in coated pits (Anderson, 1993). Components important in Ca2+ transport have also been found in caveolae (Fujimoto, 1992, 1993). Immunogold EM studies using antibodies specific to the inositol 1,4,5-triphosphate (IP3) receptor and Ca2+-ATPase revealed the presence of these proteins in smooth invaginations of the plasma membrane of a variety of cells. We saw in Chapter 7 that IP3 receptors function as a Ca2+-channel, at least inside the cell. Caveolae are involved in signaling (e.g., Lisanti et al., 1995; see Okamoto et al., 1998; Razani et al., 2000). In at least some cases, glycosylphosphatidylinositol (GPI) anchored receptor proteins become associated with caveolae only after binding to their ligand (or an antibody) ( Sevinsky et al., 1996). ). Receptor and non-receptor tyrosine kinases have been found in caveolae (see Anderson, 1998, his table 1), e.g., by immunofluorescence. Caveolins bind to signaling molecules such as heterotrimeric G protein subunits, Src kinases, and Raf,. G-protein-coupled receptors such as inositol 1,4,5-trisphosphate receptors, endothelin, bradykinin, muscarinic acetylcholine, and adrenergic receptors. Caveolae are the principal location of platelet derived growth factor (PDGF) receptors at the surface of platelets and many caveolar proteins are phosphorylated when PDGF binds to its receptor suggesting that many proteins involved in the signaling cascade are also in caveolae (Liu et al., 1996). GTP-binding proteins (Gproteins) (Sargiacomo et al., 1993), receptors and effectors appear to be enriched in caveolae. In addition to this direct role, some of the lipids and lipid-anchored proteins are sources of signaling intermediaries for the formation of ceramide, inositol trisphosphate (IP3) and inositol phosphoglycans. Many G-coupled receptors are localized or internalized at uncoated invaginations or vesicles presumed to be caveolae (Montesano et al., 1982, Raposo et al., 1987, 1989; Strosberg et al., 1991). A role of caveolae in signal transduction is also strongly supported by the observation that antibodies against different GPI-anchored proteins activate cells (e.g. Thompson et al., 1989). We saw in Chapter 6 that cross-linking by antibodies activates receptors. Mutations of the gene coding for caveolin 3 have been implicated in limb-girdle muscular dystrophy and http://www.albany.edu/~abio304/text/chapter_9.html (31 of 41) [3/5/2003 7:54:40 PM]
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in rippling muscle disease. Caveolin 3 truncation mutants have been found to inhibit signaling (Roy et al., 1999) and caveolin 3 point mutants have been found to inhibit H-Ras-dependent signaling. The inhibition is rescued by cholesterol addition (Carozzi et al., 2002). When activated H-Ras cause significant changes in cell shape and cytoskeletal organization. What is the role of caveolae in signaling? The caveolins are thought to act as scaffolds for the assembly of signaling complexes. In this way the assembled complexes can be quickly deployed where needed. In addition, caveolae may have an inhibitory regulatory function in removing these complexes from the active pool (see Okamoto et al., 1998). However, in some cases the signaling molecules can be shown to be activated in caveolae so that the assembling may actually permit a more effective activation of the complexes (e.g., see Chambliss et al., 2000; Peiro et al., 2000). The internalization of receptors requires protein kinase C (PKC)α (see Chapter 7) and serine/threonine phosphatase activity in caveolae (Smart et al., 1995a). A 90-kDa protein is the substrate for these enzymes. The carrier vesicles derived from caveolae retain much of their structure and can function as exocytotic vesicles (see Anderson, 1993). Until relatively recently, the link between caveolae and intracellular vesicles was unclear because the pinching off of vesicles had not been observed in detail and caveolin containing vesicles had not been studied sufficiently. The formation of vesicles from caveolae by a pinching off process was observed in permeabilized cells (Schnitzer et al., 1996). Caveolae can be isolated after mechanical disruption of endothelial tissues and after GTP-induced formation of vesicles (Schnitzer et al., 1995a). They have been found to contain the machinery for receptor mediated endocytosis and transcytosis as well as vesicle budding, docking and fusion (see Schnitzer, 1995a). As in the case of clathrin mediated endocytosis, the pinching off of vesicles was found to require cytosolic dynamin (see above section) and the hydrolysis of GTP (also required for formation of trans-Golgi vesicles, Jones et al., 1998). In addition, dynamin was found to be concentrated in caveolae (Oh et al., 1998; Hendley et al. 1998). The emerging picture suggests that the caveolar system behaves in an entirely analogous way to that of coated pits as represented in Fig. 7. The process of vesicle formation from caveolae has been studied by observing the endocytosis elicited by simian virus 40 (SV40) in monkey kidney normal cells (CV-1) in culture (Pelkmans et al., 2002). The movement of the various proteins was followed by tagging them with fluorescent proteins [green fluorescent protein (GFP) or yellow fluorescent protein (YFP); see Chapter 1]. After binding to the caveolae, SV40 was found to induce the disassembly of stress fibers. The actin and dynamin II were then recruited to the caveolae where their recruitment lead to actin "tail" formation. The formation of tails corresponds to an assembly of actin molecules to produce motion (see Chapter 24). Cholesterol and the phosphorylation of the proteins of caveolae by tyrosine kinases was found to be required for endocytosis to occur.
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Anderson et al. (1992) have hypothesized that caveolae are active in the endocytotic uptake of low molecular weight substances, a process referred to as potocytosis (poto meaning to drink). A role of potocytosis in the transport of low molecular weight substances is primarily based on the study of the uptake of 5-methyltetrahydrofolate. The properties of the folate transport system have been studied in folate-depleted cells. Folate binds to a specific receptors. At 0oC half of the receptors bind folate. At 37oC, however, after 1 hour, internal and external receptors were found to bind folate. The two could be distinguished because externally bound folate is released by acid wash. Eventually, all the folate was inside the cell and accumulation stopped. The release into the cytoplasm required an acid pH and was mediated by an anion transporter. The process in one distinct from endocytosis: the folate receptors have never been found in endocytotic vesicles. The receptors are segregated in caveolae and the recycling of the receptors closely matches the sequestration of materials in the caveolae. The involvement of an anion carrier is based on the observation that the release is probenecid sensitive. Probenecid is a drug which blocks tubular secretion (i.e. active transport) of many anionic drugs. The transport is thought to follow the steps outlined by the model shown in Fig. 9 (Rothberg et al., 1990). In step 1, the folate molecules are bound to their receptors. In step 2, the caveola closes and in step 3 the caveolar interior is acidified, so that the folate molecules detach from the receptors. The folate molecules are transported to the cytoplasm in step 4, and the partially empty caveola opens again in step 5.
Fig. 9 Model of receptor coupled transmembrane transport for folate in caveolae. Reproduced from Rothberg et al., Journal of Cell Biology by copyright ©1990. Reproduced by permission of The Rockefeller University Press.
Some of the clathrin-independent pathways carry lipids and GPI-anchored proteins to the Golgi apparatus ( Nichols et al., 2001). In at least some cases, caveolae are not likely to be involved because the processes are dynamin independent (e.g., Vickery and von Zastrow, 1999; Roseberry and Hosey, 2000). In view of the involvement of GPI, rafts are thought to be reponsible for this transport (Puri et al., 2001; Nichols et al., 2001). To complicate this scenario even further, different raft markers follow different pathways. http://www.albany.edu/~abio304/text/chapter_9.html (33 of 41) [3/5/2003 7:54:40 PM]
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Some GPI-anchored proteins cycle between the cell surface and the Golgi, others cycle to the endosomes (Nichols et al., 2001; Mayor et al., 1998). A study of proteins anchored to GPI (the folate receptor, the decay accelerating factor and GFP-linked to GPI) taken up by endocytosis found them recycling to an endosomal compartment by a pathway not involving either clathrin, caveolae or the Golgi apparatus, (Sabharanjak et al., 2002). The transfer was mediated by tubular-vesicular elements distinct from those involved with the Golgi. The pathway was dependent on the GTPase Cdc42 . In addition to the possible role of caveolae and rafts, other lipid specializations are thought to have a role in the localization of components. Late endosomes contain a internal membranes in their lumen. The distribution within these organelles is distinct. Some proteins are present in the internal membranes, whereas others are localized in the limiting membrane. The internal membranes form a specialized domain with a high content of lysobisphosphatidic acid 2-phosphate (LBPA) (Kobayashi et al., 1999). LBPA is also the antigen for human antibodies associated with the antiphospholipid syndrome. The membranes are responsible for the sorting of the receptors for insulin-like growth factor 2 and lysosomal enzymes. In addition, the LBPA-rich domain has a role in cholesterol transport. Cholesterol derived from LDL was found to be processed by late endosomes where cholesterol accumulates in the genetic disease Niemann-Pick type C (NPC) and in cells treated with drugs that mimic NPC (Kobayashi et al., 1999). Another lipid, phosphatidylinositol 3-phosphate has been found in limiting membranes of early endosomes and in the internal membranes of the multivesicular bodies (Gillooly et al., 2000). A variety of proteins containing the FYVE and the PX (PHOX) homology domains which bind to phosphatidylinositol 3-phosphate have been shown to be present in these endosomes and supposedly may be bound to the membranes . VI. OTHER FORMS OF ENDOCYTOSIS Phagocytosis, the uptake of large particles by cells, is most generally thought to be a property of phagocytic protozoa or phagocytic leukocytes of the mammalian immune system such as macrophages. In protozoans, phagocytosis has a nutritional role. In mammals, one of the important roles is in immunity (see Chapter 6 and Aderem and Underhill, 1999) and apoptosis (see Chapter 2). Cell surface receptors bind to the particle to initiate phagocytosis. In protozoans, the receptors involved in phagocytosis are thought to be lectin-like components (Cohen et al., 1994). Macrophages possess several phagocytic receptors that bind conserved motifs on pathogens (e.g., the mannose receptor). In addition, pathogens are recognized by receptors after coating of the particles with complement, Ig Fc or specific antibodies (opsonization). Receptors include members of the IgG Fc receptor family (Mellman et al., 1983), integrins (e.g. Isberg and Tran Van Nhieu, 1994) and lectins (Ezekowitz et al., 1991). The binding to receptors is followed by polymerization of actin in the cytoplasm close to the site of particle attachment (see May and Machesky, 2001). Various small GTPases (see Chapter 11) control this step. Phosphatidylinositol 3-kinase is recruited to the plasma membrane and is involved in pseudopodia extension and phagosome formation (e.g., Vieira et al., 2001). Basically the process takes place as
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follows. First the receptor binds to its ligand. Other receptors are recruited and lead to the formation of a protrusion without intervention of actin. This is then followed by recruitment of cytoskeletal components (e.g., the Arp2/3 complex) which in turn recruit actin and form a pseudopod. Then, the actin network moves the pseudopod around the target and the pseudopod engulfs the bound particles in a large membrane enclosed vacuole, the phagosome (see Greenberg et al., 1990, 1991). The vacuole or phagosome taken up by the macrophage undergoes maturation, which involves sequential interactions with components of the endocytic pathway, and culminates with fusion to lysosomes (see Tjelle et al., 2000) followed by digestion (e.g. Desjardins et al., 1994). The membrane component is eventually recycled. Apparently, membrane elements of the endoplasmic reticulum (Gagnon et al., 2002) fuse with the plasma membrane to provide some of the needed membrane for the formation of the vacuole. The mediation of the ER is regulated by phosphatidylinositol 3-kinase. Clathrin-dependent endocytosis is well recognized. We have also discussed evidence for an involvement of caveolae in endocytosis. Many observations indicate endocytosis also proceeds by different mechanisms. Macropinocytosis (see Swanson and Watts, 1995) was first observed in macrophages where surface ruffles (see Chapter 23) formed endocytotic vesicles. Since then similar events have been observed in other cells. Macropinocytosis differs from clathrin-mediated endocytosis in that vesicles are formed only at margins of cells at sites of ruffling. The size of the vesicles varies but can be as large as 5 µm in diameter, whereas coated pits are restricted to 85-110 nm because of their clathrin coating. The formation of macropinocytotic vesicles can be stimulated by binding of some growth factors. The role of macropinocytosis is not clear, although it might possibly contribute to the immune response. As in the case of phagocytosis, actin is likely to play a role in the formation of vesicles in macropincyotis. Ruffles are formed by outwardly directed actin filaments. The actin filaments are involved in the formation of the vesicles, possibly by enclosing the vesicles as clathrin does in the case of the endocytosis mediated by coated pits. At any rate, in a later step the vacuoles lose their actin coating and are indistinguishable from other endosomal compartments. Endocytosis of activated receptors without the involvement of clathrin has also been observed.. Interleukin 2 receptors were found to become internalized in dominant-negative mutants of eps15 (Lamaze et al., 2001) in human (HeLa)cells, mouse fibroblasts transfected with genes encoding the IL2 receptor and lymphocytes. Expression of the mutants inhibits both constitutive and ligand induced receptor-mediated endocytosis (see Benmerah et al., 1999). The receptors were not present in clathrin coated structures but were present in detergent resistant membranes. Since lymphocytes do not have caveolae the receptors are probably internalized in membrane rafts (see Chapter 4). The biochemical requirements for non-clathrin endocytosis have generally been found to be similar to those of the clathrin-dependent process. Clathrin-independent endocytosis is temperature dependent, requires ATP, and is sensitive to sulfhydryl reagents (Sandvig et al., 1991). Activated Rho, a small GTP-
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binding protein, has been shown to stimulate pinocytosis when microinjected into Xenopus oocytes (Schmalzing et al., 1995), and Ras and Rho have a role in membrane ruffling in fibroblasts (e.g. Ridley et al., 1992).The endocytosis of the IL2 receptors requires the presence of dynamin and is also controlled by GTPases of the Rho family (Lamaze et al., 2001). VII. ENDOSOMES AND LYSOSOMES: INTERACTIONS The discussion of the fate of ligands and receptors after endocytosis (section IIIC) included the digestion of material present in endosomes by lysosomal hydrolytic enzymes. The properties of either endosomes or lysosomes differ depending on their stage during their cycle. Early endosomes close to the cell surface differ from late endosomes which interact with lysosomes. It has been assumed that these differences are the result of a maturation process, and that the late lysosomes and endosomes interact by fusion. The characteristics of the endosomal compartments have been discussed above. Recently, a view has gained favor that endosomes and lysosomes form systems of stationary organelles akin to the Golgi stacks and tubular elements. Transport vesicles shuttle between the stationary elements (e.g. Storrie, 1988, Griffiths and Gruenberg, 1991). The stationary elements act as the relay stations: the endosomes receive endocytotic transport vesicles and deliver the contents, also in transport vesicles, to the lysosomes where digestion takes place. In part, the proposals find support from structural observations. The early endosomes of many kinds of cells are constituted of cisternae, tubules, and large vesicles exhibiting coated buds. Similarly, a complex lysosomal system of cisternae, tubules and large vesicles has been described. In the case of the transfer of material taken up by endocytosis (e.g., horse radish peroxidase) the process is discontinuous, and is mediated by transport vesicles possibly transported along microtubules. In addition, the properties of the vesicles derived from the endosome system are in harmony with this general concept. Early endosomes can fuse with each other in vitro, a fusion that is regulated by a GTP-binding protein which has been implicated in intracellular transport. Direct fusion between early and late endosomes does not take place in vitro. However, the apparent endosomaltransport vesicles fuse with late endosomes. In the late endosomes or MVBs , some proteins are found in the internal membranes while others are present only in the outer membrane (Griffiths et al., 1988). The internal membranes contain a unique lipid, lysobisphosphatidic acid, forming a specialized domain that is thought to segregate several proteins including lysosomal enzymes (Kobayashi et al., 1998). An antibody to this lipid interferes with the structure and function of these organelles, for example it blocks the recycling of the mannose-6phosphate receptor. The fusion of the outer membrane of the MVBs with the lysosomal membrane discharges the contents and the internal vesicles into the lysosome for degradation (Futter et al., 1996). In contrast, the proteins are not to be degraded, such as receptors that are recycled, remain in the limiting membrane of the MVB by being excluded from the inner vesicles. The sorting nexin (SNX) proteins (see Worby and Dixon. 2002) are either cytoplasmic or membrane bound. This protein family functions in intracellular membrane traffic. The SNXs are needed for transport
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to the lysosomes and recycling of endosomes. SNX3 is present in the early endosome and its action depends on its PX domain (Xu et al., 2001) which allows it to bind to phosphoinositides (see Chapter 11). Some of the SNXs contain only a PX domain. However, others contain protein-protein binding sequences (SH3 and TPR, see Table 2, Chapter 6), hydrophobic sequences that allow interaction with membranes and G-protein regulatory sequences. Some of the phospholipids are localized at particular membrane sites and can serve as targeting signals. Some of the SNXs are likely to have a role in sorting vesicles not originating from the plasma membrane. Two-hybrid interaction demonstrated a binding between SNX1 and the intracellular portion of the receptor tyrosine kinase EGFR (Kurten et al., 1996). SNX1 was implicated in the ligand-induced EGFR degradation where it could play a role in sorting EGFR to lysosomes. Similar proteins in yeast the proteins vacuolar sorting proteins (Vps) were shown to be involved in vacuolar targeting in yeast. SNX1 also binds other receptors of the tyrosine kinase family such as PDGFR, the insulin receptor, the leptin receptor and the transferrin receptor (see Worby and Dixon. 2002). The story of endocytosis is a fascinating one which has opened many windows to our understanding of cell processes. However, there are still many blank spots that will be the focus of future studies. VIII. TRANSCYTOSIS Epithelial cells form monolayers lining ducts and cavities in the body. They constitute tight barriers that allow selective transport through the cells themselves. In these cells, the surface of the plasma membrane in contact with the lumen (the apical surface) differs significantly from the rest of the surface (the basolateral surface). It has a unique protein composition and a high glycosphingolipid content. These cells are said to be polarized (see Chapter 11). The solute-pumps of the kidney, intestine and other tissues operate to transport solutes across the cells. For example, in the cortical collecting ducts of the kidney, Na+ enters the cells in the direction of its electrochemical gradient at the apical surface through Na+-channels. Simultaneously, it is actively pumped out into the interstitial fluid by the Na+, K+-ATPase of the basolateral membrane. Na+ traverses the cell by diffusion so that this transport results in a net flux of Na+ from the lumen of the ducts to the interstitial fluid. In addition to the transport of solutes, vesicle-mediated traffic carrying materials across the cells by transcytosis is continuous (see Simons and Wandiger-Ness, 1990). Generally, transcytosis occurs by receptor mediated endocytosis (see Schaerer et al., 1991; Sztul et al., 1991; Rodriguez-Boulan and Powell, 1992), followed by passage of vesicles across the cells and eventually delivery to the cell surface by exocytosis. The biosynthetic pathway which transports newly synthesized proteins to apical or basolateral surfaces is obviously related to the transcytotic system (see Chapter 11) although the connections between the two are not entirely clear.
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One of the in vitro epithelial cell system most commonly used for studying these two pathways is that of Madin-Darby canine kidney (MDCK) cells because in culture they form polarized monolayers when grown on permeable filters. A cultured human intestinal adenocarcinoma cell line (Caco-2) also forms monolayers and has been used for similar studies. The MDCK cells do not normally express receptors for polymeric immunoglobulin (pIgR) or transferrin (TRs). When these two kinds of receptors are introduced by transfection with recombinant retrovirus vectors containing the appropriate cDNAs (see Chapter 1), pIgRs transport bound dimeric IgAs from the basolateral to the apical surfaces (Mostov and Simister, 1985) whereas TRs move in the opposite direction (e.g., Knight et al., 1995; Odorizzi et al., 1996). The cytoplasmic regions of the pIgR contain domains which function as trafficking signals throughout the transcytotic pathway (Mostov et al., 1992; Casanova et al., 1990, 1991; Aroeti et al., 1993). These include the tyrosine and dileucine motifs recognized by the clathrin/AP2 system. However, basolateral targeting requires other residues (Odorizzi et al., 1996; Mellman, 1996). As discussed above, transport across the cells can reach different targets. Do the various receptor mediated transports use the same pathway? The endosomes involved in the trafficking of pIgR and IgA also contain internalized TRs (Barroso and Sztul, 1994; Apodaca et al., 1994). However TRs are returned to the basolateral surface whereas pIgR proceeds to the apical surface (Barroso and Sztul, 1994; Apodaca et al., 1994). The exocytotic vesicles that carry the TR to the basolateral surface, arise from clathrin-AP-1 coated buds associated with the endosomes (Futter et al., 1998). Clathrin-AP-1 coated buds and vesicles had previously only been found associated with the trans-Golgi network (TGN) (see Chapter 11). The various components were studied with gold labelling and electron microscopy. The apical transcytotic pathway of dIgA, pIgR at 20 o shows that these components internalized at the basolateral surface and were subsequently localized in interconnected vacuoles and tubules they share with internalized TR and epidermal growth factor receptor (EGFR) (Gibson et al., 1998). When transferred to 37o the vacuoles form long tubules in a process that depends on the cytoplasmic microtubular system. These long tubules then form distinct cup-shaped 100nm vesicles containing pIgR. The cup-shape vesicles carry dIgA and pIgR to the apical surface where they are exocytosized. In contrast, the fate of TRs and EGFR are different. TRs are selectively removed in 60 nm clathrin coated buds, whereas EGFR are removed by intraluminal vesicle of multivesicular bodies. EGFRs are ultimately targeted to the lysosomes. TRs are targeted to the basolateral surface. These results indicate that although these transported integral proteins share common early compartments eventually they are targeted separately. In agreement with the notion of an initial common pathway, a sorting station is thought to operate in polarized cells next to the TGN. This station connects the apical and basolateral transport pathways and is involved in transcytosis and the recycling of proteins (e.g., Gibson et al, 1998; Futter et al., 1998) and lipids (e.g., vanIJzendoorn et al., 1997; van IJzendoorn and Hoekstra, 1998). Current thinking considers it a separate compartment, the Golgi sub-apical compartment (SAC) related to the endosomes. The SAC appears to be involved for either apical to basolateral transport or the basolateral to apical transport (see http://www.albany.edu/~abio304/text/chapter_9.html (38 of 41) [3/5/2003 7:54:40 PM]
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van IJzendoorn and Hoekstra, 1999). The evidence that has led to the formulation of this concept is as follows: (a) basolaterally derived proteins and proteins internalized from the apical surface colocalize to the same structures (e.g., Hughson and Hopkins, 1990; Knight et al., 1995), and, (b) the transcytotic marker polymeric immunoglobulin receptor (pIgR) which mediates basolateral to apical transcytosis of IgA and IgM (Apodaca et al., 1994) is picked up by endocytosis from the basolateral surface and is targeted to the SAC before delivery to the apical membrane domain (Apodaca et al., 1994; Barroso and Sztul, 1994). IX. INVOLVEMENT OF THE CYTOSKELETON In view of the location of actin networks at the periphery of the cell, the possibility of a role of an actinmyosin system in endocytosis cannot be overlooked. This chapter frequently suggested a role of actin in most kinds of endocytosis. However, the cited evidence was very indirect. The involvement of actin in endocytosis at the molecular level is just beginning to be understood (e.g., see Riezman et al., 1997; McPherson, 2002. The formation of clathrin coated pits which takes place at certain specific sites in the cell surface involves actin (e.g., Santini et al., 2002) A major role of actin in endocytosis is well established in Saccharomyces cerevisiae (see Geli and Riezman, 1998) and Wendland et al., 1998). Mutations in actin and actin binding proteins inhibit receptormediated and fluid endocytosis. (e.g. Kübler and Riezman, 1993; Raths et al., 1993; Munn et al., 1995). In contrast, β-tubulin mutant strains showed no defect in this process indicating no major role for microtubules. Actin and cofilin mutants indicate that the rapid turnover of actin is required for endocytosis (see Lappalainen and Drubin, 1997; Belmont and Drubin, 1998). Cofilin is an actindepolymerizing factor (see Chapter 24). Among other actin-binding proteins, S1a2p (also known as End4p and Mop2p), has been identified as an actin binding protein (e.g., Wesp et al., 1997) implicated in endocytosis (e.g., Raths et al., 1993). Experiments using cytochalasin, which destabilize actin filaments and blocks endocytosis, suggest a similar mechanism for endocytosis at the apical membrane (but not the basolateral membrane) of polarized mammalian cells (e.g., Jackman et al., 1994). A role of actin in endocytosis is also confirmed by the observation that many of the proteins involved in endocytosis bind to actin and some of the components of the clathrin coat (see McPherson, 2002). Among these dynamin which binds to many endocytotic adaptor proteins as well as proteins capable of binding to actin (see Schmid et al., 1998). Dynamin the GTPase usually thought to be involved in the scission of the the vesicles from the plasma membrane (see Chapter 11), binds to proteins capable of binding actin and has a role in the production of actin tails (Orth et al., 2002; Lee and De Camilli, 2002) suggesting that it is part of the a protein machinery needed for nucleation of actin from membranes. Search in mammalian data bases (see Chapter 1) for proteins with a similar amino acid sequence to http://www.albany.edu/~abio304/text/chapter_9.html (39 of 41) [3/5/2003 7:54:40 PM]
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known yeast proteins already implicated in endocytosis (such as Sla2p), offers a way of extending these studies to the role of actin in mammalian cells. The mouse protein mHip1R was identified in this way (Engqvist-Goldstein et al., 1999) and was found to be very similar to the human huntingtin interacting protein 1 (Hip1). Huntingtin is the protein responsible for Huntington disease when the polyglutamine section is expanded to more then 35 repeats. Huntington disease is an inherited progressive neurodegenerative deficity (e.g., see Freiman and Tjian, 2002). HIP1 and the related protein, HIP1R/HIP12, have been found in the clathrin coat of clathrin coated pits and vesicles (EngqvistGoldstein et al., 1999; Mishra et al., 2001). HIP1R/HIP12 colocalizes with clathrin, AP-2, and endocytosed transferrin. Indirectly, it binds to clathrin and stimulate clathrin assembly (e.g., (LegendreGuillemin et al., 2002). At least in vitro, HIP1 does not bind to actin but does bind to clathrin and AP2, whereas HIP1R/HIP12 co-sediments with F-actin, binds to actin and but does not bind to AP2 or clathrin. However, HIP1R and HIP1 form heterodimers (Legendre-Guillemin et al., 2002) so that the complex could bind to both clathrin and actin. HIP1 and HIP1R/HIP12 contain a phosphatidylinositol 4,5biphosphate-binding motif (ENTH). Phosphatidylinositol 4,5-biphosphate has also been shown to bind to a variety of other proteins involved in endocytosis (e.g., AP2, epsin and AP180). Furthermore, phosphatidylinositol 4,5-biphosphate is present in the plasma membrane of resting neurons. The level in endocytized membranes increases following stimulation of the presynaptic cells (Micheva et al., 2001). These findings are intriguing and suggest a role of phosphatidylinositol 4,5-biphosphate in endocytosis, perhaps in the localization of the endocytotic machinery and actin in the specialized spots of the plasma membrane. In view of the involvement of actin comets tails (see Chapter 24) in vesicle movement, actin polymerization alone could result in movement associated with endocytosis (see Taunton, 2000; Taunton, 2001). However, the motor myosin could also be involved in budding and other movements (see Chapter 24). Most myosins move toward the plus-end of actin filaments (see Chapter 24), toward the periphery. The one exception is myosin VI, which is directed toward the minus-end . An experimental examination of this question made use of myosin VI-GFP constructs in polarized cells (Buss et al., 2001). The constructs were found to localize mostly at the apical surface where endocytosis takes place. In addition, they were associated with clathrin-AP2-coated vesicles, since myosin VI co-precipitates in a complex with AP-2 and clathrin. In transfected fibroblasts, the inhibition of the interaction by over-expression of mutants of myosin VI lacking the motor domain, decreased the endocytotic uptake of transferri in fibroblasts, again supporting a role of myosin VI in clathrin-mediated endocytosis. Not surprisingly clathrin coats have been found to be connected to cytoskeletal elements that play either a functional role or that link actin or microtubules to the plasma membrane . The spectrin-binding protein ankyrinR facilitates the budding of clathrin-coated vesicles from the plasma membrane ( Michaely et al., 1999). The β1 subunit of the AP-1 complex was found to bind to KIF13A, a kinesin that responsible for the transport from the TGN to the plasma membrane (Nakagawa et al., 2000). In addition, the Cdc42associated tyrosine kinase (ACK) were found to be a clathrin binding proteins (Teo et al., 2001; Yang et al., 2001). Cdc42 is a Rho GTPase involved in actin polymerization at the plasma membrane and the Golgi. http://www.albany.edu/~abio304/text/chapter_9.html (40 of 41) [3/5/2003 7:54:40 PM]
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SUGGESTED READING Bonifacino, J.S. and Weissman, A.M. (1998) Ubiquitin and the control of protein fate in the secretory and endocytic pathways, Annu. Rev. Cell. Dev Biol. 14:19-57.(Medline) Marsh, M. and McMahon, H.T. (1999) The structural era of endocytosis, Science 285:215-220. McNiven, M.A., Cao, I., Pitts, K.R. and Yoon, I. (2000) The dynamin family of mechanoenzymes: pinching in new places, Trends Biochem Sci 25:115-120. (MedLine) Parton, R.G. (1996) Caveolae and caveolins, Curr. Opin. Cell Biol 8:542-548 (Medline) Parton, R.G. (2003) Caveolae - from ultrastructure to molecular mechanisms, Nature Rev. Mol. Cell Biol. 4:162-167. (MedLine) Pishvaee, B. and Payne, G.S. (1998) Clathrin coats--threads laid bare, Cell 95:443-446. (Medline) Riezman, H., Woodman, P.G., van Meer, G. and Marsh, M. (1997) Molecular mechanisms of endocytosis, Cell 91:731-738. (Medline) Schmid, S.L. and Damke, H. (1995) Coated vesicles: a diversity of form and function, FASEB J. 9:14451453. (Medline) Travis, J. (1993) Cell biologists explore 'tiny caves', Science 262: 1208-1209. WEB RESOURCES Maniak, M. et al. Dynamic redistribution of the cytoskeleton during phagocytosis. http://www.cellsalive.com/dictyo.htm REFERENCES Search the textbook
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Chapter 9: References
Back to Chapter 9
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Chapter 9: References
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Sztul, E., Kaplin, A., Saucan, L. and Palade, G. (1991) Protein traffic between distinct plasma membrane domains: isolation and characterization of vesicular carriers involved in transcytosis, Cell 64:81-89. (Medline) Takei, K., Haucke, V., Slepnev, V., Farsad, K., Salazar, M., Chen, H. and De Camilli, P. (1998) Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes, Cell 94:131-141. (Medline) Takei, K. and Haucke, V. (2001) Clathrin-mediated endocytosis: membrane factors pull the trigger, Trends Cell Biol. 11:385-391. (MedLine) Tang, Z., Scherer, P.E., Okamoto, T., Song, K., Chu, C., Kohtz, D.S., Nishimoto, I., Lodish, H.F. and Lisanti, M.P. (1996) Molecular cloning of caveolin-3, a novel member of the caveolin gene family expressed predominantly in muscle, J. Biol. Chem. 271:2255-2261. (Medline) Taunton J. (2001) Actin filament nucleation by endosomes, lysosomes and secretory vesicles, Curr. Opin. Cell Biol. 13:85-91. (MedLine) Taunton, J., Rowning, B.A., Coughlin, M.L., Wu, M., Moon, R.T., Mitchison, T.J. and Larabell, C.A. (2000) Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP, J. Cell Biol. 148:519-530. (MedLine) Tebar, F., Sorkina, T., Sorkin, A., Ericsson, M. and Kirchhausen, T. (1996) Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits, J. Biol. Chem. 271:28727-28730. (Medline) Teo, M., Tan, L., Lim, L. and Manser, E. (2001) The tyrosine kinase ACK1 associates with clathrincoated vesicles through a binding motif shared by arrestin and other adaptors, J. Biol. Chem. 276:1839218398. (MedLine) ter Haar, E., Musacchio, A., Harrison, S.C. and Kirchhausen, T. (1998) Atomic structure of clathrin: a β propeller terminal domain joins an α zigzag linker, Cell 95:563-573. (Medline) Thompson, L.P., Ruedi, J.M., Glass, A., Low, M.G. and Lucas, A.H. (1989) Antibodies to 5-nucleotidase (CD73), a glycosyl-phosphatidylinositol-anchored protein, cause human peripheral blood T cells to proliferate, J. Immunol. 143:1815-1821. (Medline) Tjelle, T.E., Lovdal, T. and Berg, T. (2000) Phagosome dynamics and function. BioEssays 22:255–263. (MedLine)
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Tran, D., Carpentier, J.-L., Sawano, F., Gorden, P., and Orci, L.. (1987) Ligand internalized through coated and noncoated invaginations follow a common intracellular pathway, Proc. Natl. Acad. Sci. USA84:7957-7961. (Medline) Trigatti, B.L., Anderson, R.G.W. and Gerber, G.E. (1999) Identification of caveolin-1 as a fatty acid binding protein, Biochem. Biophys. Res. Commun. 255:34-39. (Medline) Trowbridge, I.S., Collawn, J.F. and Hopkins, C.R. (1993) Signal-dependent membrane protein trafficking in the endocytotic pathway, Annu. Rev. Cell Biol. 9:129-161. (Medline) Tycko, B. and Maxfield, F. R. (1982) Rapid acidification of endocytotic vesicles containing macroglobulin, Cell 28:640-651. Ungewickell, E. and Ungewickell, H. (1991) Bovine brain clathrin light chains impede heavy chain assembly in vitro, J. Biol. Chem. 266:12710-12714. (Medline) Ungewickell, E., Ungewickell, H., Holstein, S.E., Lindner, R., Prasad, K., Barouch, W., Martin, B., Greene, L.E. and Eisenberg, E. (1995) Role of auxilin in uncoating clathrin-coated vesicles, Nature 378:632-635. (Medline) Urrutia, R., Henley, J.R., Cook, T.and McNiven, M.A. (1997) The dynamins: redundant or distinct functions for an expanding family of related GTPases? Proc. Natl. Acad. Sci. USA 94:377-384. (Medline) van der Bliek, A.M. (1999a) Functional diversity in the dynamin family, Trends Cell Biol. 9:96-102. (Medline) van der Bliek, A.M. (1999b) Is dynamin a regular motor or a master regulator? Trends Cell Biol. 9:253254. (Medline) van der Sluijs, P., Hull, M., Webster, P., Male, P., Goud, B. and Mellman, I. (1992) The small GTPbinding protein rab4 controls an early sorting event on the endocytic pathway, Cell 70:729-740. (Medline) van IJzendoorn, S.C. and Hoekstra, D. (1998) (Glyco)sphingolipids are sorted in sub-apical compartments in HepG2 cells: a role for non-Golgi-related intracellular sites in the polarized distribution of (glyco)sphingolipids, J. Cell Biol. 142:683-696. (Medline) van IJzendoorn, S.C., Zegers, M.M., Kok, J.W. and Hoekstra, D. (1997) Segregation of glucosylceramide and sphingomyelin occurs in the apical to basolateral transcytotic route in HepG2 cells, J. Cell Biol. 137:347-357. (Medline)
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van IJzendoorn, S.C.D. and Hoekstra, D. (1999) The subapical compartment: novel sorting centre, Trends Cell Biol. 9:144-149. (Medline) van Meer, G., Stelzer, E.H.K., Wijnaendts-van-Resandt, R.W. and Simons, K. (1987) Sorting of sphingolipids in epithelial (Madin-Darbin Canine Kidney) cells, J. Cell Biol. 105:1623-1635. (Medline) Vasile, E., Simionescu, M. and Simionescu, N. (1983) Visualization of the binding, endocytosis and transcytosis of low-density lipoprotein in the arterial endothelium in situ, J. Cell Biol. 96:1677-1689. (Medline) Vaux, D. (1992) The structure of an endocytotic signal, Trends in Cell Biol. 2: 189-192. Vickery, R.G. and von Zastrow, M. (1999) Distinct dynamin-dependent and -independent mechanisms target structurally homologous dopamine receptors to different endocytic membranes, J. Cell Biol. 144(1):31-43. (MedLine) Vieira, A.V., Lamaze, C. and Schmid, S.L. (1996) Control of EGF receptor signaling by clathrinmediated endocytosis, Science 274:2086-2089. (Medline) Vieira, O.V., Botelho, R.J., Rameh, L., Brachmann, S.M., Matsuo, T., Davidson, H.W., Schreiber, A., Backer, J.M., Cantley, L.C. and Grinstein, S. (2001) Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation, J. Cell Biol. 155:19-25. (MedLine) Vigers, G.P., Crowther, R.A. and Pearse, B.M. (1986) Three-dimensional structure of clathrin cages in ice, EMBO J. 5:529-534. (Medline) Voorhees, P., Deignan, E., van Donselaer, E., Humphrey, J., Marks, M.S., Peters, P.J. and Bonaficino, J.S.(1995) An acidic sequence within the cytoplasmic domain of furin functions as a determinant of transGolgi network localization and internalization from the cell surface, EMBO J. 14:4961-4975. (Medline) Wang, J.Y.J. (1994) Nuclear protein tyrosine kinases, Trends Biochem. Sci. 19:373-376. (Medline) Warnock, D.E., Hinshaw, J.E. and Schmid, S.L. (1996) Dynamin self-assembly stimulates its GTPase activity, J. Biol. Chem. 271:22310-22314. (Medline) Wary, K.K., Mainiero, F., Isakoff, S.J., Marcantonio, E.E. and Giancotti, F.G. (1996)The adaptor protein Shc couples a class of integrins to the control of cell cycle progression, Cell 87:733-743. (Medline) Wells, A., Welsh, J.B., Lazar, C.S., Wiley, H.S., Gill, G.N. and Rosenfeld, M.G. (1990) Ligand-induced http://www.albany.edu/~abio304/ref/ref9.html (28 of 30) [3/5/2003 7:54:50 PM]
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transformation by a noninternalizing epidermal growth factor receptor, Science 247:962-964. (Medline) Wendland, B., Emr, S.D. and Riezman, H. (1998) Protein traffic in the yeast endocytic and vacuolar protein sorting pathways, Curr. Opin. Cell Biol. 10:513-522. (Medline) Wendland, B., Steece, K.E. and Emr, S.D. (1999) Yeast epsins contain an essential N-terminal ENTH domain, bind clathrin and are required for endocytosis, EMBO J. 18:4383-4393. (MedLine) Wesp, A., Hicke, L., Palecek, J., Lombardi, R., Aust, T., Munn, A.L. and Riezman, H. (1997) End4p/Sla2p interacts with actin-associated proteins for endocytosis in Saccharomyces cerevisiae, Mol. Biol. Cell 8:2291-2306. (Medline) Wigge, P., Vallis, Y. and McMahon, H.T. (1997) Inhibition of receptor-mediated endocytosis by the amphiphysin SH3 domain, Curr. Biol. 7:554-560. (Medline) Wilde, A. and Brodsky, F.M. (1996) In vivo phosphorylation of adaptors regulates their interaction with clathrin, J. Cell Biol. 135:635-645. (Medline) Wilde, A., Beattie, E.C., Lem, L. Riethof, D.A., Liu, S.-H., Mobley, W.C., Soriano, P. and Bordsky, F.M. (1999) EGF receptor signaling stimulated SRC kinase prhopshorylation of clathrin , influencing clathrin redistribution and EGF uptake, Cell 96:677-687. (Medline) Worby, C.A. and Dixon, J.E. (2002) Sorting out the cellular functions of sorting nexins, Nature Rev. Mol. Cell Biol. 3:919-931. (MedLine) Xu, Y., Hortsman, H., Seet, L., Wong, S.H. and Hong, W. (2001) SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P, Nature Cell Biol. 3:658-666. (MedLine) Yamada, E. (1955) The fine structure of the gall bladder epithelium of the mouse, J. Cell Biol. 1:445-458. Yamashiro, D.J., Tycko, B., Fluss, S.R. and Maxfield, F.R. (1984) Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway, Cell 37:789-800. (Medline) Yang, W., Lo, C.G., Dispenza, T. and Cerione, R.A. (2001) The Cdc42 target ACK2 directly interacts with clathrin and influences clathrin assembly, J. Biol. Chem. 276:17468-17473. (MedLine) Ybe, J.A., Brodsky, F.M., Hofmann, K., Lin, K., Liu, S.H., Chen, L., Earnest, T.N., Fletterick, R.J. and Hwang, P.K (1999) Clathrin self-assembly is mediated by a tandemly repeated superhelix, Nature 399:371-375. (Medline)
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Yoshimori, T., Keller, P., Roth, M.G. and Simons, K. (1996) Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells, J. Cell Biol. 133:247-256. (Medline) Zhang, F., Crise, B., Su, B., Hou, Y., Rose, J.K., Bothwell, A. and Jacobson, K.. (1991) Lateral diffusion of membrane-spanning and glycosylphosphatidylinositol-linked proteins: towards establishing rules governing the lateral mobility of membrane proteins, J. Cell Biol.115:75-84. (Medline) Zhang, J.Z., Davletov, B.A., Sudhof, T.C. and Anderson, R.G. (1994) Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling, Cell 78:751-760. (MedLine) Zhang, Y., Moheban, D.B., Conway, B.R., Bhattacharyya, A. and Segal, R.A. (2000) Cell surface Trk receptors mediate NGF-induced survival while internalized receptors regulate NGF-induced differentiation, J. Neurosci. 20:5671-5678. (MedLine)
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10. Biosynthesis and Cytoplasmic Trafficking: Synthesis, Packaging and the Golgi Complex
10. Biosynthesis and Cytoplasmic Trafficking: Synthesis, Packaging and the Golgi Complex I. Protein Synthesis and Intracellular Transport II. Role of the Endoplasmic Reticulum A. Signal Sequences B. Targeting C. Translocation D. Insertion of Integral Proteins E. Protein Processing and Folding III. Sorting Proteins in the Endoplasmic Reticulum and the Golgi Complex A. Different Experimental Approaches Yeast genetics used as a tool Animal viruses used as tracers Temperature blocks in normal cells Reconstitution experiments B. Export from the ER C. Passage Through the Golgi System and Transfer to the Cell Surface Sequential events and sequential sites Mechanisms of anterograde transport Retention of resident proteins in the Golgi stacks Bulk flow and sorting signals Post-Golgi signals Exocytosis and transport to the cell surface IV. Alternative Secretory Pathway and Alternative Mechanisms V. Targeting mRNAs VI. The Traffic of Lipids A. Glycerophospholipids B. Sphingolipids C. Cholesterol Suggested Reading
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Web Resources References Back to List of Chapters All cells are engaged in the synthesis of proteins and other components destined to be transported to various locations. In regulated secretion, the destination of proteins is the storage secretory granule that discharges its contents to the extracellular medium on receipt of the appropriate physiological signal, such as the release of a neurotransmitter. In all cases the discharge is by a process known as exocytosis. In exocytosis, the vesicular membrane becomes continuous with the plasma membrane and simultaneously discharges its contents (Breckenbridge and Almers, 1987). The secretory granules serve as stores in which the concentration of secretory products is greater than in the Golgi cisternae from which they originated, as much as 10 times greater in exocrine secretion and 200 times greater in endocrine secretion. Other proteins may be destined to be delivered to intracellular organelles such as lysosomes, while still others may be targeted to the cell surface, to form new plasma membrane or discharge the contents to the outside in the process of constitutive secretion. Because of the mechanism of exocytosis, the transport of integral plasma membrane proteins and constitutive secretion are facets of the same process. This question was addressed by immunoelectronmicroscopy (Strous et al., 1983). Antibodies were produced against either integral plasma membrane proteins or secreted proteins. Each distinct antibody was labelled with colloidal gold particles of distinct size. The membrane proteins and the products of constitutive secretion were found associated with the same vesicles. Recycling of components requires transport to occur in the opposite direction. The transport from the ER to the Golgi apparatus and beyond is referred to as anterograde transport. When it is in the opposite direction, it is called retrograde transport. The transfer of materials (referred to as cargo) from one compartment to another is generally thought to occur in vesicles. However, the translocation of cargo between the Golgi compartments may be by alternative mechanisms (see Section I and Section IIIC). In addition, mRNA itself may be transported and targeted to a specific location and translated there (Section V). However, most of this chapter will address the pathway that transports in vesicles, the topic of Sections I to III. Just as many proteins are transferred to their target in transport vesicles, so are membrane lipids (Section VI). The regulated secretory system was the first to be examined in detail. Its study has influenced the experiments that examined other intracellular biosynthetic protein transport. For this reason, it is introduced first. I. PROTEIN SYNTHESIS AND INTRACELLULAR TRANSPORT What is the general pattern of this synthesis and transport? The fate of a newly synthesized protein can be traced, after incubation in a medium containing a radioactive amino acid, by techniques using electron microscopy (EM) and autoradiography. The radioactivity is recorded by placing a photographic emulsion or film next to a tissue section. Generally, the autoradiograph has to be stored in the dark for weeks. Where a radioactive disintegration has taken place, a silver grain will appear. Sharp localization can be obtained using transmission electron microscopy (TEM) with components labelled with [3H]. The radiation of [3H] is of low energy and therefore cannot travel very far, permitting localizations within 0.1 to 0.2 µm. The amount of http://www.albany.edu/~abio304/text/10part1.html (2 of 24) [3/5/2003 7:55:32 PM]
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incorporation can be estimated by counting the grains. Alternatively, the radioactivity of various cell fractions can be measured after they are isolated. In early experiments, [14C]leucine was injected into guinea pigs and then various cell fractions were isolated (Siekevitz and Palade, 1960). The highest specific radioactivity (radioactivity per milligram of enzyme) was detected in the rough endoplasmic reticulum (RER, i.e., polysomes attached to the endoplasmic vesicles) at the earliest possible sampling time, and not in the detached polysomes. These observations show that the synthesis takes place in the polysomes associated with the endoplasmic reticulum. The chronological sequence of events after the initial synthesis can be followed by a pulse-chase procedure. In pulse-chase, the incubation of the cells or tissue with a radioactively labelled precursor for a short time period (pulse), is followed by the introduction of a very large excess of unlabelled precursor (chase). In essence, the chase excludes from observation any incorporation of the radioactive precursor that occurs after the pulse incubation. Sampling the cells or tissue at various times recognizes the migration of the labelled material. Autoradiographic pulse-chase studies have been carried out with slices of guinea pig pancreas. Experiments with the autoradiographic technique using L-[3H]leucine are illustrated in Fig. 1 (Jamieson and Palade, 1967). The electron micrograph of an acinar cell shows the vesicles of the RER, mitochondria, the nucleus and secretory granules, the dense spherical inclusions on the left side of the figure. The autoradiograph corresponds to an incubation of the pancreatic slice for 3 minutes, and the grains are predominantly on the RER. A summary of the autoradiographic data as a function of time is shown in graphical form in Fig. 2. In this figure, each point represents the percent of label corresponding to the incubation time shown in the abcissa. Curve 1 represents the percentage of grains over the RER. The radioactivity is incorporated into the condensing vacuoles of the Golgi complex (curve 2) and eventually the secretory granules (curve 3) release their contents into the acinar lumen (not shown). A qualitatively similar chronology was found for the incorporation of [3H]leucine in monocytes, in which the predominant proteins synthesized are sequestered in lysosomes.
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Fig. 1 Electron microscopic autoradiograph of an acinar cell at the end of pulse labeling for 3 min with L[3H]leucine. The autoradiographic grains are located almost exclusively over elements of the RER. A few grains partly overlie mitochondria. These may be associated with adjacent RER. m, Mitochondrion; N, nucleus. X17,000. Reproduced from The Journal of Cell Biology by copyright © permission of The Rockefeller University Press.
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Fig. 2 Pulse-chase of pancreatic acinar cell. See text. Taken from the data of Jamieson and Palade, 1967.
As indicated by the pulse-chase experiments just discussed, newly synthesized proteins are transferred from the RER to the Golgi apparatus. From the Golgi, various vesicles are sorted out to form either storage secretory vesicles, lysosomes, or vesicles involved in constitutive secretion. The divergent fates of the various components suggest the sorting out of the proteins or the vesicles, as already indicated in Chapter 9, for the endocytotic pathway. The Golgi system seems to have a central role in this sorting. Fig. 3A (Farquhar, 1985) summarizes a model that incorporates this and other information. For a more realistic description of the structure of the Golgi apparatus see Section III. The main features of this model are: (1) each cisterna represents a separate subcompartment with a distinctive membrane composition and internal milieu; the stacks closest to the nucleus are referred to as cis, the more distant ones are referred to as trans; (2) products move vectorially from RER to transitional elements, that include the vesicular tubular cluster (VTCs) also referred to as intermediate compartments (IC) located on the cis side of the Golgi complex, and then unidirectionally across the stacks (cis to trans), traversing the cisternae one-by-one; (3) transport along the route occurs in vesicles; and (4) the main flow of traffic, that is the reception of vesicles and their budding, is at the rims of the cisternae. The model in which vesicles are the vehicle for transport of cargo within the Golgi is supported by a wealth of data. However, there are indications that it may be an oversimplification. The maturation model of intra-Golgi transport suggests that the various cisternae of the Golgi are formed on the cis side, modified and displaced http://www.albany.edu/~abio304/text/10part1.html (5 of 24) [3/5/2003 7:55:32 PM]
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without mediation of vesicles (e.g., Bannykh and Balch, 1997; Mironov et al., 1997). In this model, the Golgi proteins that reside in the cisternae would be recovered by retrograde transport (see Allan and Balch, 1999). This model is represented in cartoon form in Fig. 3B. There is considerable evidence supporting alternative models such as this, at least for some special cases. Transport could also take place by yet another mechanism, via the tubules that connect the various Golgi cisternae. In one study, three-dimensional reconstruction of the Golgi apparatus have shown that tubules connect the cisternae (e.g., Rambourg and Clermont, 1990; Weidman et al., 1993) although a more recent study (Ladinsky et al., 1999) has not found tubular connections between successive cisternae. This tubule model of transport is displayed in Fig. 3C. For an evaluation of these models see Section IIIC. In addition to these different views in relation to the intra-Golgi transport, some experiments indicate that the transfer from post-ER compartments to the Golgi (see Section III) and from the Golgi to the cell surface (see Section IIIC), in at least some special cases, may occur through elongated large structures or large vesicles rather than the small vesicles discussed in most of this Chapter and Chapter 11. These experiments are discussed later, as indicated. The diagram of Fig. 3 represents reasonably well the process thought to take place in secretory cells (e.g., see Palade, 1975). The VTCs are in close proximity to the cis face of the Golgi stacks. However, studies using other cell lines have revealed that export from the ER can occur at multiple sites, some very far from the Golgi apparatus. A more thorough examination of the geometry of the VTCs has revealed that the ER, in the process of budding, is present in morphological units referred to as export complexes (Bannykh et al., 1996, 1998). The ER vesicles are disposed around VTCs with their COPII buds facing the center. The VTCs are separate entities containing COPI proteins (thought to be involved in retrograde transport). The coat proteins, COPI, COPII and clathrin will be discussed in more detail in Chapter 11. The various processes of sorting are discussed in the rest of this chapter. The mechanisms involving the vesicles, their targeting and fusion with the target membranes is the subject of the next chapter.
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Fig. 3A Stationary cisternae model of the Golgi complex. The membrane components that have been localized in situ, together with their most frequent localization in either cis, middle, or trans cisternae, are indicated on the right. The flow of biosynthetic products through the Golgi complex is diagrammed on the left. Sg, Secretory granules; En, endosome; Re, reticular element (Farquhar, 1985). Reproduced, with permission, from the Annual Review of Cell Biology, Volume 1, copyright ©1985, by Annual Reviews Inc. B. Representation of the maturation model of intra-Golgi transport. The cisternae themselves move from the cis to the trans direction. The appropriate resident enzymes are recovered by retrograde transport involving vesicles. C. Representation of the tubular model of intra-Golgi transport. Materials are exchanged through the tubules connecting the cisternae.
II. ROLE OF THE ENDOPLASMIC RETICULUM The previous section discussed how polysomes of the RER are responsible for the synthesis of proteins destined for secretion, or for packaging in the lysosomes. The synthesis of plasma membrane integral proteins also occurs in these polysomes. The information for the delivery of newly synthesized or nascent polypeptides into the RER vesicles resides in a discrete segment of the polypeptide, the signal sequence or leader sequence. The translocation of the polypeptides into the vesicles requires interaction of the signal sequence with receptors in the cytoplasm or in the RER membrane. The receptors have a role in targeting the protein to the RER and may have a role in its translocation into the RER lumen. In addition, a special sequence is required for integral proteins to be located in the bilayer of the RER membrane. Chapter 5 discussed the presence of certain protein domains needed to transfer proteins into the nucleus, the NLSs. The uptake and targeting of receptor proteins taken up by endocytosis, discussed in Chapter 9, also require the recognition of an amino acids sequence motif. Signal sequences represented by discrete segments of targeted proteins and the corresponding binding domains of receptors on the acceptor membrane, are also thought to play a role in the targeting of proteins synthesized in the free polysomes of the cytoplasm to mitochondria and chloroplasts (see Haucke and Schatz, 1997). The translocation reactions for secretory, lysosomal and some integral proteins have been studied in isolated systems and are discussed in some detail in the rest of this section. A. Signal Sequences As discussed in Chapter 3, protein synthesis generally proceeds one amino acid at a time along the mRNA thread from the initiation triplet in the 5' to the 3' direction of the mRNA. The process of translation is outlined diagrammatically in Fig. 4. The ribosome with attached nascent polypeptide is displaced a step at a time (corresponding to a triplet), to read the mRNA (C to F). The polypeptide attached to the ribosome becomes progressively longer as it advances along the mRNA thread. Eventually, the complex reaches the termination codon, the ribosome's subunits disassemble, and the new polypeptide detaches (G). Many ribosomes are independently and simultaneously involved in the translation of a single mRNA thread to form a native protein, the polypeptide also has to be folded and, in some cases, assembled into a larger protein. The folding and assembly will not be discussed here. http://www.albany.edu/~abio304/text/10part1.html (8 of 24) [3/5/2003 7:55:32 PM]
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Fig. 4 Diagram summarizing the events involved in the translation of a protein that is free in the cytoplasm.
The interaction between polysomes, RER membranes (forming microsome vesicles in isolated preparations), and the nascent polypeptide, chain has been studied in many cell types. Murine myeloma cells engage in the synthesis and secretion of immunoglobulin (Blobel and Dobberstein, 1975a, 1975b). The mRNA for the light chain of immunoglobulin was found exclusively in membrane-bound polysomes. Surprisingly, when this mRNA was used in a vesicle-free translation system, the product was a protein larger than the secreted light chains (Blobel and Dobberstein, 1975a). In contrast, completion of chains contained by RER vesicles produced only chains of normal length. These experiments suggest that the vesicle components are responsible for the processing needed to produce shorter mature proteins by cleavage of a short segment by a peptidase. Later experiments demonstrated that this short segment corresponds to the signal peptide. Results of experiments using mRNA, the translational system and various concentrations of microsomes stripped of polysomes, are shown in Table 1 (Blobel and Dobberstein, 1975b). The processed and unprocessed proteins were characterized by their size, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The first column indicates the addition of a preparation of stripped microsomes, and the second and third columns show the production of processed (P) and unprocessed (U) light IgG chains, respectively. In the absence of stripped microsomes (row a), there is no synthesis of processed light chains; only the unprocessed proteins are produced. However, processing does take place (rows b-e) when the stripped microsomes are added to the
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mixture. The processing is stopped by heating the membranes before the incubation (row f), as expected if the membranes play an active role in the protein processing. Increasing the concentration of microsomes beyond optimal values (rows d and e) decreases the synthesis of either processed or unprocessed protein, possibly because of nonspecific binding of needed components at the higher concentrations of membranes. Table 1 Synthesis of Processed (P) and Nonprocessed (U) light Chains of IgG in an initiation system containing light chain mRNA and either no added EDTA-Stripped Microsomes (µl RM-EDTA), increasing amounts of RM-EDTA (5, 10, 25, 50 µl), or heat Inactivated RM-EDTA (25µl).
RM-EDTA (µl)
P
U
(a)
0
0.0
3.4
(b)
5
4.9
0.8
(c)
10
4.8
0.9
(d)
25
2.8
0.4
(e)
50
0.8
0.0
(f)
25 (55o)
0.0
3.2
From G. Blobel and B. Dobberstein., "Transfer of proteins across membranes" in Journal of Cell Biology 67:852-862. Reproduced from The Journal of Cell Biology by copyright © permission of the Rockefeller University Press.
The experiment of Fig. 5 (Blobel and Dobberstein, 1975b) provides more information. A translation mixture containing intact microsomal vesicles is first incubated in the presence of radioactive amino acids, and then the vesicles are disrupted with detergent. The time of addition of the detergent corresponds to 0 time in the abscissa of the figure. Synthesis of processed protein (curve 1) continues after the disruption, from the pool of polypeptides not yet completed when the vesicles are disrupted. At about the time when the synthesis of processed polypeptides ceases, the unprocessed peptides begin to make their appearance (curve 2) and continue being produced thereafter. These experiments show that the processing by the microsomal membranes is cotranslational and the cleavage of the signal peptide takes place before the polypeptide is completed, because processed nascent chains continue to be produced even after the vesicles are removed. However, when the synthesis is initiated in the absence of membranes, the polypeptides remain unprocessed. Note, however, that posttranslational translocation can occur in mammalian systems and yeast cells exhibit both cotranslational and posttranslational translocation (see Section C, below). Signal sequences are usually on the amino terminal of nascent peptide chains and they direct the protein to http://www.albany.edu/~abio304/text/10part1.html (10 of 24) [3/5/2003 7:55:32 PM]
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translocation sites on the ER. Similar sequences fulfill much the same function in transferring proteins to the inner membrane of mitochondria and the thylakoid membrane of chloroplasts. After membrane insertion, the signal sequences are typically cleaved by a membrane bound signal peptidase. In some proteins, the signal sequence is not cleaved. Furthermore, the signal sequence can also be at the carboxy terminal or within the peptide chain (see Simons et al., 1987; Kutay et al., 1995). Characteristically, the signal sequences have a hydrophobic core (h-) region that is 6 to 15 amino acid residues long in the case of the signal sequences that are cleaved. This sector is the most important for function, as determined using mutants (von Heijne, 1990). The h- region is flanked on the carboxy terminal side by the polar c- region which contains proline and glycine residues that are known to interfere with a helical configuration. This region also has uncharged residues in positions -3 and -1 that determine the cleavage site (see von Heijne, 1990). In the amino terminal side, the signal sequences have a relatively polar n-region generally with a positive charge. The length of this sector is very variable and ranges between 15 to more than 50 amino acids (von Heijne, 1990). Although in many respects the signal sequences are interchangeable (see Gierasch 1989), many are specialized as to their target and mode of insertion (Zheng and Gierasch, 1996; Hedge and Lingappa, 1997). They appear to have information to carry out a variety of distinct functions, direct the peptide in variety of mechanisms of insertion and even contain instructions for the role of the cleaved sequence after the cleavage. Signal sequences can direct to different targeting pathways (see Ng et al., 1996; Berks, 1996) and mediate the translocation at the amino or the carboxy terminal of the protein across the membrane (Spiess, 1995). In addition, they determine whether the protein remains in the cytoplasm, is inserted in the membrane or is translocated to the lumen (Swameye and Schaller, 1997; Belin et al., 1996). What happens to the signal sequence once it is severed? Apparently, it is cleaved again and transferred to the cytoplasm (Lyko et al., 1995) where it may have other functions (Martoglio, 1997; Long, 1998; Braud et al., 1998).
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Fig. 5 Relation between processing and the presence of microsomal membranes. Densitometry of an autoradiograph was used to estimate the radioactivity. U and P designate the unprocessed and the processed light chains of IgG, respectively. See text. Reproduced from Journal of Cell Biology by copyright © permission of The Rockefeller University Press.
B. Targeting A model for the complete cycle of the translation in the RER is represented in Fig. 6A (Walter et al., 1984). As indicated in the diagram, two subunits form a ribosome at the initiation codon of the mRNA (steps A and B). As the translation starts, the signal recognition particle (SRP) binds to the ribosome and the signal sequence (B and C). In the presence of SRP, but in the absence of RER membranes, the translation is arrested, showing that synthesis of the entire polypeptide requires the complete system. The SRP is involved in the arrest of elongation. When a nascent polypeptide emerges, the SRP-ribosome complex is targeted to the membrane of the RER by an interaction of the SRP with its receptor (docking protein or SRP receptor, SR; more recently called the translocon associated protein, TRAP). The ribosomes are also attached via the ribosome receptor (step D). The ribosome-nascent polypeptide complex remains attached to the RER membrane, forming a ribosome-membrane junction where the translocation of the nascent chain takes place. The SRP and the docking protein are released to enter a new cycle. Translocation begins as the peptide is synthesized (steps E and F). The signal sequence is cleaved cotranslationally by the signal peptidase. The ribosomal subunits are freed and are ready to start another cycle. The model is based on several observations (Walter and Blobel, 1980, 1981). In salt-extracted canine pancreatic microsomes, recognition of the nascent peptide by the membrane system requires addition of the SRP. This particle includes a 300-nucleotide 7S RNA and six nonidentical polypeptides (Walter and Blobel,
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1982a). The SRP core region has a signal sequence recognition surface composed of both protein and RNA (Batey et al., 2000). The SRP receptor was first implicated in the translation-translocation system of the RER when it was found that proteolytic digestion of RER membranes blocked translocation and the activity could be reconstituted by addition of an extract solubilized by partial protease treatment (Meyer and Dobberstein, 1980; Walter et al., 1979). The active factor was subsequently shown to be a 52-kDa fragment of a 69-kDa integral membrane protein of the ER, as demonstrated using immunological and peptide mapping techniques (Gilmore et al., 1982a, 1982b). The 69-kDa protein was isolated using affinity chromatography in which SRP was conjugated to the Sepharose beads (Meyer et al., 1982b). The receptor would then remain attached to the column, because it would bind to the immobilized SRP. The 69-kDa docking protein (referred to as the SR subunit) is thought to be part of a complex with a 30-kDa protein, because the two have been found tightly bound in most preparations (Tajima et al.,1986). The SRP interacts directly with the signal sequence, as indicated by the binding of SRP to the signal peptide in the isolated translating systems. This has been shown for a signal peptide rich in lysine, where the attachment of SRP is blocked by the lysine analog β-hydroxyleucine (Walter and Blobel, 1981). In addition, in the case of the peptide hormone precursor preprolactin, photoactivated crosslinking reagents were shown to be incorporated into the amino region of the polypeptide and to crosslink to the SRP (Kurzchalia et al.,1986). One of the subunits of SRP (SRP54) contains a GTPase-domain at the amino-terminal and a carboxy-domain that binds to the signal sequence and the SRP-RNA. The two subunits of the SR both contain GTPase-domains. Current models (Fig. 6B, Bacher et al., 1996) propose that SRP (oval black particle) binds to both the ribosome and signal sequence (SS) (I, II and III in the figure). Then the binding to GTP activates the docking of the complex to the SRP receptor (IV). The SRP receptor stabilizes the binding of GTP to SRP54 and induces its dissociation from the ribosome-signal sequence complex (V). Binding to GTP facilitates the binding of the complex to the translocon (the heterotrimeric Sec61p complex discussed below). The ribosome (Bacher et al., 1996) and SRP receptor binding to SRP54 (Miller et al., 1993) increases its affinity to GTP. Then the GTP hydrolysis induces dissociation of the SRP from the membrane. In Fig. 6B, the nascent polypeptide is shown entering as a loop, as discussed below.
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Fig. 6A Protein synthesis, targeting and translocation across or into the membrane of the endoplasmic reticulum. Reproduced with permission from Walter et al. (1984), copyright ©1984 by Cell Press.
Fig. 6B The role of GTP in the targeting. I. Free SRP binding GDP. II. Complexing of SRP to the ribosome and the SS. III. Displacement of GDP by GTP. IV. binding of SS-ribosome and SRP to the SRP receptor (α and β) and attachment to the translocon, V initiation of translocation and hydrolysis of GTP on the SRP. Reproduced with permission from Nature, Bacher et al., 381:248-251, copyright ©1996, MacMillan Magazines Ltd.
A signal sequence targets a protein for delivery to the ER. The signal-anchor sequence that determines the orientation of integral proteins, serves two functions. It targets the protein to the ER and acts as an uncleaved transmembrane sequence (see section C below). http://www.albany.edu/~abio304/text/10part1.html (14 of 24) [3/5/2003 7:55:32 PM]
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This representation eventually had to be modified, amended and refined. For example, the model of Fig. 6A shows the nascent protein being transferred linearly from the amino to the carboxy terminal. As shown by the model of Fig. 6B, this is not likely to be the case. Some proteins containing a signal sequence are targeted to the ER membrane without the involvement of SRP and are translocated post-transcriptionally (Rapoport et al., 1996a). Another group of proteins is inserted independently of the translocon (Kutay et al, 1995). These proteins are inserted posttranscriptionally and at least some of them are ER proteins. C. Translocation The precise events of translocation and the involvement of membrane components are still not entirely clear (see Brodsky, 1998 for a review). In the mammalian in vitro systems, translocation is generally cotranslational, as shown by the results represented in Fig. 5. However, translation seems to be unrelated to the mechanism of translocation. This is indicated by several observations. A potential for posttranslational translocation has been demonstrated in mammalian systems. The glucose transporter protein produced in the absence of vesicles has been shown to be subsequently transferred into added vesicles (Mueckler and Lodish, 1986). Similar experiments have also demonstrated that translocation is an independent phenomenon (Perara et al., 1986). Following transcription and translation from cDNA without termination codons and in the absence of microsomes, newly formed proteins remained attached to the ribosomes. However, subsequent addition of the microsomes stripped of polysomes elicited their transfer into the vesicles. These findings suggest that the translocation machinery is part of the ER membrane and the mechanism is not part of the translational process itself. The posttranslational transfer and, presumably, the cotranslational transfer, were found to require an energy source, in these experiments supplied by ATP, GTP, and phosphocreatine in the presence of creatine phosphokinase (Perara et al., 1986). The role of phosphocreatine and phosphocreatine kinase is to replenish the terminal phosphate of ATP, hydrolyzed during the activation. As we saw, GTP is required for targeting of the ribosome-nascent polypeptide complex (section IIB, above). Many of the proteins involved in the cotranslational translocation of the nascent polypeptides were identified by a strategy in which the components were isolated after crosslinking. A protein crosslinked to the nascent chain must have been in the proximity of the translocated polypeptide. The crosslinking requires special crosslinking reagents (some of them photoactivated) or UV radiation. The components identified include: the translocon associated protein (TRAP) (previously called the signal sequence receptor, SR; Wiedmann et al., 1987; Hartmann et al., 1993), translocating chain associated protein (TRAM) (Görlich et al., 1992a), and Sec61p, the translocon complex, first discovered in yeast (Görlich et al., 1992b). The minimal complement required for in vitro translocation includes TRAP (SR), the translocon complex or channel (the Sec61p complex), and, for some proteins, TRAM (Görlich and Rapoport, 1993). Although not needed for the translocation itself, other enzymes needed to produce the mature protein may be at the translocation site. These include the enzymes involved in glycosylation, oligosaccharide transferase and the signal peptidase. So far, there is evidence for the involvement of nine or more separate proteins. For example, the oligosaccharide portion is added to a nascent protein by the oligosaccharide-transferase when only 15 residues are exposed to the lumen (Whitley et al., 1996). This indicates that the enzyme is located very close to the channel. The various proteins need not be associated with the translocon all the time. This has led to the concept of the translocon as a dynamic unit, involving any protein that interacts with the polypeptide when still attached to the tRNA (Andrews and Johnson, 1996) and involving time dependent composition, conformation and structure. http://www.albany.edu/~abio304/text/10part1.html (15 of 24) [3/5/2003 7:55:32 PM]
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The signal peptidase was shown to be predominantly in the RER lumen, suggesting that the excision of the signal sequence is in the lumen. TRAP was also identified as a protein that is in close proximity to the signal sequence during the initial SRP-dependent targeting, by crosslinking. Antibodies to the cytoplasmic carboxyterminal of the α-subunit of TRAP block translocation and, therefore, also implicate this protein in the process (Hartmann et al., 1989a). Two integral proteins, named ribophorins, were found associated with the ribosomes of the rough endoplasmic reticulum, suggesting a role in the process of translocation. Recently, the ribophorins have been shown to be oligosaccharide transferases (e.g., Kelleher et al., 1992). Reconstitution of the system, including membrane bound elements, has demonstrated the involvement of the TRAP (SR) and TRAM in translocation. In the study of Migliaccio et al., (1992), TRAP (SR) and TRAM were removed from detergent extracts using immunoaffinity columns. Without affecting ribosome binding, depletion of TRAP (SR) resulted in a failure to release the system from elongation and, in addition, arrest, failure in the targeting and failure to translocate the protein. At least in systems reconstituted using proteoliposomes, the translocation of a few specific secretory proteins does not require the presence of TRAM. The need for TRAM depends on the nature of the signal sequence (Voight et al., 1996). Generally, in vitro, cotranslational translocation can be carried out with only TRAM, the heterotrimeric Sec61p, and the TRAP (SR) (Görlich and Rapoport, 1993). In Saccharomyces cerevisiae, the translocation of proteins into the ER can be either posttranslational or cotranslational and not all preproteins require a functioning SRP for translocation into the ER. Mutants were found that specifically impaired the translocation of SRP-independent preproteins in vivo and in vitro, without affecting the SRP-dependent pathway. Which pathway is followed by the preprotein depends on the amino acids present in the hydrophobic core of the signal sequence (Ng et al., 1996; Zheng and Gierasch, 1996). Some preproteins were found to be able to use either the SRP or the SRP-independent route. Furthermore, some proteins distribute between the cytoplasm and the secretory pathway as a function of the nature of the signal sequence (Belin et al., 1996). In postranslational translocation in yeast, instead of the SRP related system, the Sec62p-Sec63p complex is involved (e.g., Ng et al., 1996). The translocation system can be reconstituted in proteoliposomes containing the tetrameric Sec62p-Sec63p complex, the trimeric Sec61p (translocon) complex and, in addition, the Sbh1p protein (in yeast, equivalent to BiP) (Panzner et al., 1995). The SRP-pathway is regulated by three GTPases, the 54 kDa SRP protein and the α β subunits of the SRP receptor. These are not necessary for the SRP-less pathway. The two pathways converge at the Sec translocon assembled from Sec61p complex and involving BiP (Rapoport et al., 1996b; Hamman et al., 1998). The ER lumenal protein BiP, involved in posttranslational translocation, is a chaperone (see Chapters 15 and below) of the Hsp70 family. In yeast, it is required for the efficient import of precursors in vivo and in vitro. Mutations in the gene for BiP (KAR2) prevents early translocation before the precursor protein reaches the channel (Sanders et al, 1992; Lyman and Schekman, 1995). Like other chaperones, BiP functions with a partner protein (Sec63p) in Saccharomyces cerevisiae (DnaJ in E. coli). Homologues of DnaJ have been found in several eukaryotic compartments including the ER (see Cyr et al., 1994). Most DNAJ-like proteins are soluble
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or loosely attached to membranes. In contrast, Sec63p is an integral protein (Feldheim et al., 1992) with a sector in the lumen homologous to the J domain. A mutation in the conserved residue of the luminal sector of Sec63p causes a defect in precursor translocation (Rothblatt et al., 1989). The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon (Corsi and Schekman, 1997). BiP is also thought to act in closing the translocon channel (see below). How is the nascent peptide transferred into the RER? The hydrophobicity of the signal peptide suggests that at least the initial insertion occurs through the hydrophobic part of the membrane. The capacity of these peptides to insert in the bilayer is supported by the spontaneous insertion of isolated signal sequences into phospholipid bilayers (e.g., McKnight et al., 1991). However, the signal sequences crosslink to protein components (Robinson et al., 1987), arguing for a more complex mechanism. The passage of an entire polypeptide through the RER membrane suggests the involvement of a channel. Electrophysiological techniques implicate a channel (Simon and Blobel, 1991). In these studies, RER vesicles were fused to planar bilayers that separated two chambers containing 45 mM potassium glutamate. In this procedure, the cytoplasmic faces of the vesicles were exposed to only one side of the bilayer. The addition of puromycin to the cytoplasmic side, increased the conductance of the bilayer and in some cases induced single channel activity of about 220 pS. Puromycin by itself did not produce channels. Puromycin combines with nascent peptides and releases them to the lumen side; this process presumably would leave open the channel needed for the transfer. Extraction of the ribosomes at high KCl concentrations closed the channels, suggesting that the ribosomes themselves are involved in gating the channels. These experiments are consistent with the presence of a water-filled channel, and the walls of the channels that have been studied so far have invariably been protein in nature. Experiments using fluorescent probes have provided more decisive evidence for this idea (Crowley et al., 1993; 1994). Nascent chains can be synthesized in the presence of photoreactive probes attached to Lys-tRNA. The fluorescent Lys derivative is incorporated in the position of a lysine codon. The position can be varied by using mRNA of various lengths or by the choice of protein, so that the probes can be positioned at any point in the pathway. Stable conformational intermediates were produced by the use of truncated mRNA for secreted proteins lacking termination codons, so that the selected piece of the nascent chains remained bound to the ribosomes. The probes do not interfere with translation or translocation and can serve as indicators of the polarity of their environment. The probe used was 6-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)amino hexanoic acid (NBD) attached to the side chain of Lysine. NBD derivatives have been shown to differ in fluorescence lifetime depending on whether they are present in an aqueous (1.4 ns) or hydrophobic (7-10 ns) medium. In addition, the fluorescence in an aqueous solution is quenched by the presence of I-. The fluorescent lifetimes of the NBDlysines in the ribosome and in the membrane, were those expected from an aqueous medium. Since there was no quenching by iodide from the cytoplasmic side, the channel is sealed at the cytoplasmic end by a tight membrane-ribosome-nascent peptide complex. I- quenching from the vesicle side (by making them leaky with the addition of streptolysin O) indicated that the channel opens in the ER lumen only after the nascent chain has grown to approximately 70 amino acids. With the ER lumen gate open, I- from the ER side was able to quench probes located inside the ribosome, showing that the water-filled channel is continuous. Later image reconstruction using the EM confirmed this conclusion. Cryo-electron microscopy of the ribosomeSec61 complex and its three-dimensional reconstruction (Beckmann et al. 1997) show that the Sec61 oligomer http://www.albany.edu/~abio304/text/10part1.html (17 of 24) [3/5/2003 7:55:32 PM]
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is attached to the large ribosomal subunit by a single connection. The pore of the Sec61 oligomer is aligned with a channel traversing the large ribosomal subunit. These reconstructions favor a mechanism in which the nascent peptide is transferred from ribosome to the ER lumen via this continuous composite channel. A recent study has indicated that in the mammalian system BiP seals the nontranslocating and newly targeted translocons (Hamman et al., 1998) As already mentioned, the minimal apparatus for cotranslational translocation required in reconstituted vesicles (Görlich and Rapoport, 1993) includes the SRP receptor of two subunits (probably required only for targeting) and the Sec61p complex (the heterotrimeric translocon). TRAM is also required only for some proteins and transiently at the beginning of the translocation of the peptides. Cross-linking experiments using single photoreactive groups located at various positions in the peptide showed that, before cleavage, the entire polypeptide segment extending from the ribosome is almost exclusively in contact with Sec61 (Mothes et al., 1994), which is then likely to constitute the walls of the channel. Sec61 probably spans the membrane 10 times, and the spanning segments have several hydrophilic amino acid residues. After passage through the channel, the peptide comes in contact with many other proteins, explaining the cross-linking observed in other experiments. Sec61p purified from mammalian and yeast cells in the presence of detergent, forms a cylindrical structure of 3 to 4 oligomers approximately 8.5 nm in diameter with a central pore of approximately 2 nm (Hanein et al., 1996). These structures are also present in ER membranes and reconstituted proteoliposomes. The movement is thought to occur by a Brownian ratchet mechanism, in which the nascent polypeptide chain can only move in one direction because it is restricted in its passage by the walls of the channel and the ribosome that closes one end of the channel. In addition, interaction with BiP, other lumenal RER proteins and glycosylation reactions, makes the process vectorial by trapping the protein on the inside of the vesicle. The transfer and cleavage of the signal sequence require some special features (see Section IIA). These are beginning to be understood in detail. All signal sequences have in common a variable stretch of hydrophobic amino acids, a short positively charged amino terminal region and a polar carboxy terminal region which contains the site that is generally cleaved. After insertion and before cleavage, the carboxy terminal faces the ER lumen and the amino terminal, attached to the nascent polypeptide, remains on the outside. The various steps in the translocation and eventual cleavage of the signal sequence have been revealed by several studies. During cotranslational translocation, the signal sequence of the nascent protein first encounters a subunit of the SRP that contains a hydrophobic segment lined with flexible methionine side chains which are capable of binding the highly variable central hydrophobic region of the signal sequence (Keenan et al., 1998). The signal peptide is then inserted in the lipid-exposed area of the Sec61α two transmembrane helices (Plath et al., 1998; Mothes et al., 1998), probably opening the channel for polypeptide transport. This coincides with the formation of a tight seal between the ribosome and the translocation channel (Hamman et al., 1998). As already discussed, the arrangement produces a continuous channel, in this case, traversed by the signal sequence from the ribosome through the large ribosomal subunit, the translocation channel and finally arriving at the ER lumen (Beckmann et al., 1997), as revealed by electron microscopy. The signal peptide has its carboxy terminal
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end facing the ER lumen and the hydrophobic portion (now in a helical conformation; Plath et al., 1998) exposed to the lipid phase. The mechanism for cleavage by the peptidase can be deduced from the structure of an E. coli signal peptidase, determined by X-ray crystallography (Paetzel et al., 1998). The peptidase has two transmembrane domains. One of these contains the active site which remains exposed to the lumenal side. Apparently, the active site of the peptidase comes in contact with the carboxy terminal portion of the signal sequence, held in place by the its anchoring hydrophobic region. Cleavage at this site follows. D. Insertion of Integral Proteins The translocation of integral proteins differs from proteins destined to be delivered to the ER lumen. Integral proteins have to be inserted in the lipid bilayer during or after translocation. In addition, for proteins with several transmembrane domains (polytopic proteins) the protein must be properly folded. Note that because of the mechanism of exocytosis (see below), the portion of the molecules located in the face of the ER lumen will be on the external face of the cell when delivered to the plasma membrane. The eukaryotic and bacterial systems are very similar and will therefore discussed together. However,in bacteria, the incorporation is in the inner membrane. The SecY and Sec E of E.coli are homologous to Sec61α and Sec61β (see Rapoport et al., 1996a). Four heterodimers of SecY and SecE from the translocation channel. However, in bacteria the translation and membrane insertion need not be coupled. For proteins that have a single transmembrane segment, the orientation may differ (see Spiess, 1995) (see Fig. 7). The amino terminal (type I and type III) or alternatively the carboxy terminal (type II) may be in the ER lumen. For type II and III, the signal sequence in the midportion of the molecule (corresponding to the transmembrane (TM) segment), is not cleaved. For type IV integral proteins (not shown in Fig. 7), the amino terminal is in the cytoplasmic phase and most of the carboxy section of the molecule is in the transmembrane segment. For this latter case, the signal sequence is also not cleaved. In addition, some integral proteins have both terminals in the cytoplasmic compartment (diagram 6, Fig. 7). Still others span the membrane repeatedly. How do the transmembrane proteins reach their proper orientation and folding? Some insights have been gained in recent years. The targeting to the ER membrane first requires a signal-sequence (Blobel and Dobberstein, 1975) and generally, in cotranslational synthesis, the interaction between signal sequence, SRP and SRP receptor (see Section IIA). However, SRPs are not needed for the integration of tail-anchored proteins. At least in yeast, a type II signal-anchor protein can be incorporated independently from SRPs (Ng et al., 1996). In type II proteins, a non-cleaved signal-anchor need not be at the amino terminal. For type I proteins, insertion is initiated by a cleavable signal sequence at the amino terminal. The translocation is terminated by a hydrophobic sequence, the stop-transfer sequence. Typically, cleaved signals have a positively charged short segment followed by a hydrophobic domain of 7 to 15 residues. Cross-linking studies show that the transmembrane domain comes in contact with at least three different protein environments, suggesting that the process involves at least three steps (Do et al., 1996), suggesting a complex process rather than a simple partitioning in the lipid bilayer. http://www.albany.edu/~abio304/text/10part1.html (19 of 24) [3/5/2003 7:55:32 PM]
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The translocation of the integral proteins follows the same path as that of secreted proteins. The channel protein Sec61 and, in some cases, TRAM are involved. However, cross-linking experiments also implicate phospholipids (Martoglio et al., 1995). The details of the translocation are not known. For peptides that span the membrane only once, models that consider the insertion of the peptide as a loop (e.g., Engelman and Steiz, 1981) can explain the orientation of the proteins. The loop could be inserted in the membrane with one site interacting with a hydrophobic sector of the sequence (the zigzag domain in the diagrams)and another site with the polar sector of the protein (see Fig. 7, middle diagram 1). Release of the polar sector to the lumen of the ER would produce type II orientation (Fig. 7, diagram 4). Interaction with the hydrophobic sector only could produce type III (Fig. 7, diagram 5) orientation, as it does for type I (Fig. 7, diagram 3)
Fig. 7 Diagram showing the insertion of an integral protein so that either the amino (1, 2 and 3) or the carboxy terminal (1 and 4) are in the lumen of the ER (see below) .
In eukaryotes, one of the factors in the orientation of type II (carboxy group in ER) and III (amino group in ER)
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proteins appears to be the charge difference between the residues flanking the signal-anchor (more positive on the cytosolic side) (e.g., Hartmann et al., 1989b). Experiments using mutagenesis to alter the sequence support this view. The type III protein cytochrome P-450 was converted to type II by insertion of a positively charged domain at the amino-terminal (e.g., Sato et al., 1990). Similar results were obtained with asialoglycoprotein receptor subunit H1 and paramyxovirus hemagglutinin-neuraminidase, two type II proteins induced to orient in type III orientation by mutation of flanking sequences (e.g. Parks and Lamb, 1993). However, in general the distribution was not unique (e.g., Andrews et al., 1992) suggesting that additional requirements have to be met. The hydrophobic segment also has an influence in the orientation. The longer sequence favored type III orientation, whereas shorter segments favored type II orientation (e.g., Sakaguchi et al., 1992, Sato et al., 1990). Wahlberg and Spiess (1997) studied the role of hydrophobicity and the length of the apolar domain of a type II signal anchor sequence. Long hydrophobic domains promote amino-terminal translocation (producing type III orientation). Short segments promote carboxy-terminal translocation (producing type II orientation). Wahlberg and Spiess conclude that the topology is determined by the combination of three factors: (a) the charge distribution in the vicinity of the signal sequence, (b) the presence or absence of folded NH2 segments and (c) the hydrophobicity of the hydrophobic segment. The folding state of the amino-terminal hydrophilic domain has a role, since the translocation is facilitated by the absence of folding (e.g., Denzer et al., 1995). A nascent polypeptide destined to be located across the membrane must be recognized and moved laterally into the bilayer. This suggests that the channel of the translocon allows access to the phospholipid bilayer. We saw that phospholipids are photo-crosslinked to nascent chains when these contain a photoactive probe in the middle of either the signal or signal-anchor sequence (Martoglio et al., 1995). For integral proteins with a single transmembrane domain (Type I proteins where the an amino terminal signal sequence is cleaved, see Fig. 7), some studies indicate that the TM domain is integrated into the lipid phase after termination of translation and disassembly of ribosomal-channel assembly (Borel and Simon, 1996; Do et al, 1996). The experiments of Do et al. (1996) indicate that the TM sequence leaves the aqueous pore formed by Sec61α, TRAM, and other proteins during the cotranslational integration of the protein into the membrane. The TM sector traverses three different protein environments (adjacent to Sec61p and TRAM and two adjacent to TRAM). However, the TM sequence is retained by the TRAM site and moves in the bilayer only after translation terminates. For single-spanning proteins where the signal sequence is the TM domain and is not cleaved (type I or type III), the results are different. The TM domain enters the lipid domain well before translation has ended (e.g., Mothes et al., 1997). For a type III protein, the Sec61p channel in conjunction with the TRAM protein allows the transmembrane domain of the nascent protein to enter the hydrophobic interior of the membrane without coming in contact with the polar head groups of the lipid bilayer (Heinrich et al., 2000). The process depends on the hydrophobicity of the TM domain and the length of the polypeptide segment attached to the ribosome. Initially, SRP targets the TM domain to the Sec61p channel at a chain length of 61 residues. This is similar to secretory proteins where the targeting and membrane insertion takes place between 50 and 60 residues (Jungnickel and Rapaport, 1995; Mothes et al., 1998). As soon as the length of the sector is long enough to span the membrane, the TM enters the lipid phase. (18-23 amino acids of the TM) and immediately leaves the translocation site. http://www.albany.edu/~abio304/text/10part1.html (21 of 24) [3/5/2003 7:55:32 PM]
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The translocation and insertion of polytopic proteins (with several transmembrane domains) is more complex (see Bibi, 1998; Dalbey, 2000 and Chin et al., 2002). Considerable progress has been made indicating that the translocation and pattern of insertion of these proteins differ significantly. Several studies suggest that in some cases there are interactions between all or some of the transmembrane domains. Folding and the insertion into the bilayer may be two sides of the same coin. However, to facilitate discussion, the two will be approached separately. In some cases, the transmembrane segments of polytopic membrane proteins are inserted in the membrane sequentially following the amino-terminal. However, there are many variations (see Dalbey, 2000). For the cystic fibrosis transmembrane conductance regulator (CFTCR), a cAMP-regulated chloride channel, the first transmembrane segment can act as a non-cleaved signal sequence and the second as a stop-transfer sequence. Alternatively, the role of these two can be reversed. The Shaker-K+ cannel spans the membrane six times and any of the transmembrane segments can initiate translocation acting as a non-cleavable signal sequence . There are many other factors as well, some indicating interactions between membrane-spanning segments. In the case of the bacterial tetracyclin-export protein, the insertion of the odd numbered membrane spanning segments requires the presence of the even numbered segments, suggesting the formation of hair-pin sectors and interaction between the transmembrane sectors on each side of the hairpin. The association of helical segments probably depends on van der Waal forces, interhelical polar interaction (see Popot and Engelman, 2000) and hydrogen bonding between Asn, Asp, Gln and Glu residues of the side chains (Zhou et al, 2001; Gratkowski et al., 2001). Cofactor binding is sometimes needed to reach the final folded state (e.g., see Lu and Booth, 2000). Insertion into the bilayer part of the membrane is a closely related topic. In vitro experiments indicate that hydrophobicity is the main factor determining entry of α-helical segments into the bilayer (von Heijne,1996) The lipids in the membrane have also been found to play an important role. A bilayer structure is probably required. The absence of phosphatidylethanolamine (PE)was shown to produce misfolding of LacY in E. coli (see Bogdanov and Dohan, 1999). The role of the lipid is thought to be chaperone-like. Synthesis of LacY in the absence of PE, allowed examining the properties of lipids needed for refolding. Primary amines (either PE or phosphatidylserine, PS) were found to be most effective, whereas phosphatidylcholine (PC) was ineffective. In addition, monoacyl phospholipids were ineffective and diacyl phospholipids had to contain at least one saturated fatty acid with a preference for chain lengths above 14 carbons. The insertion of several transmembrane sectors of the same protein into the bilayer could occur one at a time. Alternatively, the entire multispan protein could be transferred simultaneously after being packed together in the tranlocon. Although one of the conformations of the translocon is too narrow for allowing more than one peptide at a time (Beckmann et al., 2001), some studies indicate a channel that during translocation can acquire a diameter of 40-60 Å (Hamman et al., 1997). In the case of the multidrug resistance protein (P-glycoprotein), the evidence supports a cooperative release (Borel and Simon, 1996). In these experiments, abbreviated nascent chains containing up to five transmembrane sectors, still attached to ribosomes, were studied. The abbreviated chains were selectively extracted from the membranes with urea, at moderate salt concentrations. This treatment should affect only proteins in an aqueous environment. These findings suggest that all the sectors are not integrated into lipids one at a time, but are maintained in a polar environment, stabilized by electrostatic http://www.albany.edu/~abio304/text/10part1.html (22 of 24) [3/5/2003 7:55:32 PM]
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interactions (Borel and Simon, 1996). The subsequent release of the nascent chains from the ribosomes made the peptides resistant to urea extraction, suggesting that they have been transferred to the bilayer. The nature of the transmembrane segments seems to make a difference in the transfer to the bilayer. A strongly hydrophobic segment enters the lipid environment almost immediately. A less hydrophobic segment is retained and could be cross-linked to TRAM and Sec61 α under the same conditions, suggesting that the less hydrophobic segments can be retained in the the translocon (Heinrich et al., 2000). E. Protein Processing and Folding As the proteins gain access to the lumen of the RER, they are modified. The signal peptide is cleaved, disulfide bonds form, and glycosylation of the amino terminal takes place. However, cleavage by the signal peptidase is not required for translocation, as indicated by secreted proteins such as ovalbumin, which lack a hydrophobic signal removed during processing (Palmiter et al., 1978). In addition to covalent modification, newly synthesized proteins in the ER go through a series of folding and unfolding reactions and assemble into complexes. These rearrangements are catalyzed by ER-specific chaperones that prevent nonproductive or irreversible folding errors (Rothman, 1989; Gething and Sambrook, 1992). Unfolded or unassembled proteins generally remain in the ER that acts as quality control device for newly synthesized proteins (see Section IIIB and Hurtley and Helenius, 1989). However, they are also degraded (see Chapter 15) The accumulation of unfolded proteins produces aggregates in the ER. Signals selectively activate transcription of all the genes encoding the chaperones of the glucose regulated proteins (GRPs) family (see below) as well as other ER-localized proteins such as PDI ( Kozutsumi et al., 1988; Dorner et al. 1989). This response has been termed the unfolded protein response (UPR) (also discussed in Chapter 7). Although the longer term regulation is transcriptional, the immediate response to the accumulation of unfolded proteins in the ER occurs at the translational level where translation initiation is inhibited and, therefore, further accumulation of unfolded proteins is prevented. The inhibition results from the phosphorylation of the subunit of eukaryotic translation initiation factor 2 (eIF-2). Another function of UPR is to coordinate the synthesis of lipids and new membrane structures. In S. cerevisiae, the UPR is also activated by lipid and sterol deprivation. Sterol deprivation in the ER membrane signals to induce transcription of sterol biosynthetic genes. Chaperones are present not only in the ER but also in different organelles and in the cytoplasm. They were first discovered as heat shock proteins (Hsps) which were induced by heat shock (see Jolly and Morimoto, 2000). In addition to UPR, in mammals they also were found to be induced by environmental stresses including the conditions produced by oxidative stress, exposure to heavy metals, or pathologic states (e.g., inflammation, tissue damage, infection). Some members of the family are constitutively expressed, others are inducible. Hsp genes contain heat shock elements (HSE) and in vertebrates heat shock transcription factors are transiently bound to the HSEs. In addition to their induction by UPR and stresses, genes encoding Hsps are transcriptionally regulated during a variety of biological processes such as cell proliferation (e.g., Jerome et al., 1993) or differentiation ( Galea-Lauri et al., 1996). Chaperones also have a role in initiating apoptosis, triggered for example, by the inhibition of N-linked glycosylation or disruption of ER calcium stores (see Kaufman, 1999), although in some cases they can block programmed cell death caused by Ca2+ depletion (e.g.,
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Reddy et al., 1999; Miyake et al., 2000). Chaperones have also been found to have a role in transcriptional regulation by the disassembly of transcriptional regulatory complexes (Freeman et al., 2002) and in particular, those associated with intracellular hormone receptors. The GRP family of the endoplasmic reticulum are a class of chaperones (see Lee, 1992; Lee, 2001). The immunoglobulin binding protein BiP is a GRP. GRPs can be induced in cell in culture by glucose starvation although they are also induced by other stresses (see Lee, 1992; Little et al., 1994). The GRPs, located in the endoplasmic reticulum, are transferred to the nucleus when induced by stress. In the absence of stress they are posttranscriptionally modified into biologically inactive forms ( Little et al., 1994). The promoters of two grp genes have a high level of redundancy ensuring their expression. The grp genes are expressed constitutively, however, the expression is increased under stress conditions (e.g., low glucose or oxygen). As in the case of other Hsps, GRPs are also thought to have a role during development (see Lee, 2001).
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10. Biosynthesis and Cytoplasmic Trafficking: Synthesis, Packaging and the Golgi Complex
Back to Part 1
III. SORTING PROTEINS IN THE ENDOPLASMIC RETICULUM AND THE GOLGI COMPLEX The passage of proteins through the intracellular vesicular transport system is complex and involves some posttranslational synthetic steps as well as the movement of materials. This section examines the steps that were summarized in Fig. 3 in some detail. Many proteins are partially glycosylated in the ER, and both glycosylated and unglycosylated proteins are transported out of the ER at variable rates. The Golgi system has a role in the posttranscriptional Nglycosylation of integral membrane proteins and in sorting the various proteins. The Golgi complex of animal cells generally has three to eight flattened cisternae (see also Section IIIC). In the complex, the displacement of newly synthesized proteins with time, through the various cisternae, is accompanied by stepwise processing by enzymes. These enzymes occupy specific locations in the system in an organization analogous to an assembly line. Depending on their position, the Golgi cisternae are referred to as cis, medial, or trans components, where cis corresponds to the elements closest to the RER (see Fig. 3). The tubular-vesicular elements found in the cell's periphery or at the cis-face of the Golgi, have been termed VTCs or intermediate compartments (IC). A network of tubular vesicles, the trans Golgi network (TGN), has been observed in the trans side of the system. Analogous to the trans system, the term cis Golgi network (CGN) has been adopted more recently to include cis elements. The transfer from the ER to the VTCs is thought to occur via vesicles. What is responsible for the transport of cargo from VTC to the Golgi stacks? Small vesicles, possibly coated with COPI, could carry the cargo. Alternatively, the VTC units themselves could be transferred. Presley et al. (1997) tagged the VSV G protein of a temperature sensitive mutant with the green fluorescent protein by introducing the appropriate chimeric cDNA into the cells (see Chapter 1). The mutant is unable to fold the VSV protein at 40oC, producing an accumulation of fluorescence in the ER of these cells. As we shall see below (Section IIIB), a protein can leave the ER only when folded. The preparation was then maintained at 15oC, a procedure that enlarges pre-Golgi structures. When the preparation was subsequently shifted to the permissive temperature (32oC), the fluorescent protein was able to move out of the ER. The fluorescent structures transported to the Golgi were larger than single vesicles (often greater than 1.5 µm in diameter) and frequently acquired tubular shapes. The 15oC-step used to facilitate the observations was not essential. When the cells were transferred directly to the permissive temperature, the fluorescent vesicular elements were smaller, however, the process was similar. These finding suggest that the transport from pre-Golgi elements to the Golgi takes place in relatively large vesicles.
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In the experiments just discussed, the structures were transported toward the Golgi probably using the minus-end motor, dynein (see Chapter 24), where dynactin serves as an adapter to bind to vesicles (Schroer et al., 1996; Gaglio et al., 1996). In these experiments, an excess of dynactin was found to block the movement suggesting an involvement of dynein. Immunofluorescence showed that the vesicles colocalized with β-COP, a component of COPI coated vesicles (see Chapter 11, Section I and Table 1). The Golgi cisternae do not separate out when the plasma membrane is disrupted or after micromanipulation, suggesting that they are held together by some adhesive molecules. An Nmyristoylated Golgi protein of 65 kDa, GRASP65, has been identified in Golgi cisternae (Barr et al., 1997). GRASP65 is likely to be an important player in the formation of stacks. This is indicated, for example, by experiments in which antibodies against GASP65 were shown to block the in vitro assembly of stacks without interfering with the formation of cisternae. The central position of the Golgi is attributed to its tendency to move along the microtubules in the direction of their minus end, toward the microtubular organizing center (MTOC), which is the centrosome in almost all mammalian cells. The microtubules begin to assemble at this center, generally located to one side of the nucleus. All proteins processed by the Golgi complex, regardless of eventual destination, can be found throughout the cisternae, as shown, by immunocytochemistry. Sorting must therefore occur when the proteins leave the Golgi at the trans end. The TGN has been proposed to play a special role in the sorting (Griffiths and Simon, 1986). Several distinct strategies have been used to examine the mechanisms of these intracellular pathways. These are discussed in the first section (A), followed by a discussion of the information presently available (sections B and C). A. Different Experimental Approaches Most of the techniques discussed in previous chapters were used in the study of secretion, including isolation of cell components, EM, and immunoelectronmicroscopy. Some unique strategies were also applied. These are the subjects of this section. Yeast genetics used as a tool The yeast Saccharomyces cerevisiae functions in many ways like other eukaryotes. In yeast, the central vacuole corresponds functionally to the mammalian lysosome; yeast also secrete proteins into their periplasmic space, i.e., the space between the plasma membrane and cell wall. The presence of a structurally distinct Golgi with several compartments is still to be demonstrated. However, the functional evidence for its presence is incontestable (see below). Yeast have the advantage of permitting the application of genetic techniques so useful in elucidating the molecular biology of cell functions. http://www.albany.edu/~abio304/text/10part2.html (2 of 9) [3/5/2003 7:55:42 PM]
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The temperature sensitive (ts) yeast secretion mutants can be grown at the permissive temperature. They malfunction at the non-permissive temperature and the mutant cells can be readily isolated (Novick et al., 1980). The accumulation of a secretory product progressively increases the mass of the cells, so that they can be isolated by centrifugation techniques. Twenty-three complementation groups were isolated in this fashion. Generally, (but not always, because other strategies and conventions were used) each gene known to be involved in the secretory pathway is referred to as SECn, where n is an arbitrarily assigned number. Lower case letters indicate a mutant. A protein encoded by an SECn gene is referred to as secnp. Mutants defective in steps that take place in the ER required a different strategy, referred to as [3H] mannose suicide (Newman and Ferro-Novick, 1987). The initial glycosylation of proteins takes place in the ER. Therefore, in the presence of excessive concentrations of radioactive mannose at the temperature restrictive for the mutant, wild type yeast will be destroyed. In contrast, the mutants will remain viable because they are unable to incorporate the radioactive mannose. They can be subsequently grown at the permissive temperature and in the absence of radioactive mannose. This approach demonstrates that a minimum of 11 genes contribute to the passage from the ER to the Golgi system. In addition to the mutants isolated by these direct approaches, several yeast genes involved in vesicular traffic were identified because of their homology to the genes of other organisms. Generally, their products were recognized by their cross-reactivity to antibodies to the homologous protein. The yeast Arf1p and Arf2p were recognized from the mammalian Arf1p, which had been discovered earlier. Arf1p is a Ras-like GTP-binding protein (see Chapter 11). Subsequently, Arf-1p-deficient yeast cells were found to be defective in the transit through the Golgi apparatus. The genetic approach can also reveal physiological interactions between gene products by the use of double mutants. sec18, sec17 and sec22 were found to accumulate vesicles produced by the ER (Kaiser and Shekman, 1990). The vesicle accumulation is blocked by four other mutations: sec12, sec13, sec16 and sec23. This observation indicates that the proteins coded by the last three genes control an earlier step (or steps). Mutations in either one of the two sets are lethal, but only when in combination (e.g., sec17 and sec18). This dependence indicates some sort of cooperation between the various gene products in both vesicle budding and fusion. Suppression of a mutant by another mutant gene is also likely to indicate an interaction of their products at the molecular level. In agreement with this idea, when bacterially produced Sar1p (using recombinant DNA technology) was added to an in vitro membrane system from sec12 cells, normal function was re-established (Oka et al., 1991). Sar1p is a Ras-like GTPbinding protein (see section IID). As illustrated by this example, analysis of the mechanisms first suspected from genetic studies requires additional studies involving biochemical techniques. In addition, the location of the proteins in the cell is most readily revealed using antibodies to the proteins (labelled for cytochemistry or electron microscopy), generally produced against the bacterially expressed proteins. Animal viruses used as tracers http://www.albany.edu/~abio304/text/10part2.html (3 of 9) [3/5/2003 7:55:42 PM]
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Viruses use the synthetic machinery of infected cells. During completion of the synthesis of new animal virus, the viral envelope containing its own characteristic integral proteins is generated from the plasma membrane of the host cell by a process of budding. The fate of the newly synthesized viral coat proteins therefore can be used as a model for the pathway followed by plasma membrane integral proteins. Vesicular stomatitis virus (VSV) and Semliki Forest virus (SFV) have been particularly useful. Semliki Forest virus, which infects mosquitoes, is related to yellow fever virus and is named after a forest in Uganda. Vesicular stomatitis virus is a mild pathogen of cattle. Generally, cells in culture such as Chinese hamster ovary (CHO) cells and baby hamster kidney (BHK) fibroblasts, have been used. Temperature blocks in normal cells Apart from the localization of components using EM immunocytochemistry, normal cells in culture provide a good deal of useful information because the various steps are differentially affected by temperature. Lowering the temperature to 20oC slows the exit from the TGN so that many proteins pile up at this step (Griffiths and Simon, 1986). Lowering to 15oC, blocks at an earlier step, causing an accumulation of proteins in the CGN (Saraste and Kuismanen, 1984). Reconstitution experiments In vitro reconstitutions, greatly simplified the molecular dissection of the intracellular transport system. At first, cells with damaged membranes or impure fractions were used. These studies were then followed by others using purer cell fractions (e.g., see Pryer et al., 1992). Blocking a step by a mutation, a chemical inhibitor, or the removal of a key component, should result in an accumulation of vesicles or intermediates. These can be readily recognized. Addition of the missing component, should re-establish function. This strategy would not only identify components of the system, but also reveal the order of the reactions. Experiments that identified the NEM-sensitive factor (required for vesicle fusion) and recognized the need for ATP and cytosolic components, used this approach. Replacement of a missing component in an extract of cells from one organism with a protein from an unrelated organism, establishes the presence of a functionally equivalent factor for both. Some of these experiments will be discussed in the rest of the chapter. B. Export from the ER The ER plays a role in regulation of the intracellular transport by controlling the duration of residence of the various proteins, each having a characteristic half time of residence (e.g., Fries et al., 1984). Presently available evidence indicates that proteins have to be folded and assembled before leaving the ER. For example, the retinol-binding protein cannot be transported unless it binds its ligand (Ronne et al., 1983) and, similarly, the heavy chain of immunoglobulin M (IgM) accumulates in the ER unless it is able to bind to the light chain to form the complete antibody molecule. These observations suggest that the http://www.albany.edu/~abio304/text/10part2.html (4 of 9) [3/5/2003 7:55:42 PM]
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sorting signal for leaving the ER corresponds to patches in the protein molecule that are conformation dependent. Unfolded or unassembled chains appear to be retained in the ER until they are assembled. In some cases, unfolded proteins, such as mutant proteins of influenza hemagglutinin, have been found to be associated with a 77-kDa protein (heavy chain-binding protein, BiP; a resident chaperone, involved in protein folding), in a pattern that suggests an association during an intermediate step of folding (Gething et al., 1986). Unfolded and unassembled proteins have a tendency to form aggregates and may be unable to proceed through transport from the ER unless they acquire their native conformation. The disulfide isomerase (PDI) is needed for correct disulfide bond formation, and the BiP protein would facilitate these processes (Pfeiffer and Rothman, 1987). BiP is released from immobilized immunoglobulin heavy chains on addition of ATP (Munro and Pelham, 1986), suggesting a possible energy-requiring step in which proteins are disaggregated so that their transport from the ER can proceed. However, this cannot be the only mechanism for retention in the ER. BiP, PDI, glucose regulating protein (GRP94) and other soluble proteins residing in the ER, have a role in the initial steps of maturation of secretory proteins (see Pelham, 1990). These proteins must be present in a functional form and be properly folded, yet they are retained; therefore, they must be distinguished from other proteins that are transported rapidly through the Golgi complex. Although retention must play a role, an alternative mechanism, the retrieval of proteins leaving the ER by retrograde transport, has also been shown to be important. As we have seen a number of times, special amino acid motifs in a protein are likely to be involved in targeting. Why not examine whether special motifs have a role in the retention or retrieval of proteins of the ER? This approach can be used by first determining the amino acid sequence of the proteins and then looking for common sequences, perhaps with computers using available data banks (see Chapter 1). The identification of common sequences would also open the way for manipulating protein structure using the bag of tricks supplied by molecular biology, using modified DNA inserted in vectors (Chapter 1). A resident protein from which the suspected sequence is missing should proceed to another compartment. Conversely, a protein generally targeted to another location should acquire residence in the ER when the recognition sequence is attached to the molecule. The search for common motifs revealed common or similar sequences at the carboxy-terminal of resident proteins corresponding to a tetrapeptide [in mammals, usually Lys-Asp-Glu-Leu (KDEL) and in yeast, Hist-Asp-Glu-Leu (HDEL)]. When expressed in monkey COS cells (transformed kidney cells from the African green monkey), BiP lacking this sequence was secreted (Munro and Pelham, 1987). In contrast, addition of the last 6 amino acids of BiP to secretory, lysosomal, or vacuolar proteins, caused their localization in the ER. Similar results were obtained with other mammalian cells, plants and yeast. The KDEL or HDEL sequences are not always the ones used for recognition. Although some mammalian liver esterases sometimes use similar tetrapeptides, they frequently have different coding sequences. Residence in the ER is also favored by a double lysine motif in the cytoplasmic tail of a protein (Jackson et al., 1993).
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These sequences are retrieval signals, as has been demonstrated ingeniously by adding the KDEL retention sequence to the lysosomal enzyme cathepsin D (Pelham, 1988). Lysosomal enzymes are processed in the Golgi stacks (see sections IIIC and G), in this case by the addition of Nacetylglucosamine-1-phosphate (G1cNAc-1-phosphate). The cathepsin was found in the ER, but with the addition of N-acetylglucosamine (GlcNAc), which must have taken place after cathepsin left the ER. This experiment suggests that a membrane receptor for the retained protein is activated in a post-ER component and the complex is then returned to the ER. Yeast mutants unable to retain the HDEL signal have been isolated, and a putative receptor for post-ER recognition has been found (Semenza et al., 1990). As mentioned above, many proteins such as enzymes are required in their folded form to perform the tasks of the ER. Is retrieval the only mechanism to retain these resident proteins in the ER? The proteins could be immobilized by binding to receptors excluded from the buds in the process of forming transport vesicles. Removal of either the KDEL sequence or the double lysine motif, permits the secretion of ER resident enzymes. However, the rate of loss is very low (Pelham, 1989) and generally vesicles budding from the ER in an in vitro yeast system do not contain resident proteins (Barlowe et al., 1994, Bednarek et al., 1995, Füllerkrug et al., 1994). These observations suggest that, in these cases, retention is likely to play the predominant role. In addition to retaining needed or defective proteins in the ER, at least some of the exported proteins are selected. The cargo proteins are loaded by selective binding as demonstrated by the fact that they are concentrated in the anterograde (COPII) vesicles about 10-fold. A concentration of this kind suggests a binding to specific binding sites. The presence of such binding sites is supported by the need of certain domains such as AspXGlu in the carboxy-terminal cytosolic domain of certain transmembrane proteins (e.g., Nishimura and Balch., 1997). In the case of proteins recycling between the ER and the Golgi, the anterograde transport depends on two phenylalanine residues close to the carboxy terminal (e.g., Dominguez et al., 1998) required for binding to coat components of vesicles (the Sec23p/Sec24p complex). The binding between cargo proteins and coat subunits (in this case COPII) should be demonstrable in vitro. As expected, complexes have been found in yeast ER extracts containing COPII subunits and cargo molecules (Kuehn et al., 1998). However, resident proteins were absent. In mammalian ER extracts, components of the COPII vesicles [the GTPase Sar1p (expressed as a glutathione S-transferase fusion protein) and Sec23p/Sec24p] formed a complex in ER vesicles that contained VSV-G cargo glycoprotein (Aridor et al., 1998). Again, a resident protein, ribophorin, was found to be absent. A variety of known signals for transport and retrieval are displayed in Table 2 (Rothman and Wieland, 1996). These are involved in anterograde and or retrograde transport. Table 2 Examples of transport signals. Reproduced from Rothman and Wieland, 1996, by permission. Copyright ©1996, American Association for the Advancement of Science.
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Signal
Location in protein and with respect to membrane
KDEL
COOH-terminus, luminal
Retrieval of proteins from Golgi to ER
KKXX
COOH-terminus, luminal
Retrieval of membrane proteins from Golgi to ER
XXRR
NH2-terminus, in cytoplasm
Retrieval of membrane proteins from Golgi to ER
Propeptide
NH2-terminus, luminal
Transport from Golgi to endosomes or lysosomes
Mannose-6-phosphate
Asn-linked saccharides, luminal
Transport from Golgi to endosomes or lysosomes
Tyrosine-rich dileucine
Cytoplasmic domain
Transport from Golgi to endosomes or lysosomes
YQRL
Cytoplasmic domain
Transport from cell surface to Golgi
NPXY (and similar)
Cytoplasmic domain
Transport from cell surface to endosomes
GPI anchor
COOH-terminus, luminal
Transport from Golgi to apical cell surface in polarized cells
Fate specified
What happens to the proteins that are retained in the ER because they are defective, unassembled or misfolded? The ER possesses what has been called "quality" control (see Kopito, 1997; Ellgaard et al., 1999) and these proteins are degraded (see Bonaficino and Klausner, 1994). In addition, the same degradation system regulates the activity of ER-resident proteins and plasma membrane proteins. The degradation apparently involves a translocation system that removes the soluble (McCracken and Brodsky, 1996) or integral membrane proteins (Wiertz et al., 1996a) from the ER to the cytoplasm. In the cytoplasm, they are degraded by the proteasome system (e.g., Wiertz et al., 1996a) (see Chapter 15). At http://www.albany.edu/~abio304/text/10part2.html (7 of 9) [3/5/2003 7:55:42 PM]
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least in some cases, polyubiquination is required (e.g., Hiller et al., 1996) (see Chapter 15, Section IIB). The proteasomes cover the cytoplasmic face of the ER membrane in secretory cells (see Rivett, 1993) and, at least in yeast, the ER membrane contains ubiquitin conjugating enzymes. (Sommer and Jentsch, 1993). The translocation from the interior of the ER to the cytoplasm involves Sec61 protein, the translocon component discussed above and, in addition, BiP and Sec63p. The latter two are also involved in posttransciptional import (Plemper et al. 1997) as demonstrated using various yeast mutants. Import into the ER vesicles and reverse translocation may therefore involve, at least in part, the same pathway (Wiertz et al., 1996b). A 97 kDA ATPase, valosin containing protein (VCP; also called CDC48 or p97) serves as a link between the export of the proteins from the ER and their degradation in the proteasome. VCP is one of the proteins of the ATPase with multiple cellular activities (AAA family). VCP forms a homohexamer that binds to a number of other proteins with a function in the transfer. The VCP complex binds to polyubiquitin chains while these are in the translocon and delivers most of them to the proteasome (e.g., Jarosch et al., 2002; Braun et al., 2002). The ER-proteasome apparatus may not be the only mechanism used by cells to control the quality of its proteins. A resident protein of the TGN, furin, is degraded by lysosomes. The transfer to the lysosomes is apparently controlled by the state of aggregation of the furin (Wolins et al., 1997). The production of the misfolded or defective proteins may exceed the capacity of the proteasomes. In these cases, the intracellular deposition of misfolded protein can produce aggregates that form ubiquitinrich cytoplasmic inclusions, sometimes characteristic of neurodegenerative diseases (see Mayer et al., 1991). The cystic fibrosis transmembrane conductance regulator (CFTR), an integral protein, is a transporter molecule that functions as a Cl--channel in the plasma membrane. Mutations of the corresponding gene produces cystic fibrosis, which causes severe chronic bronchopulmonary malfunction and pancreatic insufficiency. Most cystic fibrosis patients carry an allele of CFTR which interferes with the normal folding so that the mutated CFTR is retained in the ER and rapidly degraded. The degradation can be prevented by inhibiting the proteasomes (Ward et al., 1995; Jensen et al., 1995). The mutant CFTR surprisingly can function normally if it were delivered to the cell surface (Cheng et al., 1991). Presenilin-1 (PS1) is another integral protein predominantly present in the ER. PS1 is present in neurofilament-rich cytoplasmic inclusion bodies in Alzheimer disease (e.g., Busciglio et al., 1997). Both CFTR and PS1 have multiple transmembrane segments. The fate of CFTR and PS1 molecules were studied in transfected human embryonic kidney or Chinese hamster ovary cells where proteasome activity was overexpressed or inhibited (Johnston et al., 1998). The accumulation was in the form of high molecular weight, detergent-insoluble, multiubiquitinated forms in distinct pericentriolar structures that have been called aggresomes. The formation of aggresomes redistributes the intermediate filament protein vimentin (see Chapter 24) so that it forms an enclosure for the protein. Interference with microtubules blocks the formation of aggresomes.
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The retention of mutant misfolded Wilson protein is the basis for Wilson disease, an inherited disorder of copper metabolism resulting in neuronal degeneration. The Wilson protein is a Cu transporter localized in the trans-Golgi. The mutant form is eliminated by the action of the ER quality control machinery (Payne et al., 1998). Current thinking suspects inefficient ER quality control to be responsible for prion diseases such as bovine spongiform encephalopathy (BSE) or Creutzfeldt-Jakob disease (see Dobson and Ellis, 1998). Apparently, the transmission of the diseases depend on the expression and accumulation of abnormal variants of the prion protein PrP (e.g., see Prusiner, 1997; Hegde et al., 1998.
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10. Biosynthesis and Cytoplasmic Trafficking: Synthesis, Packaging and the Golgi Complex
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C. Passage Through the Golgi System and Transfer to the Cell Surface We have seen that the Golgi apparatus is made up of stacks of cisternae. Seen in three-dimensions, the details of the Golgi apparatus differ considerably from cell type to cell type (e.g., see Rambourg and Clermont, 1990, 1997). Some of the early studies have indicated that many of these cisternae are interconnected by tubules as revealed by stereoscopic EM techniques (see Chapter 1) (Rambourg and Clermont, 1997) (however, see below). The imaging of cisternae isolated from mammalian cells in culture using freeze etching, (Weidman et al., 1993) indicates that generally the cisternae have two domains: a central biconcave circular domain bounded by an irregular peripheral domain containing a reticulum of interconnected tubules (referred to as fenestrations) and tubules. The peripheral domain has buds with a coating suggesting the presence of coat proteins (see Chapter 11). The central domain is thought to contain oligomers of resident enzymes (Nillson et al., 1993) (see below section on retention). Tubular connections between cisternae are frequent (Weidman et al., 1993). More recently, a partial three-dimensional reconstruction of the Golgi apparatus from rat kidney cultured cells (Ladinsky et al., 1999) and pancreatic β cells (Marsh et al., 2001) have been carried out and present a picture very similar to that from previous studies. However, some of the details differ significantly. In the study with rat kidney cells, the specimens were subjected to ultrarapid cryofixation and freezesubstitution [where freezing at very low temperatures (e.g., -174oC) is followed by fixation in an organic solvent also at low temperatures (e.g., -90oC)]. The observations were carried out with a high-voltage EM using thick sections and then the appropriate tomographic techniques were applied. (see Chapter 1). The study used dual-axis tomography (Mastronarde, 1997). With this technique, the specimen were tilted around two orthogonal axes. The cisternae appeared closely apposed in stacks (the compact region) or loosely connected laterally by bridging tubules (the non-compact region) at equivalent levels, but not at nonequivalent levels. All cisternae were found to be fenestrated and to display buds: the trans-most cisternae displayed clathrin coated buds. Other cisternae displayed non-clathrin coated buds and others were uncoated. Tubules with budding profiles were found at the margins of all cis and trans cisternae. Vesicles filled holes were found at the cis and lateral sides of the stacks and these "wells" may be involved in vesicle transport between the stacks. The stacks of the Golgi were found between cis-ER and the trans-ER which is very close to the trans-stacks of the Golgi. Between the cis-ER and the Golgi, there are many tubular and flattened sac structures. The idea that anterograde transport depends on transient tubular connections between successive cisternae (see Section I, above) is not supported by this study or the study of pancreatic β cells (Marsh et al., 2001), since such connections between successive cisternae were not found. Some tubular projections reaching outside of the Golgi were found and they sometimes bypassed cisternae. The results with cultured pancreatic β cells ( Marsh et al., 2001) provides a http://www.albany.edu/~abio304/text/10part3.html (1 of 30) [3/5/2003 7:55:54 PM]
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reconstructions with a resolution of approximately 6 nm. The observations are similar to those of the previous study. However, in the reconstructed sector the ER is a single continuous compartment in close contact with mitochondria, trans-Golgi cisternae, and endosomal and lysosomal elements. The Golgi cisternal openings permit the ER traversing the Golgi ribbon from one side to the other. Microtubules are in close contact with the cis-Golgi, the ER, and elements of the endo-lysosomal system. Sequential events and sequential sites The Golgi system is the site of synthesis of many sphingolipids, such as sphingomyelin and glucosyl ceramide. Furthermore, the Golgi modifies newly synthesized proteins and lipids. Golgi enzymes glycosylate proteins or trim them by removing carbohydrate components. Still others, add sulfates to tyrosines, attach palmatoyl groups and cleave certain proteins. The reactions are carried out stepwise in various cisternae. The enzymes needed for these reactions are not distributed evenly, but generally in the order of the reactions from the cis to the trans direction. Eventually the Golgi system sorts out the different cargos into vesicles destined to different targets. The passage of proteins through the Golgi system can be traced by initiating the synthesis of a protein at a specific time and following its travel through the cell's compartments. This can be accomplished by viral infection. The viral glycoproteins enter the Golgi complex at the cis face and exit through the trans face (Bergmann and Singer, 1983, Saraste and Hedman, 1983). A finished glycoprotein requires several posttranscriptional stepwise enzymatic additions to arrive at its mature structure. The specific locations of the necessary enzymes, mirroring the order of the necessary steps, are evidence of vectorial processing akin to an assembly line. The distribution of enzymes in the ER and the Golgi system is shown in Fig. 8 (Goldberg and Kornfeld, 1983). The evidence for this distribution comes, in part, from the experiments in which the components of CHO cells were fractionated in sucrose gradients and the glycoprotein-processing enzymes were found in different fractions. Enzymes acting earlier in the processing pathway were found in the heavier fractions. This was confirmed in more detail using mouse lymphoma cells, in which the order of fractionation in the sucrose gradients coincided with the order of processing. Although this evidence shows that the location is vectorial, it is somewhat harder to correlate the presence of an enzyme with an actual location inside the cell. Immunocytochemistry has confirmed the location of some of these enzymes in the appropriate Golgi compartment. Experiments involving a complementation assay that requires cell fusion, confirmed that proteins are transferred from one Golgi stack to another in a unidirectional manner (Rothman et al., 1984a, 1984b). In these experiments, VSV-infected CHO cells were fused to noninfected cells. The Golgi complexes of the two types of cells maintained their integrity. Three clones of cells were used. The fusion of the various types of cells was accomplished by short exposure of the cells to low pH.
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Fig. 8 Steps in the processing of asparagine-linked oligosaccharides and their presumptive intracellular site. Steps for addition of M6P to lysosomal enzymes are indicated in the side branch (3-5). 1; Glucosidase I, 2, glucosidase II; 3, lysosomal enzyme, N-acetylglucosaminylphosphotransferase; 4, lysosomal enzyme, phosphodiester glycosidase; 5, mannosidase I; 6, GlcNAc transferase I; 7, mannosidase II; 8, GlcNAc tranferase II; 9, fucosyltransferase; 10, GlcNAc transferase IV; 11, galactosyltransferase; 12, sialyltransferase. ( ) Glucose; ( ) GlcNAc; ( ) mannose; ( ) galactose; ( ) fucose; ( ) sialic acid; P, phosphate. Note: ER mannosidase is not indicated in the diagram. Reprinted with permission from Goldberg and Kornfeld (1983).
The experiments can be explained most simply by using arbitrary symbols to indicate the direction of the reactions, which occur in separate stacks as follows : A → B → C → D. In the first experiment, infected cells which were able to carry out the first reaction A → B but could not carry out B → C, were fused to non-infected cells which could not carry out the reaction A → B. The virus G-protein was now appropriately modified by reaction B → C. This could only have happened if the viral G protein was transferred from the stacks of one cell to that of the other as shown in Fig. 9.
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Fig. 9 Cartoon showing the design of an experiment demonstrating the transfer of materials between two different Golgi stacks.
In experiments following the same strategy, the fusion of the two different kinds of cells was carried out either immediately or after delays of different duration. As indicated in Fig. 10, the longer the delay, the lesser the transfer. In this figure, the ordinate shows the extent of the reaction (in this case the incorporation of [3H]GlcNAc into the G viral protein) and the abscissa, the time after the introduction of the labelled material, before fusion was allowed to take place. These results indicate (a) the transfer between Golgi stacks can take place even when separated in space and (b) the passage must be in a single direction (that is vectorial), otherwise it would not follow the pattern shown in Fig. 10.
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Fig. 10 Kinetics of galactosylation of G-protein labelled with [3H]GlcNAc in VSV-infected clone 13 cells after fusion to uninfected clone 1 15B cells (Rothman et al., 1984a). Reproduced from The Journal of Cell Biology, by copyright © permission of the Rockefeller University Press.
Experiments with a somewhat different design have been used to study the vectorial nature of the transfer directly. Infected cells from a clone unable to carry out the reaction C → D but able to carry out the reactions A* → B* and Bo → Co (where the two suprascripts indicate a different kind of radioactive probe) were fused to infected cells able to carry out reaction B → C and subsequent reactions, but not A → B. The design is shown in Fig. 11. The cells in which the viral G-protein was labelled with *, were able to produce E* to a significant extent. In contrast, the viral G-protein labelled with o at a later step were unable to incorporate the o label and produce Eo. This indicates that the G-protein labelled at an earlier step was able to be transferred more readily, as expected for a vectorial transfer. The protein labelled in a later step did not have enough time to proceed to the next step.
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Fig. 11 Cartoon illustrating the experimental design in which the ability to transfer to the same Golgi stack from two different sequential Golgi steps.
Experiments with cell-free systems of CHO cells provide similar evidence (e.g., see Balch 1989). The transport between stacks requires the presence of cytosol extract, ATP, and a protein in the surface of the Golgi. In general, intercompartment transport of G-protein requires sequential steps probably involved in budding and fusion. ATP is required for each step, and cytosol is required for all except the last step. The experiments just described have been interpreted to mean that in the Golgi, the transport of cargo is vectorial and that the anterograde transport is carried out in packets, such as vesicles. As we shall see in the next section, an involvement of vesicles is contradicted by the results of other studies and this issue is still far from being resolved. Mechanisms of anterograde transport Three different mechanisms of transport within the Golgi cisternae have been considered recently: transport mediated by vesicles, the maturation and displacement of cisternae and the transport through the tubules that interconnect the various Golgi elements (for a recent evaluation of the various models, see Pelham, 1998; Allan and Balch, 1999; Pelham, 2001). Present information is insufficient to reach a firm conclusion in relation to the vesicular and maturation mechanisms. In addition, the two models are not mutually exclusive and both may be operating.
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Transport via tubules that connect the various cisternae is highly unlikely. We have already indicated that three-dimensional reconstruction of the Golgi apparatus did not find connecting tubules in the cis-to-trans direction (see above). There is some other evidence that bears on this question. In the tubule-transport model, resident proteins, all of them integral proteins, could be excluded from some Golgi compartments because of oligomerization and possibly binding of their cytoplasmic domains to an underlying matrix (Nilsson et al., 1993) (see discussion of retention that follows). As noted the reactions of the Golgi take place sequentially and the various Golgi compartments differ in biochemical composition as indicated diagrammatically in Fig. 8. There must be some mechanism that keeps the various components from mixing. Studies with chimeric resident integral Golgi proteins linked to the green fluorescent protein (GFP) (see Chapter 1) have been carried out to examine the mobility of proteins residing in the Golgi ( Cole et al, 1996) using either photobleaching recovery (FRAP), (see Chapters 1 and Chapter 4) or fluorescence loss in photobleaching (FLIP). In FRAP, the fluorescence of a small area is bleached by a laser flash. The rate of recovery is then measured using an attenuated beam. The return of fluorescence can be interpreted as the exchange of non-fluorescent and fluorescent proteins; under most circumstances bleaching can be considered irreversible. In contrast, FLIP measures the disappearance of the fluorescence from the whole structure after photobleaching a small area repeatedly. Both methods indicate that the proteins are free to move laterally and are not bound. However, the results cannot be interpreted to mean that the membranes of the Golgi system are continuous. Since the Golgi compartments are biochemically specialized, the chimeric proteins used (β-1-4-galactosyltransferaseGFP, mannosidase II-GFP and KDEL-receptor-GFP) are likely to be present in individual compartments of the Golgi (e.g., laterally connected cisternae) and not be distributed throughout the Golgi. In addition, the transport through tubules would require some means of propelling the cargo through the connections. To our knowledge, this aspect has not been addressed so far. A transport via the small vesicles curently implicated, cannot readily explain the transport of large structures, such as algal scales (Becker et al., 1995), casein micelles in in epithelial mammary cells (Clermont et al., 1993), procollagen in fibroblasts (Bonfanti et al., 1998), and albumin in hepatocyes (Dahan et al., 1994). These cargos progress through the Golgi apparatus but are too large to fit into vesicles (see Mironov et al., 1997, 1998; Glick and Malhotra, 1998). In addition, these components have been found in all Golgi stacks, but not in vesicles. One of the present maturation models proposes that in anterograde transport the cisternae move from the cis to the trans direction and simultaneously acquire the appropriate enzymatic complement from the more advanced cisternae by retrograde vesicular transport. In fact, some resident Golgi enzymes are known to move retrogradely through the Golgi stacks (e.g., see below and Harris and Waters, 1996). That a vectoral movement of Golgi compartments take place is indicated by a study of the flagellate Triconympha (Grimstone, 1959) in which the Golgi stacks (forming a so called parabasal body) disappear in the absence of food and reappear when the food is reintroduced. The results are in harmony with the interpretation that the cisternae are formed in the cis side and disappear on the trans side. Studies of the progression of procollagen in the Golgi apparatus of chick fibroblasts, support the http://www.albany.edu/~abio304/text/10part3.html (7 of 30) [3/5/2003 7:55:54 PM]
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maturation model. The translocation from cis-to-trans structures proceeds without showing a presence in COPI vesicles (implicated in intra-Golgi transport). This component, remains within the compartment from which it was transported from the ER (Bonfanti et al., 1998; Mironov et al., 2001). Furthermore, the compartment undergoes a change while moving from a cis-to-trans position. In the experiments of Bonfanti et al., 1998, the exit from the ER was synchronized first by inhibiting proline hydroxylation (needed for procollagen folding and hence release from the ER) followed by removal of the inhibitor. The polarity of the stacks was determined using clathrin buds as a marker of trans-Golgi, either with serial sections or immunogold using two different sizes of gold particles identifying procollagen and clathrin and, in addition, the visualization of COP coats. Mannosidase II, a medial Golgi enzyme was visualized using an immunological method with peroxidase as a marker (Rabouille et al., 1995). The movement from the ER to the cis-Golgi was in tubular-saccular structures greater then 300 nm in length. These were interpreted to correspond to precursors of the cis-Golgi cisternae that then progress through the stacks by a maturation process. The passage from trans-Golgi to the plasma membrane occurs in vesicles. The cisternal maturation model of anterograde transport across the Golgi apparatus requires a continuous retrograde movement of resident proteins to compensate for their loss. Therefore, retrograde-directed vesicles must contain these proteins and the retrograde transport must equal the anterograde movement. The demonstration of vesicular retrograde transport of Golgi components supports the maturation model. Under steady-state conditions in mammalian cells in culture (Love et al., 1998), a fraction of resident Golgi enzymes was found in vesicles that were depleted of secretory cargo (and therefore not involved in anterograde transport) and could be separated from cisternal membranes. They were capable of binding to and fusing with isolated Golgi. Furthermore, after fusion, their enzymatic complement was able to process newly acquired cargo. Other experiments support the presence of recycling of processing enzymes (Hoe et al., 1995; Harris and Waters, 1996; Wooding and Pelham, 1998). In the experiments of Wooding and Pelham (1998), green fluorescent protein (GFP)-chimeras (see Chapter 1) were used to visualize late and early resident Golgi markers. These are present in distinct sets of scattered moving cisternae. In temperature-sensitive mutants, after shifting to the permissive temperature, late Golgi markers dispersed into vesicle-like structures within minutes. These results agree with the notion that resident Golgi components have to undergo retrograde transport. One of these moved quickly to the ER, suggesting that it cycles between Golgi and the ER. The results of other experiments are also in harmony with the maturation model of transport between the Golgi stacks. This model of Golgi transport predicts the presence of resident proteins in the peri-Golgi vesicles (i.e., edges of the cisternae and neighboring vesicles) to be recycled to their original stack for the process to continue. In contrast, the vesicular model predicts that anterograde cargo would be contained in these vesicles. Resident proteins were in fact found in the peri-Golgi vesicles. In contrast in the cells expressing VSV-G, a transmembrane protein, used as anterograde marker found this protein mostly absent from these vesicles (Martínez-Menárguez et al., 2001). In the maturation model, large aggregates (such a procollagen) and other cargoes (such as VSV-G) should be found in the cisternae of the Golgi and be transferred at the same rate. Procollagen aggregates were found to be transferred through the Golgi complex without leaving the lumen of the cisternae in agreement with the maturation model (Mironov et al., 2001) and to be transferred at the same rate as the G protein of the VSV. Futhermore, the two were http://www.albany.edu/~abio304/text/10part3.html (8 of 30) [3/5/2003 7:55:54 PM]
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found to be transported without entering vesicles. In addition, COP-I vesicles were found to contain Golgi resident proteins but minor amounts of anterograde cargo (Lanoix et al., 2001). The early Golgi proteins were in vesicles distinct from those containing proteins of the medial Golgi stacks as might be expected from the retrograde component of the maturation model. Some in vitro experiments lend strong support to models of transport not dependent on vesicle formation. The production of coated vesicles from Golgi vesicles requires the presence of the small GTPase, ARF (Orci et al., 1993; Taylor et al., 1994). At least in vitro, anterograde transport through the Golgi complex can take place when the formation of coated vesicles is blocked by the absence of ARF (Taylor et al., 1994; Happe and Weidman, 1998). The rate of transport was found to be about the same, with or without endogenous ARF (Happe and Weidman, 1998). However, the density of coated vesicles was reduced fifteen-fold in the ARF-depleted system. Some observations challenge the maturation model by suggesting that the rate of retrograde transport does not match the rate lf the anterograde transport. Immunoelectronmicroscopy techniques using gold particle markers (see Chapter 1) with human cells in culture were used to localize two proteins, residents of the medial Golgi (Orci et al., 2000). In contrast to the studies just discussed, the resident proteins were found to be excluded from buds and vesicles suggesting that the progression of the Golgi cisternae is much slower than the anterograde protein transport. These observations do not exclude the interpretation that the cisternae do progress in the anterograde direction, but they suggest that this mechanism is not a major player in anterograde transport. Other recent experiments suggest an alternative explanation for the transfer of large particles, in harmony with the vesicular transport model: the transport seems to occur in very large vesicles (Volchuk et al., 2000). Expression of a construct of a self-aggregating mutant of a binding protein was used (Rollins et al., 2000) in a human cell line. The construct is delivered into the ER and normally would be secreted, if maintained in folded and soluble form. A permeant ligand which allowed the protein to remain in this condition was used. However, the transport was blocked by maintaining the cells at 15o C. When the ligand was removed, protein aggregates ranging up to 400 nm in diameter were formed within the ciscisternae of the Golgi stack. After shifting the cells to 20o C, the huge particles were transported across the Golgi stack within 10 min. An EM study using serial sections showed that during the peak of the transport, about 20% of the aggregates were in megavesicles originating from the rims of the cisternae. These experiments suggest that, at least in part, vesicle size may depend on the size of the cargo. A strong argument in favor of the vesicular model comes from studies of Orci et al. (1997) which show the presence of anterograde vesicles (proinsulin and VSV G protein) and retrograde vesicles (KDEL receptor, see Chapter 11) budding throughout the Golgi. Segregation of the two sets of proteins can also be demonstrated in vitro. The VSV-G protein and the KDEL proteins are packaged in separate vesicles. The maturation and tubular transport proposals are unlikely to explain the evidence obtained from experiments using the fusion of cells to demonstrate a vectorial unidirectional anterograde transport (see http://www.albany.edu/~abio304/text/10part3.html (9 of 30) [3/5/2003 7:55:54 PM]
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previous section) or the demonstration of the presence of both anterograde and retrograde vesicles associated with the Golgi system. As we saw a role of tubules is highly unlikely. However, it is entirely possible that both the maturation and vesicle-transport mechanisms operate, depending on circumstances. Retention of resident proteins in the Golgi stacks The individual components of the Golgi system differ in composition, including the lipid elements. This individuality could conceivably result from a steady state where a variety of products would flow through the system, but the composition of each compartment would remain the same at any one moment. A steady state mechanism such as this, is part of any maturation model. There is evidence to support the view that structural elements and the enzymatic machinery of the Golgi, the glycosidases and glycosyl transferases are retained or retrieved to stationary compartments. The resident proteins of the Golgi, unlike those of the ER, are integral membrane proteins or peripheral proteins on the cytoplasmic face of the cisternae (see Munro, 1998). The characteristics of the proteins are shown in Table 3. Table 3 Localization of the domains of Golgi proteins
PROTEINS
TRANSMEMBRANE DOMAIN (TMD)
LOCATION IN GOLGI
Glycosylation enzymes
At amino terminal
Lumen
Internal sequence
Amino portion lumen, carboxy end in cytoplasm
At carboxy terminal
Amino portion in cytoplasm; carboxy terminal in lumen
None
Amino terminal portion bound to cytoplasmic face
TGN proteases/ TGN 38
SNARES
Peripheral proteins
If there is a continuous movement of cargo between Golgi compartments and from the Golgi to target sites, what is responsible for the retention of resident proteins? Certain sequences or domains have been http://www.albany.edu/~abio304/text/10part3.html (10 of 30) [3/5/2003 7:55:54 PM]
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found to be responsible for localization in the Golgi. The localization may be the result of retention or retrieval, and frequently both (e.g., Bryant et al., 1997). Presently available evidence is sometimes insufficient to allow us to generalize. For glycosylating enzymes, the transmembrane domain (TMD) is an important determinant of localization (see Colley, 1997). However, for some of the enzymes, the sequences flanking the TMD and the lumenal portion of the proteins also play a role. The TMD also is important in the localization of the SNAREs, Sed5p and Sft1p (Banfield et al., 1994) to tubules of the cis-Golgi and viral proteins targeted to the Golgi, although cytoplasmic domains are also needed. Many of the TGN proteins are recycled continuously, from the cell surface and endosomes and are returned to the TGN (e.g., Bos et al., 1993; Molloy et al., 1994). For the proteases of the TGN (e.g., furin in mammalian cells, and in yeast, DPAP-A, Kex1p and Kex2p) as well as TGN38, short sequences in the cytoplasmic tail specify location to the late Golgi probably by retrieval signals (Wilcox et al., 1992; Nothwehr et al., 1993; Bos et al., 1993, Schäfer et al., 1995). In the case of furin and TGN38, the sequences are short motifs containing tyrosine and for DPAP a ten amino acid sequence containing phenylalanine. What is the mechanism of retention? The TMDs are thought to be resposible for retention by an anchoring mechanism. In contrast to secreted or integral plasma membrane proteins, some of the proteins of the trans-Golgi and TGN are not found at the cell surface (Teasdale et al., 1994; Wong et al., 1992). Therefore, it would seem that they do not enter the vesicles exiting the TGN. One of the mechanisms proposed is the kin recognition model. In this model, like molecules recognize each other and form oligomers. The complexes are then excluded from vesicles. Supporting this view are the observations that two enzymes of the medial Golgi, N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II, are tightly associated in vivo (Nilsson et al., 1994). In addition, viral proteins that localize in the Golgi are known to form homo-oligomers (Weisz et al., 1993). However, the association of NAGT I and mannosidase II appears to depend on the lumenal domains of these two proteins and not the TMDs responsible for their retention (Munro, 1995b; Nilsson et al., 1996). The presence of lipids and sterol in specialized plasma membrane domains, offers another possible explanation for the retention of proteins. The presence of sphingolipids and sterols thicken membrane bilayers (Ren et al., 1997). These domains could exclude the shorter transmembrane domains of resident proteins that are unable to straddle the bilayer. Supporting this view, the TMD of Golgi enzymes are, on the average, five residues shorter than those in the plasma membrane and contain a higher proportion of phenylalanine (Bretscher and Munro, 1993; Munro, 1995a). The role of the length of the TMD was put to a direct test by adding hydrophobic amino acids to the TMD of certain proteins. The TMD of sialyltransferase or galactosyltransferase were lengthened. These modifications were found to produce a reduction of retention. The experiments found that with a synthetic http://www.albany.edu/~abio304/text/10part3.html (11 of 30) [3/5/2003 7:55:54 PM]
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TMD of 17 leucines, the proteins were retained. However, when 23 leucines were added, the proteins were no longer retained (Munro, 1991; Masibay et al., 1993: Munro, 1995a). As already noted, retrieval frequently plays a role for localization in any part of the Golgi. In yeast there is evidence of rapid cycling of an early Golgi enzyme, Och1p (Harris and Waters, 1996) The localization signal for this protein is not known, but for other medial enzymes, TMDs have a role, although lumenal domains and cytoplasmic domains are also involved (Burke et al., 1994; Graham and Krasnov, 1995). Similarly, a protein can achieve a predominant cis-Golgi localization by recycling between the cisternae and the ER, as in the case of the yeast protein Emp47p Schroder et al, 1995) and the KDEL receptors Erd2p and Sec12p (Sato et al., 1996; Füllerkrug, 1997). An avian coronavirus, infectious bronchitis virus (IBV), has provided information to define the retention signal of the cis compartment. IBV E1 is an integral protein which spans the membrane three times; the retention information is thought to be in the first intramembrane span (Machamer and Rose, 1987). When expressed from cDNA in animal cells, this glycoprotein is trapped in the cis Golgi membranes where the uncharged polar residues Asn, Thr, and Gln appear to be the significant feature of the retention signal (Machamer et al., 1990). This premise can be tested with proteins not normally retained in the cis compartment. When this amino acids motif is added to proteins normally transferred to the plasma membrane, they remain in the cis Golgi (Swift and Machamer, 1991). Bulk flow and sorting signals All proteins transported in the anterograde direction share the same pathway until their final packaging in the appropriate vesicle in the TGN. What determines their final cellular destination? The different proteins could be coded by separate sorting signals analogous to the signal domains (or peptides) discussed in the previous sections. Each sorting signal could then mark the destination of the protein. Alternatively, all proteins not containing a sorting signal could be transported through the various compartments, culminating with arrival at the cell surface. These proteins would include those that are constitutively secreted (they do not have to be stored) and the integral proteins of the plasma membrane. This non-specific targeting by default has been termed bulk flow. A sorting signal would control a destination to the remaining compartments, such as lysosomes or storage secretory vesicles. Recognition of the signal is likely to require interaction between the signal (i.e., some specific sequence or configuration of the protein molecule) and its corresponding receptor. Therefore, the selective pathways should be saturable, and when the protein is overproduced, it should follow the nonselective (bulk flow) route even when possessing the appropriate signal. Present evidence suggests that all destinations have a corresponding targeting signal recognized by a corresponding receptor. However, transport via bulk flow is still possible, although it is likely to be much slower (see discussion in Rothman and Wieland, 1996). Experiments carried out with yeast have been very enlightening in illustrating both the involvement of a receptor in targeting and its saturability by excess of the targeted protein (e.g., Stevens et al., 1986). In http://www.albany.edu/~abio304/text/10part3.html (12 of 30) [3/5/2003 7:55:54 PM]
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yeast, the vacuolar compartment corresponds to the lysosomes of other cells. The recognition signal in yeast, however, is a polypeptide segment, in contrast to the carbohydrate portion of the glycoprotein, which serves as one of the signals for lysosomal localization (see below). One such protein is carboxypeptidase Y (CPY). Before reaching the vacuole, CPY is present in the cell as proCPY, which contains the sorting signal at its amino terminal. Processing to the mature form of the enzyme takes place in the vacuole. In these cells, overproduction of the vacuolar enzyme results in constitutive secretion to the periplasmic space. In experiments testing the effect of overproduction, the CPY structural gene (PRC 1) was cloned to produce plasmids containing multiple copies, which were used to transfect yeast cells. The results are summarized in Table 4 (Stevens et al., 1986). In these experiments, the increased gene dosage overproduced CPY (in the form of proCPY), presumably saturating the processing system and resulting in constitutive secretion. This interpretation is supported by the observation that deleting the amino-terminal of proCPY leads to constitutive secretion. Fusion of sequences that code for the amino-terminal portion of CPY to the gene that codes for the secretory enzyme invertase (Inv) has allowed study of the sorting signal (Johnson et al., 1987). Fifty amino acids of the CPY NH2-terminal appear to be sufficient to code for the transfer to the vacuole. Twenty of these correspond to the signal peptide (which initially targets the protein to the RER), so the 30 remaining are likely to contain the vacuolar sorting signal. In fact, deletion of this segment in the CPY leads to secretion of this protein. Table 4 Overproduction of CPY Results in Secretion of the Protein
Plasmid
Estimated PRC1 gene copy number
Relative CPY synthesis
CPY secreted %
2µ
1
1
6
CEN-PRC1
2
2
12
2µ-PRC1
5
6-8
50-55
Reproduced from the Journal of Cell Biology 102:1551-1557, ©1986, by copyright permission of the Rockefeller University Press
In animal cells, as in yeast, when proteins are produced in excess or when the receptor or targeting signal (for lysosomal enzymes, mannose-6-phosphate, M6P) is absent, the proteins spill into the bulk flow pathway. This is the case, for example, in I cells, cultured animal cells which are unable to attach M6P to the lysosomal enzymes (Hasilik and Neufeld, 1980). Similarly, treatment of AtT-20 cells (a line derived
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from pituitary cells) with chloroquinone which blocks the storage route, causes constitutive secretion of the ACTH precursor (Moore et al., 1983). Since in exocytosis the membranes of the secretory vesicles become continuous with the plasma membrane, we would expect integral membrane proteins and constitutively secreted cargo to be part of the same vesicles as in fact found (Strous et al., 1983). Furthermore, in yeast, the two processes are biochemically linked, so that a mutation blocking secretion also blocks membrane growth (Tschopp et al., 1984) The targeting principles just discussed are summarized and illustrated in Fig. 12 (Rothman and Wieland, 1996). Part A illustrates the packaging of a cargo protein (the open circles) with a transport signal domain (the full rectangle) bound to a receptor, which is shown as part of the coat (indicated by the heavy straight lines). The cargo protein is an integral protein and its signal sequence is in the cytoplasmic domain of the protein. The direct binding of a signal domain to the coat proteins has been shown in a number of cases (e.g., Cosson et al., 1996). The transport signal need not be in the cytoplasmic domain, in which case it may be bound to an auxiliary integral protein acting as a bridge between transport signal and receptor in the coat. Part B shows how retention might take place. The retention signal prevents the protein (full circle) from entering the bud either because it is firmly bound to a special lipid patch (as implied in the figure) or because it is attached to a fixed receptor protein in the membrane (not shown). The transport by bulk flow is shown for proteins (indicated by the open lollipops in Part C) that do not bind to a receptor. The various coats: clathrin, COPI and COPII are discussed in more detail in Chapter 11. Cargo proteins that span the membrane can interact directly with coat components such as adaptor subunits (see Chapter 11). Proteins that do not span the membrane have to bind to separate cargoreceptors, integral proteins containing a motif capable of binding the cargo-protein and another to bind to a coat component. In many cases, currently available data strongly suggest that the adaptors have a role in the recognition of signal-motifs. If this were the case, because of the variety of targets yet to be elucidated, we would expect the presence of many more adaptors. New adaptor complexes have been found. One in neuronal cells (βNAP) is related to β-COP and β-adaptin and is a phosphoprotein associated with transport from the soma to the axon terminals (Newman et al., 1995; Pevsner et al., 1994), (Simpson et al., 1996). This coat protein is involved with TGN and plasma membrane transport. In turn, another complex has been found related to the neuronal complex (Dell'Angelica et al., 1997). These complexes have been referred to as AP-3 and have been localized in the TGN and endosomes (Simpson et al., 1996; Dell'Angelica et al., 1997). The SDYQRL sequence of TGN38, an integral protein, binds to the µ2 chain of the plasma membrane clathrin-binding AP-2 complex (Ohno et al., 1995) during endocytosis. The same motif also interacts with the AP-1 complex of the Golgi. These observations suggest that the medium chain of the clathrin associated adaptor complexes are the signal recognition molecules for sorting in the TGN or endocytosis. http://www.albany.edu/~abio304/text/10part3.html (14 of 30) [3/5/2003 7:55:54 PM]
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The proteins of the p24 family of transmembrane proteins have been proposed to act as cargo receptors, selecting the proteins that go into the COPI and COPII vesicles (Schimöller et al., 1995; Stamnes et al., 1995 and Fiedler et al., 1996). Currently, 16 homologous proteins of this family have been recognized. Chop 24a is a component of the Golgi and COPI. A mutation in the analogous yeast component (yp24A) causes a defect in vesicle transport (Stamnes et al., 1995). Similarly, Emp24p is a component of the ERderived COPII vesicles. In yeast, mutants in Emp24p show a defect in the transport of certain proteins from the ER to the Golgi (periplasmic invertase and a glycosylphosphatidyl inositol-anchored plasma membrane protein) and not others (α-factor, acid phosphatase and two vacuolar proteins), supporting the view the the various cargos need different specific receptors (Schimöller et al., 1995). Each cargo receptor also has to bind one or more subunits of the coat proteins. The cytoplasmic tail of the p24 protein family binds to the coatomer (COPI) (Fiedler et al., 1996, Sohn et al., 1996). The dilysine motif of hp24d and yp24c binds to the α-, β'- and ε-COP. Three p24 lacking the dilysine motif in their tails but containing a phenylalanine motif, bind preferentially to β-, γ- and ζ-COP. The coatomer can bind two different segments of the p24 tails. One segment binds the dilysine motif, a retrograde transport motif. Another segment binds to a phenylalanine containing motif, which is an anterograde signal. The same p24 could therefore direct a protein in the antero- or retrograde direction depending on the conformation of the coatomer.
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Fig. 12 Models that explain the fate of proteins (see text). A. Selective removal of protein by concentration during budding. B. Retention of protein in donor compartment by exclusion from vesicles, and C. Transport of proteins by default through the bulk phase. Reproduced with permission from Rothman,J.E. and Wieland,F.T., Science, Protein sorting by transport vesicles, 272:227-234, Copyright ©1996, American Association for the Advancement of Science.
Several sorting signals are listed in Table 2 (above). Certain proteins have to be targeted to more than one target. Furin is an enzyme that catalyzes the proteolytic maturation of many preproteins in the endocytotic and exocytotic pathway. A 56 amino acid cytoplasmic tail is required for TGN concentration and intracellular targeting (Jones et al., 1995). Interestingly enough, different sections of this tail are required for each step in the routing. An 11 amino acid segment containing 2 serines is required for TGN localization. Internalization requires 34 amino acids. The serine residues of the cytoplasmic tail of furin are phosphorylated by casein kinase II. Mutagenesis of these residues reduces endocytosis of furin from endosomes to the TGN, suggesting that phosphorylationdephosphorylation regulates furin recycling. Phosphorylation of a 9 amino acid sector localizes furin to the cell surface or early endosomes, whereas removal of the phosphate localizes the furin to the periphery of the TGN. A 280 kDa actin-binding-protein binds tightly to the tail of furin. The recognition of the sorting signal of a cargo protein may result from a much more subtle mechanism than those already discussed. In the trans-Golgi the specific capture into vesicles may depend on the kinetics of the GTP hydrolysis in COPI-ARF1 complexes and not merely the binding to a specific receptor. Vesicle formation in the Golgi requires the assembly of COPI and the GTPase-ARF1 (see also Chapter 11 and Rothman and Wieland, 1996). COPI is released when GTP is hydrolyzed so that COPI becomes available for additional vesicle formation. Therefore ARF1 controls the availability of COPI. The GTP-hydrolysis is coupled to the recognition of the sorting signal (Goldberg, 2000). Different sorting signals have different effects on this reaction, so that the reaction provides specificity to the packaging. hp24a (a p24 protein which are putative cargo receptors discussed above) inhibits coatomer-dependent GTP hydrolysis whereas the dilysine retrieval signal (recognized by COPI and involved in retrograde Golgi to ER transport) has no effect despite the fact that the two compete for the same binding site on COPI (Harter and Wieland, 1998). A cargo sorting signal that inhibits the GTPase reaction increases the probability that the cargo protein will be included in a vesicle. Conversely, a cargo sorting signal that permits the rapid hydrolysis of the GTP bound to ARF1 will not be taken up into a vesicle because the assembly of the coat will be disrupted before a vesicle is formed. Another small GTPase, Cdc42, may play a role in the dynamics of ARF1-COPI interactions. Cdc42 is present in the Golgi and its GTP-bound form attaches to the γ-COPI subunit through its dilysine motif (Wu et al., 2000). This interaction could well be in competition with the attachment of other cargos containing the dilysine signal. Cdc42 has a prominent role in the assembly of actin filaments and elements involved in the transport of vesicles (see van Aelst and D’Sousa-Schorey, 1997).
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The secretion of some proteins is constitutive, that is the proteins are secreted continually. The vesicles originating in the TGN are targeted to the plasma membrane. Alternatively, in regulated secretion, the secretory granules are stored in the cytoplasm and their contents released in response to a signal. Regulated mature secretory granules are formed after the aggregation and sorting out of the proteins in the TGN followed by budding of vesicles. The secretory vesicles fuse to form mature secretory granules. The prohormone or immature protein in these vesicles are then cleaved at dibasic amino acid residues by prohormone convertases (see Zhou et al., 1999.). In regulated secretion, the targeting and formation of secretory granules probably depend on more than one mechanism and these mechanisms may play different roles in different cell types (see Tooze et al, 2001). In some cases, sorting in the TGN involves a disulfide-bonded amino terminal loop in the protein (Glombik et et al., 1999) supposedly segregated by binding to a membrane receptor as we discussed above for other cargos. Aggregation of the regulated secretory proteins is also thought to magnify the effect by capturing more proteins at the membrane sites (e.g., see Thiele and Huttner, 1998). The amino terminal loop has been found in many proteins which are secreted by a regulated pathway. In addition, in some cases when the loop is disrupted by reducing the disulfide bridge, the protein is constitutively secreted (Chanat et al., 1993). Conversely, inserting the loop in a constitutively secreted protein transforms the protein into one that segregates as a regulated protein (Glombik et al., 1999). The loop structure also has a role in multimerization. Multimerization and aggregation also may act as mechanism independent of the loop structure. Other secretory proteins being transported may also play a role by serving as nuclei for binding proteins at a membrane site. A role for specialized spots in the membrane, the so-called lipid rafts (see Chapter 4), is also suspected. The secretory proteins following the regulated pathway have also been found associated with membranes (e.g., see Pimplikar and Huttner, 1992) possibly by attaching to the lipid rafts ( Martin-Belmonte et al., 2000). Several regulated secretory proteins were found associated with membrane fractions thought to correspond to rafts (e.g., Dhanvantari and Loh, 2000). Furthermore, interfering with cholesterol (Wang et al., 2000) or sphingolipids (Blásquez et al., 2000), both components of rafts, inhibited the formation of regulated secretory granules. Post-Golgi signals As we saw in Chapter 9, tyrosine signals in the cytoplasmic tails of integral proteins serve as sorting signals in endocytosis and in targeting to post-Golgi compartments. Among these, the NPXY or YXXθ (where θ stands for bulky, hydrophobic amino acids) combinations have been found frequently (see Chapter 9; see Marks et al., 1997). YXXθ sorting signals have a role in the localization to endosomes, lysosomes, basolateral plasma membrane of polarized cells, the TGN and special organelles. Many of these signals utilize pathways that are common, at least part way, as indicated by saturability experiments (Marks et al., 1996). The YXXθ-signals have common capacities. They all can direct proteins from the plasma membrane to the endosomes. However, subsets of these motifs can have very narrow specificity. This specificity may rest in the sequence itself (e.g., the nature of the X-amino acid) or in sequences around the consensus sequence (e.g. Ohno et al., 1996). However, in other cases, the signal's effectiveness
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is unaffected by the adjacent amino acids (Collawn et al., 1990). A glycine preceding tyrosine increases the targeting of acid phosphatase to lysosomes (Peters et al., 1990) and the exact position of the motif may determine its recognition (e.g., see Ohno et al., 1996). For example, the displacement of the signal by one amino acid in the lysosomal glycoprotein lamp-1 (Rohrer et al., 1996) eliminates lysosomal targeting. However, this is not always the case. In some instances, the location of the sequence does not matter; the signal may be present in any part of the cytoplasmic domain (e.g., Collawn et al., 1990). Other signals can modify the effect of the targeting signal. For example, a lysosomal avoidance signal has been reported for the M6P receptor (MPR) (e.g., Pond et al., 1995a, Rohrer et al., 1995). In some cases, multiple signals are present. In the case of the cation-dependent MPR, dileucine is required for efficient entry in the TGN. Two signals are needed for endocytosis, one includes Phe 13 and 18. A second involves Tyr 45. In addition, the incorporation of fatty acid chains (e.g., by palmotoylation) was found to be needed, suggesting that anchoring to the lipid bilayer is needed (see Schweizer et al., 1996). In some cases, the dileucine motif is needed for endocytotic targeting. However, dileucine motifs can also mediate internalization and targeting to lysosomes or the basolateral surface of polar cells. Another independent signal is contained in a methionine adjacent to a leucine. However, delivery into a large endosome compartment requires the presence of additional acidic amino acid residues (see Pond et al., 1995b). Apparently, the targeting by the dileucine pathway is through different recognition mechanisms than that of the tyrosine signals. Although the dileucine signals use a saturable pathway (Marks et al., 1996), they do not compete with tyrosine signals. As discussed below, the sorting depends on adaptor complexes. µ1 and µ2 do not bind to the dileucine motif (Ohno et al., 1995), although in at least one case, they can be recognized by AP-1 and AP-2 complexes (Heilker et al., 1996), possibly using other binding regions of the adaptor complex. Other types of signals include a cluster of acid amino acids (Pond et al., 1995b, Voorhees et al., 1995, Jones et al., 1995), the dilysine signal KKFF (Itin et al., 1995) present in the protein VIP36 the ER protein ERGIC-53 that cycle between the plasma membrane and the Golgi. ERGIC53 cycles in this way only when overexpressed. Ubiquitin added to lysine residues in plasma membrane proteins, also serves as an internalization signal (Hicke and Riezman, 1996; Strous et al., 1996). Coat proteins may be involved in these processes as well. The acidic cluster in the cation-dependent-M6Preceptor, favors recruitment of AP-1 to the TGN in vitro (Mauxion et al., 1996). Many studies suggest the presence of novel coat structures (Narula and Stow, 1995; Stoorvogel et al., 1996). The targeting of lysosomal enzymes requires two distinct signals at the carboxyl terminus of the cytoplasmic domain of the cation-dependent-MPR that are different from tyrosine-based endocytosis motifs (e.g., Voorhees et al., 1995). The first is a casein kinase II phosphorylation site. This site is is required for high affinity binding of AP-1, needed for cation dependent-MPR sorting in the trans-Golgi network (Mauxion et al., 1996). However, an adjacent di-leucine motif not involved on AP-1 binding, is required for a downstream sorting event (Pond et al., 1995b). Exocytosis and transport to the cell surface The release of the secretory material occurs after the secretory vesicle and the plasma membrane fuse and the vesicle opens to the outside. This process is known as exocytosis. In regulated secretion, exocytosis occurs in response to an external stimulus that often triggers an increase in cytoplasmic Ca2+. Exocytosis http://www.albany.edu/~abio304/text/10part3.html (18 of 30) [3/5/2003 7:55:54 PM]
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in relation to synaptic transmission is discussed in Chapter 22. A role of lysosomes in exocytosis is discussed below (Section IV). Exocytosis also has a very important function in bringing needed integral proteins to the plasma membrane. The effect of the hormone vasopressin, depends on the insertion in the plasma membrane of the water channel AQP2 (see Chapter 19). One of the effects of insulin requires the recruitment of the glucose transporter GLUT4 from an intracellular pool to the cell surface (see Chapter 19). The specific targeting of secretory vesicles depends intimately on the complexes and structures that establish polarity. These are discussed in Chapter 11. Current evidence suggests that exocytosis occurs at specialized plasma membrane domains. Specialized domains in the plasma membrane, the so called-lipid rafts (see Chapter 4), have been shown to selectively assemble specific proteins. There is evidence for two different kinds of rafts. The most studied is recognized biochemically by being insoluble in the detergent Triton X-100. An additional kind of raft has been found which is soluble in Triton X-100 but insoluble in Lubrol WX ( Roper et al., 2000). A role of these domains in exocytosis is suggested by the finding that many proteins associated with vesicle fusion with the plasma membrane (see Chapter 11) have in fact been found in the Triton-X100 insoluble rafts, [e.g., tSNARE proteins (syntaxin 1A), synaptosomal-associated protein of 25 kDa (SNAP-25) the SNARE vesicle-associated membrane protein (VAMP2)]. However, VAMP2 was found associated with the Lubrol insoluble rafts (Chamberlain et al., 2001; Lang et al., 2001). Furthermore, syntaxins and (SNAP)25 were found to be concentrated in 200 nm cholesterol-dependent clusters where secretory vesicles preferentially dock and fuse (Lang et al., 2001). However, these rafts correspond to the Triton X-100soluble domains and do not co-localize with Triton X-100 insoluble raft markers. Cholesterol depletion disperses these clusters and sharply reduces the rate of secretion. Studies of cell surfaces of living cells using atomic force microscopy (see Chapter 1) (e.g., Schneider et al., 1997) and conventional transmission EM (Jena et al., 2003) have revealed structures that have been implicated in exocytosis. A variety of cells such as pancreatic acinar cells (e.g., Schneider et al., 1997), growth hormone secreting pituitary cells (Cho et al., 2002a) and chromaffin cells (Cho et al., 2002b) exhibit circular pits about 0.4-1.2 µm in diameter with depressions 100 to 150 nm in diameter and a depth of 15-30 nm. These were called fusion pores. The depressions increase in size with exposure to a secretagogue, however, their numbers remain unchanged (Cho et al., 2002d) suggesting that they are docking sites for secretory vesicles that fuse and release the secretory vesicular contents. An involvement of these structures in secretion was shown using immunogold EM techniques with antibodies to amylase establishing the depressions as sites of fusion in pancreatic acinar cells (Cho et al., 2002c) and growth hormone antibodies establishing the same for somatotrophs of the pituitary (Cho et al., 2002b). Furthermore, immunochemical studies demonstrated that t-SNAREs, NSF, actin, vimentin, α-fodrin and the calcium channels α 1c and β 3 are associated with the fusion complex providing the machinery for docking and release of secretory products from intracellular vesicles (Jena et al., 2003). Exocytotic vesicles are likely to be present in three distinct pools: those already docked at the plasma membrane, those forming an easily recruitable pool (moved to the surface using myosin as a motor) and a http://www.albany.edu/~abio304/text/10part3.html (19 of 30) [3/5/2003 7:55:54 PM]
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more slowly recruited pool (delivered to the easily recuitable pool using kinesin as a motor). This distribution in three distinct pools is supported by a study in which local burst of cytoplasmic Ca2+ were delivered from the medium to the cytoplasm of sea urchin eggs by reversibly damaging the cell surface using laser beams (Bi et al., 1997). The extracellular phase was made bright using rhodamine dextran. The exocytotic vesicles were identified from confocal fluorescence images (see Chapter 1) as bright disks occurring against a dark intracellular background. These images indicate vesicles that have become continuous with the external medium. Microinjected anti-kinesin antibody targeted to the motor domain of the molecule or kinesin tails (that act as inhibitors of kinesin) were without effect on an early exocytotic burst. However, they inhibited a slow phase exocytotic burst. The myosin inhibitor butanedione monoxime (BDM) inhibited both the slow and the fast release. A peptide derived from the Ca2+-calmodulin dependent protein kinase II was also microinjected. This peptide has the property of blocking the native enzyme (presumably by competing with it). The peptide also inhibited the two phases. This kinase has been implicated in facilitation of transmitter release in the squid giant synapse (Llinas et al., 1991). These findings suggest that kinesin and myosin may mediate two sequential events. The results are consistent with myosin affecting an event downstream from that controlled by kinesin (t1/2~10 s). Either block did not affect all of the injury-induced exocytosis. This BDM insensitive pool was found to be fastest and was referred to as immediate and would represent docked vesicles and lasted only for a few seconds (t1/2~2-4 s). These experiments suggest that regulated secretion involves three separate pools of secretory vesicles as proposed from other studies. As discussed in Chapter 22 (Section V B), unphosphorylated synapsin is attached to neurotransmitter vesicles and probably secretory vesicles of other cell-types so that they cannot participate in exocytosis. Synapsin is a fibrous molecule as long as 33 nm that is associated with the cell surface. Phosphorylated synapsin is unable to bind to the vesicles. Phosphorylation via Ca2+-calmodulin dependent protein kinase II would therefore be the trigger for exocytosis from the "immediate" pool. Exocytosis has been studied in some detail in mast cells of a mutant mouse (beige mouse) (Spruce et al., 1990). In these cells, exocytosis can be initiated by introducing guanosine-5-O-thiotriphosphate (GTPS) to the cell interior. This observation suggests an involvement of GTP-binding proteins, which have a very important role in vesicle fusion (see Chapter 11). These cells have extremely large secretory vesicles, 1-5 µm in diameter, facilitating electrophysiological studies of the plasma membrane and its fusion with the vesicles. Surface capacitance is proportional to membrane surface area, and, therefore, provides a convenient assay of fusion. Capacitance (C) is the charge (Q) per unit voltage (V), so that (C) = Q/V . The electrical studies were carried out with a pipette containing a salt solution in contact with the cell's interior. Upon fusion, there was an outward current, indicating the discharge of the vesicle's membrane potential suggesting the opening of a pore. This was followed by a capacitance increase of the mast plasma membrane. The capacitance increase occurred stepwise, representing the fusion of individual vesicles. During this period (within the first 100 µs) the conductance of the membrane also increased, first by a few hundred pS and then by progressively higher conductances, indicating an enlargement of the pore. The approximate pore diameter calculated from the conductance is 2 to 2.5 nm.
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A possible connection between a family of proteins, the annexins, and exocytosis is intriguing (see Creutz et al., 1992). Annexins form part of a family of proteins that, in the presence of Ca2+, foster the aggregation of secretory vesicles (usually studied in adrenal medullary chromaffin granules). Considering the pore behavior exhibited by cells undergoing exocytosis, a particularly interesting feature of annexin V is its probable tertiary structure, deduced from the amino acid sequence. These reconstructions are compatible with the presence of a channel. In addition, this annexin and annexin VII (synexin) have been shown to form voltage sensitive channels when reconstituted in planar lipid bilayers (Rojas et al., 1990; Karshikov et al., 1992). Annexin II (also known as calpactin) forms a tetramer consisting of two molecules of annexin and two of another protein (p10) (Glenney et al., 1986). Chromaffin granuleaggregation of this complex requires as little as 1 µM Ca2+. As discussed later (Chapter 22), the local concentration of Ca2+ may reach even higher levels. Other molecules similar to p10 may be associated with other annexins. Addition of cis-unsaturated fatty acids increases fusion dramatically (Creutz, 1981). This may be relevant because arachnoic acid is liberated by membranes when secretion is stimulated. An involvement of annexin II in secretion is shown by immunoelectronmicroscopy using colloidal gold, a technique which shows a localization of annexin in chromaffin cells activated for secretion (Nakata et al., 1990). The annexin molecules were found to be associated with the inner face of the plasma membrane and to be conspicuous between plasma membrane and adjacent chromaffin secretory vesicles. Annexins also have a role in endocytosis, as suggested by the requirement of annexin VI in an in vitro study of the formation of endocytotic vesicles. Transport to the cell surface has been studied with a cell-free system from cells infected with influenza virus (Woodman and Edwardson, 1986). In this study, the transfer of the viral neuraminidase to the plasma membrane was followed. To detect its arrival, an acceptor fraction was prepared by binding [3H]sialic acid-labelled Semliki Forest virus to cell surfaces before cell rupture. The observed production of free [3H]sialic acid served as an assay of the fusion of exocytotic vesicles containing the enzyme with the labelled acceptor preparation. This reaction requires ATP and a variety of proteins. Most of the evidence discussed so far, indicates that the transport between the Golgi and the cell surface involves small vesicles. However, there are studies that implicate larger structures perhaps for certain special cases. The use of VSVG-green fluorescent protein chimeric protein (see Chapter 1) allowed following the protein traffic through the various compartments of the secretory pathway (Hirschberg et al., 1998). This approach, applied to the passage from the ER to the Golgi, is discussed in Section III. A temperature mutant was used where the proteins misfold and are retained in the ER at 40oC. However, when shifted to 32o, they are moved synchronously to the Golgi complex and from there to the plasma membrane.This study found that the protein molecules move from the Golgi complex in large irregular tubular structures that detach from the Golgi in a cytochalasin B sensitive manner, suggesting an involvement of actin in this process. The movement to the periphery was in rounder structures and was sensitive to nocadazole, http://www.albany.edu/~abio304/text/10part3.html (21 of 30) [3/5/2003 7:55:54 PM]
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suggesting that it takes place on microtubular tracks. Photobleaching experiments outside the Golgi region indicate that 60% of the traffic is in these large structures. Similar experiments (Toomre et al., 1999; Polishchuk et al., 2000) reach similar conclusions. In the experiments of Polishchuk et al (2000), both light and immunoelectronmicroscopy were used to follow individual vesicles in mammalian cells in culture. The fusion of large vesicles to the plasma membrane was found to be direct. The kinetics of the discharge of secretory vesicles can also be followed using fluorescent proteins. The cargo proteins can be labelled by the transfection with vectors containing DNA coding for protein-GFP conjugates (see Chapter 1). The lumen of granules can also be labelled with fluorescent weak basic dyes that accumulate inside acid compartments (e.g., acridine orange). Similarly, a membrane component can be visualized when an integral protein is conjugated to GFP (e.g., phogrin-GFP; Tsuboi et al., 2000). Events occurring at the cell surface such as exocytosis have been studied using confocal fluorescence microscopy (see Chapter 1 ) as well as evanescent field fluorescence microscopy (also referred to as total internal reflection microscopy). This latter technique is ideally suited for observing events at the cell surface (see Chapter 1). The membrane of secretory granule was found to remain intact at the cell surface for several seconds and in some cases it was endocytized after the discharge ( Tsuboi et al., 2000). Secretory vesicles from neuroendocrine cells (rat pituitary prolactin secreting cells) and their components have been shown to be recovered in endocytotic vesicles after exocytosis ( Angleson et al., 1999). In constitutive exocytosis of epithelial cells many of the secretory vesicles were found to be tubular as well as spherical ( Schmoranzer et al., 2000; Toomre et al., 2000) . In addition, the membrane of the tip of the tubular containers fused only temporarily with the plasma membrane and then closed their fusion pore. IV. ALTERNATIVE SECRETORY PATHWAY AND ALTERNATIVE MECHANISMS There are many indications that the translocation and targeting of proteins do not always follow the mechanisms discussed in most of this chapter. Several proteins are secreted from cells although they lack a typical signal sequence required for entry into the ER. Furthermore, pharmacological agents, such as monensin or brefeldin A, which perturb Golgi function, do not interfere with the release of these proteins (e.g., Florkiewicz et al., 1995). The possibility that they are exported via a different mechanism from the Golgi pathway is likely, and is referred to as "nonclassic secretion". It is not known whether all these proteins follow the same pathway. Cleves et al. (1996) explored the alternative pathway by expressing in yeast, the small (14 kDa) galactose binding mammalian lectin, galectin-1. The transfected yeast secreted galectin-1 even when the conventional secretory pathway was blocked. Protein kinases and phosphatases have a central role in the regulation of cell activity, including the activity of enzymes, the processes of transcription and translation, and various events accompanying the cell cycle. Although these enzymes must act in a very specific manner, in vitro they are very unspecific, http://www.albany.edu/~abio304/text/10part3.html (22 of 30) [3/5/2003 7:55:55 PM]
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reacting even with non-physiological substrates. Apparently, the specificity is determined by the precise localization at special sites (see Hubbard and Cohen, 1993). The localization depends on the presence of targeting domains in the enzyme molecule or, in the case of some heteromeric enzymes, in targeting subunits. The translocation of polypeptides or proteins through membranes, may involve proteins of the ATPbinding cassette (ABC) superfamily (see Higgins, 1992; Kuchler and Thorner, 1992). These transporters are present in many intracellular membranes and participate in the transport of many substrates including drugs, ions, metabolites, peptides and proteins. In Saccharomyces cerevisiae, 27 genes have been identified that encode proteins containing the ABC cassette. ABC transport systems are involved in transport of peptides into the ER lumen, part of the immunological system (see Williams et al., 1996). The ER degradation of defective protein involves cytoplasmic proteasomes, suggesting that the ER membrane can translocate these proteins to the cytoplasm (Hayes and Dice, 1996; Cuervo and Dice, 1996). The uptake is powered by ATP hydrolysis. Several studies suggest that lysosomes are involved in exocytosis where they function as regulated secretory vesicles. The increase in intracellular Ca2+ to 1-5 µM initiates the exocytosis of lysosomes in several mammalian cell lines (Rodríguez et al., 1997) indicating that this might be a general phenomenon (for a general discussion of the role of Ca2+, see Chapter 7 and for a role in exocytosis in the nervous sysem see Chapter 22). A role of lysosomes in exocytosis is most prominent in hematopoietic cells (see Stinchcombe and Griffiths, 1999). Generally, the targeting of the secreted products follow the usual lysosomal pathway (see Page et al., 1998). However, at least in some cases, in hematopoietic cells, there are distinct pathways and targeting apparently to specialized lysosomes for some of the proteins (Bossi and Griffiths, 1999). In part, the role of lysosomal exocytosis might be to dispose of discarded materials from the cell (e.g., Swank et al., 1998), as long recognized for certain protozoans such as Paramecium. The exocytosis elicited by such low calcium concentration is likely to involve a Ca2+-binding protein, binding with high affinity. Such a protein could be synaptotagmin (Syt), a protein that has been implicated in neurotransmitter release (see Chapter 22 and Sudhof and Rizo, 1996). Syt proteins constitute a family of at least twelve isoforms with a unique amino-terminal domain and a conserved carboxy-terminal domain (see Schiavo et al., 1998; Craxton and Goedert, 1999). Although most Syt found so far are in neuronal tissue, the appropriate mRNAs are detected at low level elsewhere (Butz et al., 1999; Craxton and Goedert, 1999). The isoform most likely to play a role in lysosomal secretion is Syt VII whose action (binding to syntaxin and phospholipids) has the appropriate Ca2+-dependence (Li et al., 1995). Syt VII was found in dense lysosomes in rat kidney fibroblasts and GFP-SytVII is targeted to lysosomes after transfection. Antibodies against a Syt VII domain and fragments of the same domain inhibit lysosomal exocytosis (Martinez et al., 2000). http://www.albany.edu/~abio304/text/10part3.html (23 of 30) [3/5/2003 7:55:55 PM]
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V. TARGETING mRNA A variety of cytoplasmic proteins are distributed unevenly in the cytoplasm. This uneven distribution is likely to play an important role in development (see Chapter 2) and in the function of a variety of cells, for example, neurons (e.g., Kuhl and Skehel, 1998). We have seen that many proteins are transported in vesicles that are targeted to various locations. Free protein molecules can also be targeted directly. However, the unequal distribution of some proteins is the result of the transport of mRNA and not that of the translated protein. So far, as many as 90 mRNAs have been found to be localized to specific regions in the cell (e.g., see Jansen, 2001). mRNA localization is probably more efficient than the transfer of protein molecules since a single mRNA molecule can give rise to many protein molecules. The protein generated in place will be in very high concentrations. In situ translation has also the advantage of assembling cotranslationally large complexes. In embryonic development, the location of mRNA appears to play an important role in establishing gradients of proteins that determine cell fate. For example, in Xenopus and Drosophila oocytes (see Gavis, 1997) the mRNA distribution establishes a special protein distribution in the animal and vegetal poles. The unevenly distributed proteins and RNAs are then further fractionated into separate cells by cell divisions. These components could then play a role in differentiation, perhaps by being involved in gene expression. In axons and dendrites, the mRNA of microtubule associated proteins (MAPs) is targeted directly and it has a role in determining the packaging of microtubules (Chen et al., 1992), thereby playing a primary role in the laying down of structure. The localization of specific mRNAs appears to be a general phenomenon. In polarized rat fetal enterocytes, the mRNAs was shown with light microscopic techniques (by hybridization with the appropriate cDNA; see Chapter 1) to localize in the same region of the cell as the corresponding enzyme (shown immunologically) (Rings et al., 1992). The mRNA of the β-subunit of F1-ATPase was found by electronmicroscopy [using an hybridization and recognition of the cDNA with an immunogold technique (see Chapter 1)] to localize in clusters, in close proximity to mitochondria whereas the mRNA for the αsubunit was found to be dispersed throughout the cytoplasm. (Egea et al., 1997). The mRNA is thought to form complexes with certain proteins in the nucleus and then to be transported in the cytoplasm in the form of very large ribonucleoprotein particles. In oligodendrocytes, the transport particles or mRNA granules are as large as 0.7 µm in diameter. Besides containing different mRNA, the granules have been found to contain components the protein synthesizing machinery (e.g., see Barbarese et al., 1995). In neurons, the transport of RNA also occurs in granules which contain various proteins, mRNAs and densely packed clusters of ribosomes. The granules are not active in translation and therefore can be regarded as storage units. Interestingly, when the neuron is depolarized, many mRNAs, including those involved in neuronal plasticity (see Chapter 22), leave the granules and are found in polysomes where translation can take place (Krichevsky and Kosik, 2001). Plants have been shown to transport mRNA over long distances through their vascular system ( Xoconosle-Càzares et al., 1999) and the transfer of mRNA from one cell to another may well occur in other systems.
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How can mRNA be targeted to special areas? Conceivably, the mRNA or granules containing the mRNA could diffuse randomly and be trapped at a particular location, or they could be targeted through the cell's motor system along cytoskeletal elements. The direct visualization of the transport using fluorescently labelled mRNA injected into cells, could offer important insights. Ainger et al., (1993) injected fluorescently labelled mRNA encoding myelin basic proteins into oligodendrocytes. The mRNA was found in particles that undergo unidirectional transport, suggesting a mechanism similar to that of the transport of vesicles. Furthermore, the particles moved on either microtubules or actin filaments at a rate similar to that of vesicles. The mRNA particles were found in close proximity to microtubules, suggesting that these are responsible for the transport. During Drosophila oogenesis, bcd-mRNA coding for the bicoid protein (Bcd) becomes localized to the anterior pole of the oocytes ( Schnorrer et al., 2000). Bicoid is a protein whose gradient determines the anterior pattern of the Drosophila embryo. The transport of the bcd-mRNA to that location is mediated by a protein with an RNA binding domain (Swa). The transport is powered by the microtubular motor, dynein (see Chapter 24) which also binds to Swa. The directed transport of RNA involves both the actin network for short distances and the microtubules for long range transport (see Palacios and St. Johnston 2001). For example, β-actin mRNA and the binding protein Zipcode (ZBP1) are found to be transported in granules in a microtubule dependent pathway in axons ( Zhang et al., 2001). Actin has also been found to be involved in mRNA transport, for example, in the movement of mRNA to the leading edge of fibroblasts (Hill and Gunning, 1993). ZBP1 proteins have an additional role in determining RNA stability and translational control and are found mostly in the cytoplasm, although they posses a nuclear localization and an export sequence (see Chapter 5, Sections I B and I C), suggesting that they cycle between the two sites. Another protein of 92 kDa (ZBP2) ( Gu et al., 2002) has been found to bind to actin mRNA . ZBP2 is predominatly nuclear but has also been found in the leading edge of fibroblasts or neuronal growth cones. The two ZBP proteins (ZBP1 and 2) may function in tandem where ZBP1 receives the mRNA from ZBP2 and transfers it to the appropriate location These observations can be used to construct a model in which an RNP particle is formed, translocated through the microtubular or actin system of the cell, anchored at a specific location, and translated. Obviously once synthesized, the proteins must also be anchored to maintain their localization. As might be expected, each mRNA docks by a different mechanism. In some cases the translation of the protein coded by the mRNA is needed for docking. In some cases, short untranslated RNA (UTRs) sequences complementary to the small portions of the mRNA are thought to anchor the mRNA. Certain localization sequences in the 3'UTR or the mRNA are required for targeting and anchoring and they require mRNA binding proteins (see Bashirullah et al., 1998). Presumed targeting signals include several hundred nucleotides in some of the mRNAs, suggesting that there may be multiple signals, each mediating different localization steps, as in the mRNA of the oskar gene (see Kim-Ha et al., 1993). The oskar gene is required in Drosophila for posterior body patterning and germ cell determination. How could the mRNA be anchored to its target? There are suggestions that the mRNA is attached to
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cytoskeletal elements, because it is not solubilized by the detergent Triton X-100 (Yisraeli et al., 1990). Furthermore, actin is likely to be involved because the mRNA is dispersed after cytochalasin treatment. Cytochalasin, a fungal product, is known to interfere with actin polymerization. In summary, mRNA-containing particles are targeted to structures where they remain anchored. Similarly, the proteins, newly translated at these locations, remain anchored. The microtubular system seems to be involved in the transport of the mRNA particles, whereas its anchoring is likely to involve actin. VI. THE TRAFFIC OF LIPIDS In Chapter 4, we discussed lipids in relation to the structure and function of cell membranes. Aside from their insulating and structural roles, recent research has recognized a wide range of lipid functions. Like many glycoproteins, certain glycolipids at the cell surface play an important role in cell recognition. Lipids can also serve as anchors for proteins. Some membrane lipids act as precursors of second messengers. Furthermore, lipid composition is also thought to play a role in the regulation of exocytotic vesicle transport. We have already examined (section III) how newly synthesized proteins transported in vesicles are targeted to different cell compartments or the plasma membrane. These processes result in a flow of membranes toward the cell surface. The processes culminating in exocytosis produce a flow of membranes, which would augment the surface area of the plasma membrane. Some of this flow must be compensated for by an inward flux of membranes, so that they are primarily recycled in endocytosis and subsequent events. Although we concentrated on the protein components of the membrane, obviously the same problems of targeting and maintaining the characteristics in each compartment applies to the lipid components of membranes as well. In Chapter 4, we saw that the dynamics of the plasma membrane cause it to maintain an asymmetry, so that the external leaflet differs from the inner leaflet of the lipid bilayer. This section will examine what is known about the processes responsible for the targeting of lipid components to the various subcellular compartments (generally in their membranes) and the plasma membrane. The fate of phospholipids has been followed using several approaches. Arrival at a target organelle can be followed by monitoring the metabolic conversion of the lipid. The use of fluorescent phospholipids offers a more direct and general technique. Phospholipid analogues containing dipyrromethene difluoride (BODIPY) have been used successfully as lipid tracers (Pagano et al., 1991). The sorting of cholesterol has been difficult to follow because few convenient techniques are available. However, pulse labelling with [3H] acetate in cells cultured in LDL-deficient medium will label newly synthesized cholesterol in minutes. Similarly, [3H]cholesteryl linoleate can be incorporated into LDL. The acetate labelling would reveal the fate of newly synthesized cholesterol, whereas the cholesteryl labelling would reveal the fate of exogenous cholesterol. Generally, in these studies the membranes of the various vesicles or the plasma membranes were isolated and the cholesterol extracted. http://www.albany.edu/~abio304/text/10part3.html (26 of 30) [3/5/2003 7:55:55 PM]
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A. Glycerophospholipids PC, PE, PS and PI are synthesized in the cytosolic surface of the ER (Dennis and Vance, 1992). They rapidly distribute laterally in the ER membrane and they can flip to the inner leaflet (Devaux, 1993). Newly synthesized PC is rapidly transferred to the plasma membrane and the outer mitochondrial membrane, possibly by transfer proteins that would interact with lipids in the membrane, removing them from one location and delivering them to another (Kaplan and Simoni, 1993). PE is transferred to the plasma membrane more slowly (Yaffe and Kennedy, 1983). PI synthesized in the ER, is phosphorylated by PI- and PIP-kinases en route to the plasma membrane and the nucleus (Helms et al., 1991; Banfi et al., 1993). The asymmetric distribution of glycerolipids in the plasma membrane is thought to be produced by the aminosphingolipid translocase, an ATP-dependent pump that transfers PS and PE to the inner leaflet of the plasma membrane (Berr et al., 1993, Devaux, 1993). However, the asymmetry is not easily explained by models based on the translocase activity alone. B. Sphingolipids Ceramide, the precursor of all complex sphingolipids, is synthesized in the ER. After being transported to the Golgi, ceramide is converted into sphingomyelin (SM) (Kallen et al., 1993) and glycosphingolipids (Collins and Warren, 1992; Moreau et al., 1993). In the previous section we saw that proteins that are constitutively secreted proceed via vesicles from the ER and through the Golgi system, to eventually reach the cell surface. It follows that in this case these proteins and the membrane lipids must move together. The cotransport of glycolipids and glycoproteins has been shown in in vitro systems, and the rate of this transport is consistent with bulk flow (Young et al., 1992). Experiments supporting this view have shown that inhibitors of sphingomyelin biosynthesis slow down intracellular movement (Rosenwald et al., 1992). Taken together, this information strongly suggests that both glycoproteins and sphingolipids within the Golgi system are transported in vesicles. The vesicles are then delivered to the plasma membrane. Other information indicates that from the cell surface they are recycled by endocytosis (e.g., Kok et al., 1992) and may be degraded in lysosomes (van Eichten and Sanhoff, 1993). Different sphingolipids are targeted to different cell surfaces (see van Meer, 1993). As already discussed, at the surface they may form special lipid domains, which may play a role in the distribution of proteins anchored by glycosyl-phosphatidyl inositol (GPI). Although a sharing of the pathway and the targeting by glycoproteins and glycolipids is likely, other http://www.albany.edu/~abio304/text/10part3.html (27 of 30) [3/5/2003 7:55:55 PM]
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lipids may be transferred by a different pathway, as discussed below for cholesterol and glycerophospholipids. Sphingolipids can be redistributed in polarized cells (see Chapter 11) by transcytosis. A cell system that has been found useful in the study of polarized cells is that of polarized hepatocytes, HepG2. In these cells, the apical and the basolateral domains are well defined. The fate of lipids can be followed using fluorescently tagged sphingolipids (e.g., Pagano and Sleight, 1985). The localization of glucosylceramide (GC) and SM is distinct. SM is localized in the basolateral surface, whereas GC is localized in the apical surface. When the cells were incubated in the presence of the labelled compounds (van IJzendoorn et al., 1997) both lipids were found in punctate fluorescent bodies presumed to be vesicles and, in addition, at the apical (in this case the bile canicular surface) and the basolateral cell surface. Treatment of the cells with bovine serum albumin in a saline solution removed the fluorescence from the basolateral but not the apical surface. After incubation, the lipids were found in their characteristic localization. SM-was rapidly transferred by transcytosis from the apical to the basolateral surface. The transfer system was found not to involve the Golgi apparatus, but to occur via vesicles seen below the apical surface. C. Cholesterol Mammalian cells can acquire cholesterol from extracellular sources in the form of low density lipoprotein (LDL), which is taken up by receptor-mediated endocytosis (Chapter 9). The cholesteryl ester core is hydrolyzed to free cholesterol in lysosomes. [3H] cholesterol reaches the plasma membrane within minutes of the hydrolysis of [3H] cholesteryl linoleate (Brasaemle and Attie, 1990). The Golgi system is thought to be involved in the movement because the cisternae are enriched in cholesterol when normal cells are incubated in LDL for long periods (Blanchette-Mackie et al., 1979). Furthermore, the transport from lysosomes to the plasma membrane is monensin-sensitive, implicating vesicular transport. There is no direct information on the distribution of cholesterol between the two leaflets of the lipid bilayer of the plasma membrane. However, cholesterol is known to interact primarily with sphingomyelin and, therefore, may be predominantly in the outer leaflet. In mammals, the enzymes that synthesize cholesterol reside in the ER (see (Reinhart et al., 1987). However, some studies have also implicated peroxisomes (Krisans, 1992). From the the ER, cholesterol is translocated to other cellular destinations, in most cells the plasma membrane being the major recipient (65-90% of the cellular cholesterol, Lange et al., 1989; Warnock et al., 1993). In hepatocytes, cholesterol is needed for lipoprotein and bile acid synthesis, both initiated in the ER. In steroid-producing cells, cholesterol may be transported directly to the mitochondria, the site of synthesis of the hormones. Transport to the plasma membrane is rapid and involves lipid-rich vesicles (see Liscum and Munn, 1999). The transfer is energy dependent and is blocked when the cells are incubated at 15oC. At this temperature the cholesterol accumulates in vesicles. However, the Golgi apparatus is probably not involved and the vesicles are distinct from those involved in secretion. For example, VSV-G protein and cholesterol are in http://www.albany.edu/~abio304/text/10part3.html (28 of 30) [3/5/2003 7:55:55 PM]
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different vesicles (e.g., Urbani and Simoni, 1990). Furthermore, monensin or Brefeldin A, which disrupt the Golgi system and block the delivery of VSV-G protein to the surface, are ineffective in blocking the cholesterol movement. Other agents that interfere with the cytoskeletal organization, and therefore block vesicular traffic, also do not interfere with cholesterol transport. There is some evidence that a sterol carrier is involved in cholesterol transport (protein-2, SCP-2 of 13.2kDa; Puglielli et al., 1995) and furthermore that there are two separate pathways for transport of cholesterol inside the cell and that the SCP-2 pathway is the major normal pathway for the transport of cholesterol. In normal cells, cholesterol transfer was found to be rapid, cytoskeleton-independent, and Golgi-independent in normal cells. However, in SCP-2-deficient cells it was slower, required a functioning cytoskeleton and Golgi. In these experiments, the need for the various mechanisms was tested using brefeldin or mononesin to disrupt Golgi, colchicine to disrupt microtubules and cytochalasin B to disrupts actin filaments. An involvement of caveolin (see Chapter 9) in cholesterol transport is also indicated (Uittenbogaard et al., 1998). Similar experiments confirm some these findings (Heino et al., 2000). The transport of newly synthesized cholesterol were compared to that of influenza virus hemagglutinin (HA) from the endoplasmic reticulum to the plasma membrane. Brefeldin A which disrupts the Golgi apparatus was found to completely block the passage of HA. In contrast, the cholesterol transport was not affected. However, the exposure to nocodazole which disrupts microtubules was found to block the transport of both cholesterol and HA, contradicting some of the earlier findings. The transport of cholesterol was studied in more detail and found the incorporation into low-density detergent-resistant membranes assumed to be specialized membrane elements or rafts (see Chapter 4). Interference with cholesterol transport (by lowering the temperature) decreased the amount of cholesterol in the presumed rafts. The results suggest that the transport of cholesterol involves membranes containing rafts and possibly caveolae (see Chapter 4 and Chapter 9) as suggested by an involvement of caveolin. SUGGESTED READING Bannykh, S.I., Nishimura, N. and Balch, W.E. (1998) Getting into the Golgi, Trends in Cell Biol. 8:21-25. (Medline) Brodsky, J.L. (1998) Translocation of proteins across the endoplasmic reticulum membrane, Int. Rev. Cytol. 178:277-328. (Medline) Ellgaard, L., Molinari, M. and Helenius, A. (1999) Setting the standards: quality control in the secretory pathway, Science 286:1882-1888. (Medline) Farquhar, M.G. and Palade, G. (1998) The Golgi apparatus: 100 years of progress and controversy, Trends in Cell Biol. 8:2-10. (Medline)
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Higgins, C.F. (1993) Introduction; the ABC transporter channel superfamily-an overview, Seminar in Cell Biol. 4:1-5. Kreis, T.E., Goodson, H.V., Perez, F. and Rönnholm, R. (1997) Golgi apparatus-cytoskeleton interactions, in The Golgi Apparatus (ed. Berger, E.G. and Roth, J.), Birkhäuser Verlag, Basel, Boston, pp.179-193. Marsh, B.J. and Howell, K.E. (2002) The mammalian Golgi--complex debates, Nature Rev. Mol. Cell Biol. 3: 789-795. Martin, T.F.J. (1997) Stages of regulated exocytosis, Trends in Cell Biol. 7:271-276. Plemper, R.K. and Wolf, D.H. (1999) Retrograde protein translocation: ERADication of secretory proteins in health and disease, Trends Biochem. Sci. 24:266-270. (Medline) Rambourg, A. and Clermont, Y. (1997) Three-dimensional structure of the Golgi apparatus in mammalian cells, in The Golgi Apparatus Birkhäuser Verlag, Basel, Boston, Berlin, (ed. Berger, E.G. and Roth, J.) p.37-61. Rapoport, T.A., Rolls, M.M. and Jungnickel, B. (1996) Approaching the mechanism of protein transport across the ER membrane, Curr. Opin. Cell Biol. 8:499-504. Rothman, J.E. and Wieland, F.T. (1996) Protein sorting by transport vesicles, Science 272:227234.(MedLine) Schatz, G. and Dobberstein (1996) Common principles of protein translocation across membranes, Science 271:1519-1526. (Medline) Valee, R.B. and Sheetz, M.P. (1996) Targeting of motor proteins, Science 271:1539-1544. WEB RESOURCES ER to Golgi transport: Quick time movie sequence (see Presley et al., 1997) http://dir.nichd.nih.gov/CBMB/pb1labob.html REFERENCES Search the textbook
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Chapter 10: References
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11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport
11. Biosynthesis and Cytoplasmic Trafficking: Membranes, Vesicles and Intracellular Transport I. Transport Vesicles A. Assembly of Coats, Budding and Formation of Vesicles Adaptors and receptors Involvement of phosphatides B. Targeting of Proteins and Vesicles; SNAREs C. Fusion NSF SNAPs D. The GTPases E. ER and Golgi Transport ER to Golgi Transport Transport from Golgi Stacks II. Recognition of Targets A. Cell Polarity Maintenance of polarity Components of tight junctions B. C. D. E. F.
Targeting of Plasma Membrane Proteins Targeting in Secretion and Transcytosis Transport of the Vesicles Recycling of the Plasma Membrane Formation of Lysosomes and Storage Vesicles Lysosomes Secretory storage granules
G. Synaptic Vesicles
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Suggested Readings References Web Resources Back to List of Chapters Understanding the intricate details of the interactions between membranes and membrane bound compartments is a challenging task. Electronmicroscopy using immunological methods together with genetic approaches and molecular studies of reconstituted systems are beginning to provide the needed framework of information. This chapter focuses on some of these processes. I. TRANSPORT VESICLES As we saw in Chapter 10, most intracellular transport is thought to be mediated by small vesicles, although an alternative mechanism for the transfer from the VTCs to the cis-Golgi (see Chapter 10, Section III) is likely and, in addition, transport between Golgi stacks may involve alternative mechanisms (see Chapter 10, Section IIIC). The transport can occur in either direction: from the ER to the periphery of the cell or in the opposite direction. The transport processes are discussed in relation to endocytosis in Chapter 9, in relation to the motility of cell components in Chapter 23 and the discussion of neuronal transport can be found in that Chapter and in Chapter 24. Chapter 9 discussed the involvement of clathrin in the formation of endocytotic vesicles. Some other transport vesicles are also clathrin-coated. They carry proteins originating from the trans-Golgi system (see Brodsky, 1988). They include transport vesicles destined to the lysosomes (e.g., SchulzeLohoff et al., 1985) containing acid hydrolases and mannose-6-phosphate receptors, and those destined to storage vesicles containing densely packed secretory products (see Section II F, below). Vesicles coated with proteins other than clathrin (non-clathrin coated) represent another set of transport vesicles. These are involved in the translocations within the Golgi stack, between the ER and the Golgi, from the TGN in the constitutive secretory pathway and in retrograde transport (e.g., from Golgi to ER) The present conventional view maintains that non-clathrin coated vesicles are involved in the transport between ER and Golgi and between Golgi stacks. The transport from the ER to the cisGolgi is carried out by COPII vesicles (see Barlowe, 1998). In contrast, retrograde transport from Golgi to ER takes place in coatomer protein (COPI) coated vesicles (Cosson and Letourneur, 1997; Gaynor et al., 1998) for proteins either with KKXX, KKXX-like (see below) or the KDEL amino acid motif (see Chapter 10) but not by glycosylated Golgi enzymes or Shiga toxin (Girod et al., 1999). Forward and retrograde transport between Golgi stacks is also thought to be mediated by COPI (Orci et al., 1997). In this latter case, electron microscope immunocytochemistry shows that both anterograde-cargo and retrograde-cargo are present in separate COPI-coated vesicles budding from all stacks of the Golgi. Packaging of anterograde and retrograde cargo into separate vesicles can also be demonstrated in vitro even when budding is driven by highly purified coatomer and a recombinant, small GTPase, ARF. http://www.albany.edu/~abio304/text/chapter_11.html (2 of 41) [3/5/2003 7:56:56 PM]
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All in all, at this time four different coat proteins are clearly defined, including two clathrin proteins and COPI and COPII (listed in Table 1, Rothman and Wieland, 1996). In Table 1, the two different clathrin coats are shown to function in conjunction with different adaptor molecules (see below). All these coats are associated with GTP-binding proteins (or GTPases) (see below). Their origin and initial destination are listed in the fourth column. Besides the four coat proteins listed in the Table, more coat proteins have been demonstrated and others are likely to be revealed by further studies. A lace-like coat has been described in the TGN (Ladinsky et al., 1994; Narula et al., 1995), a neuronal variant of COPI has been found (Newman et al., 1995) and another is thought to be associated with endosomal vesicle traffic (Whitney et al., 1995). A distinct coating for vesicles involved in endosome-to-Golgi retrograde transport of sorting receptors is operative in yeast (Seaman et al., 1998). The vesicles are slightly larger than the COPI vesicles. In Saccharomyces cerevisiae, a novel vesicle 30-40 nm in diameter transferring fructose-1,6-bisphosphatase to the vacuole for degradation, has been described (Huang and Chiang, 1997) . There is growing evidence for other coat proteins similar to clathrin. A gene (HC22) has been identified with the potential of coding for a protein similar to the clathrin heavy chain. mRNA corresponding to HC22 is expressed predominantly in skeletal muscle and alternative transcripts of H22 are expressed in a tissue specific fashion (see Brodsky, 1997). Table 1. Coat proteins or vesicle shuttles (from Rothman and Wieland 1996, reproduced by permission)
Type of coated vesicle
Subunits of coat
GTPase
Origin-destination
AP-1 clathrin
Clathrin, AP1 adaptor
ARF
TGN-prelysosomes
AP-2 clathrin
Clathrin, AP2 adaptor
ARF?
Plasma membraneendosomes
COPI
Coatomer (COPI proteins)
ARF
ER-Golgi; bidirectional within Golgi; Golgi-ER
COPII
COPII proteins
SAR
ER-Golgi
It is now recognized that the process of targeting and interaction between components occurs in many steps starting from the translocation of vesicles to their target and ending with their fusion to their target. The translocation of the vesicles (see Section IID and Chapter 24, section on cytoplasmic dynein and kinesin) has not been elucidated in detail. Upon arrival processes involved in membrane recognition (tethering) leaves the two sets of membranes at some distance from each http://www.albany.edu/~abio304/text/chapter_11.html (3 of 41) [3/5/2003 7:56:56 PM]
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other. The binding of SNAREs (docking) leaves the membranes in close proximity and is followed by fusion. One kind of SNARE, SNAREv,resides on the surface of the vesicle being transported and SNAREt resides on its membrane target (see Section C). Tethering is thought to be the primary step in determining specificity. Rab GTP-binding proteins have been implicated as well (see Pfeffer, 1999; Novick and Zerial, 1997; Zerial and McBride, 2001). However, a variety of proteins and protein complexes have been found to play essential roles in targeting and to be specific for individual steps. Before fusion, priming events must take place (Klenchin and Martin, 2000) and a fusion trigger, most frequently Ca2+, is needed (e.g., Heidelberger et al., 1994; Peters and Mayer, 1998; Beckers and Bach, 1989; Colombo et al., 1997). The priming includes ATP-dependent steps, such as the NSF-mediated priming of SNARE protein complexes, the ATP-dependent synthesis of phosphoinositides, and protein kinase-mediated protein phosphorylation. The protein munc 13 is also involved in the priming. The intracellular transport of proteins in vesicles raises a number of issues: (a) how the appropriate proteins are selected in the formation of specific vesicles, (b) the nature of the mechanism of budding, (c) how the vesicles are targeted unidirectionally and in an orderly fashion, and (d) the mechanism of fusion of the targeted vesicles. Unfortunately, only partial answers are known. The first issue, (a), was discussed in Chapter 10. The formation of vesicles (Section A), their targeting (Section B) and fusion to their target membranes (Section C) and the special role of the GTPases (Section D) will be discussed first, followed by an examination of some of the details of the various pathway segments (Section E). A. Assembly of Coats, Budding and Formation of Vesicles This and the following sections will be referring to several components: NSF, SNAP and SNARE. As in many other cases in this book, these acronyms stand for certain appellations that are only of historical importance. NSF stands for N-ethyl maleimide sensitive factor, SNAP for soluble NSFattachment protein and SNARE for SNAP receptors. The acronysms will be used in the text that follows. The process of vesicle formation and their delivery to a target are summarized in the diagram of Fig. 1 which will serve as the basis of this discussion. This representation reflects the bare bones, socalled SNARE hypothesis. As discussed below some of the present evidence indicates that SNAREs are responsible for docking and act downstream from tethering. The assembly described in Fig. 1A typified for COPI vesicles, occurs stepwise with (i) the binding of the small GTP-GTPase first (see Section E) (in this case ARF; open spheres: GDP-GTPase; closed spheres: GTP-GTPase) where GDP has been replaced by GTP (i-ii), followed by activation by GTP and formation of coated bud by recruitment from the cytoplasmic pool. The pinching off of the vesicle requires acyl-CoA binding (not shown, Ostermann, et al., 1993) and the GTPase, dynamin (not shown, e.g., Takei et al., 1995; Hinshaw and Schmid, 1995). Dynamin is thought to assemble around the neck of endocytotic invagination and participates in pinching off the vesicles (see below and Chapter 9). Subsequently, the hydrolysis of GTP induces the detachment of the GTP-binding protein, now bound to GDP (Melançon et al., 1987; Orci et al., 1989) (iii-iv). Finally, dissociation of the coat follows (v) . The
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steps diagrammed in Fig. 1B and C are discussed in more detail below. Basically, the uncoated vesicles bind to the target protein on the target membrane (part B of the diagram). The binding is mediated by the v-SNARE and its cognate, t-SNARE, in the target membrane (see Section C). The fusion (part C of the diagram) requires ATP, GTP, AcylCoA, the NEM-sensitive-factor (NSF), SNAPs and other factors. NSF catalyzes the hydrolysis of ATP which disrupts the SNARE complex (see Section C), and initiates fusion. This model is supported by experimental observations: (a) the presence of coated vesicles coincides with biochemically defined transport (Orci et al., 1986); (b) coated vesicles containing cargo proteins, in this case G-VSV-protein, transfer from donor to acceptor stacks of the Golgi and can be trapped at this stage by the presence of GTPγS (Orci et al., 1989); (c) after a GTPγS block is reversed the vesicles disappear; (d) budding requires fatty acylCoA; (e) addition of NEM (or AlF4-, which acts as a phosphate analog) causes a buildup of uncoated 75 nm vesicles at the Golgi complex in intact cells; and (f) the accumulation of uncoated vesicles produced by NEM is reversed by the addition of a soluble cytoplasmic factor (NSF) (Malhotra et al., 1988).
Fig. 1 Model of the vesicle shuttle (see text) (A) Vesicle budding. (B) Targeting of vesicles. (C) Fusion. Reproduced with permission from Rothman, J.E. and Wieland, F.T. (1996) Protein sorting by transport vesicles, Science 272:227-234, copyright ©1996, American Association for the Advancement of Science.
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As implied by Fig. 1A, several features in the assembly of coated vesicles are common to all three systems that have been studied. They all first require the recruitment of small GTP-GTPases. Later binding of coat proteins induces a deformation of the membrane to form a bud. Transmembrane proteins of the donor membranes recruit the proteins that will form the coat (see Fig. 12A of Chapter 10). In addition, they can function in the selection of cargo. However, there are significant differences. The assembly of COPI-coated vesicles recruits a pre-assembled coat present as a soluble complex ( e.g., Zhao et al., 1997, 1999), whereas COPII proceeds stepwise. The recruitment of clathrin is also nucleotide dependent and requires ARF1. However, unlike COPI vesicles, in clathrin vesicle assembly, ARF1 only recruits AP-1 (an adaptor protein, see below) (e.g. Zhu et al, 1998) followed by formation of the coat. In addition, clathrin disassembly in neurons requires the heat shock protein Hsc70 together with the cofactor auxilin and proceeds with the hydrolysis of ATP. In other cells cyclin G-associated protein kinase (GAK) acts similarly to auxilin. The disassembly of the clathrin coat (e.g., Ungewickell et al., 1995; Umeda et al., 2000) differs from the dissociation of the other two coats. The ability of clathrin to self assemble into polyhedral cages (Woodward and Roth, 1978) has suggested that budding occurs by a stepwise assembly of coat structure from subunits in the cytoplasm. In this model, each addition progressively deforms the membrane to eventually form the bud and then a detached vesicle (see Le Borgne and Hoflack, 1998). However, the process is best understood for the cases of budding from the ER (Barlowe et al., 1994) and the Golgi cisternae that do not involve clathrin. As already mentioned, the budding process is regulated by GTP-binding proteins (see Table 1, Fig. 1A), which initiate the process (Donaldson et al., 1992; Helms and Rothman, 1992). Then the coat proteins can begin to be assembled. In contrast, the hydrolysis of the bound GTP to form bound GDP, initiates the release of the coat (Tanigawa et al., 1993). An enzyme in the donor compartment catalyzes the exchange of GTP for GDP (Barlowe and Shekman, 1993). The components of the COPII coat needed for vesicle assembly can be shown by genetic approaches in yeast. In addition, ER membranes can be used in an in vitro assay after extraction of peripheral proteins. The stripped membranes produce vesicles by budding after the addition of cytosol extracts and GTP. The active ingredients of the cytosol extracts correspond to a 700 kDa complex of Sec31p/Sec13p, a 400 kDa complex of Sec23p/Sec24p and the small GTPase, Sarp1 (Barlowe and Sheckman, 1993). Sar1p-GDP is normally in the cytoplasm. It is recruited to the ER membrane by Sec12p, an integral membrane protein of the ER. Sec12p also functions as a guanine exchange factor (GEF) (Barlowe and Sheckman, 1993), thereby facilitating the exchage of GTP for GDP. In contrast, the disassembly, required for fusion, involves GTP hydrolysis activated by Sec23p (see Kaiser and Ferro-Novick, 1998). Another protein of 240 kDa, Sec16p, is tightly bound to the ER and acts as a scaffold for the assembly. Sec23p, Sec24p, Sec31p and Sed4p (a homolog of Sec12p) are bound to different sites in the Sec16p molecule (e.g., Shaywitz et al., 1997). The minimum system to function in the formation of COPII vesicles and buds has also been http://www.albany.edu/~abio304/text/chapter_11.html (6 of 41) [3/5/2003 7:56:56 PM]
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determined by reconstituting purified proteins in entirely synthetic liposomes (Matsuoka et al., 1998). By adding 5'guanylyl imidodiphosphate (GMP-PNP), a non-hydrolyzable analog of GTP, Sec12p (which functions as a nucleotide exchange protein) is not required. After Sar1p-GMP-PNP is bound to the membrane, the Sec23p/Sec24p and Sec31p/Sec13p complexes are sequentially attached in that order. The process differs somewhat from the native process suggesting that other factors (e.g., the presence of cargo) may facilitate the process. Apparently a minimum of 10% acidic phospholipids is required (Matsuoka et al., 1998). Sorting of integral membrane proteins is thought to require binding of the cytoplasmic domain of the protein to the Sec23p-Sec24p complex (Aridor, et al., 1998). Sorting of soluble proteins requires transmembrane receptors. These receptors would require at least one transmembrane domain, a lumenal domain that can bind to the cargo and a cytoplasmic exposed domain that would bind to coat subunits. One of these is the KDEL receptor that binds to a carboxy-terminal KDEL peptide and retrieves the proteins that have escaped from the ER (Lewis and Pelham, 1992). Two proteins of the p24 family are receptors that have been found in COPII-coated vesicles (Schimoller et al., 1995) and are needed for the secretion of certain proteins. Their cytoplasmic domain contains a diphenyl and a dibasic motif at the carboxy terminals. In order to fuse to their target the vesicles must contain v-SNAREs (see Section B). vSNAREs have been demonstrated in COPI and COPII coats. Two ER to Golgi v-SNAREs, Bet1p and Bos1p, interact specifically with Sar1p, Sec23p, and Sec24p in a guanine nucleotide-dependent fashion (Springer and Sheckman, 1998). The assembly of COPI-coated vesicles proceeds as follows. First the GTPase ARF1 (ADPribosylation factor 1) binds to the membrane. This requires binding to its GEF. ARF1 is myristoylated. The myristoyl moiety is exposed when the GTPase binds GTP, allowing ARF1 to bind to lipids. The hydrolysis of GTP leads to the retraction of the meristoyl-moiety into a pocket of the ARF1 molecule, so that the GTPase is no longer able to attach to lipids. The assembly of the coat takes place by recruitment of a preassembled coat via its β and γ-COP subunits, resulting into a deformation of the membrane to form the bud (Zhao et al., 1997; 1999). Adaptors and receptors The assembly of coated vesicles probably involves many more interactions than those considered so far. The adaptor molecules AP-1 (present in the clathrin coated vesicles that originated from the TGN) and AP-2 (from the clathrin coated vesicles originating during endocytosis) appear to have a key role in the processes involved in vesicle formation. Their structure and function have been recently reviewed (Traub, 1997; Robinson, 1997). In addition to AP-1 and AP-2, a third adaptor protein has been identified, AP-3 (Dell'Angelica et al., 1997). AP-3 is likely to function in transport to the lysosomes (in yeast the vacuole). Deletion of any of the subunits, leads to mistargeting of some of the vacuolar proteins but not others (Cowles et al., 1997; Stepp et al., 1997; Vowels and Payne, 1998). Similarly, in mammalian cells, antisense oligonucleotides for the AP-3 gene (see Chapter 1) also send lysosomal glycoproteins to the cell surface without affecting AP-1 mediated transport, such as that of the mannose 6-phosphate receptors (Le Borgne et al., 1998). An additional
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function is suggested by the observation that in vitro synaptic vesicle can be formed from endosomes in the presence of AP-3 (Faundez et al., 1998). AP-1, AP-2 and AP3 are heterotetramers [two large δ subunits of 160 kDa, and two β3A (120 kDa) subunits associated with µ3 (47 kDa) and σ3 (22 kDa)].The β-subunits of these adaptors promote clathrin-cage formation (e.g., Gallusser and Kirchhausen, 1993). AP-1 and -2 also bind in vitro to the cytoplasmic domains of membrane receptors (e.g., Pearse, 1988, Glickman et al., 1989) and clathrin (e.g., Ahle and Ungewickell, 1989; Shih et al., 1995; Traub et al., 1995, Schröder and Ungewickell, 1995). In addition, they bind the tyrosine or di-leucine sorting motifs (Heilker et al., 1996) important for endocytosis and lysosomal targeting (see Sandoval and Bakke, 1994). The µ-1 and µ-2 subunits of AP-1 and AP-2, bind to the tyrosine endocytotic motif (Ohno et al., 1995). The adaptors are therefore responsible for the selectivity of the vesicle as well as their assembly. The receptor molecules, such as the mannose 6-phosphate receptors (MPRs) sorted out in the TGN, were found to be needed for the recruitment of AP-1 to the clathrin vesicles in an in vitro system (Le Borgne et al., 1996). The amount of AP-1 recruited by clathrin coated vesicles of the TGN was found to depend on the presence of the MPRs and the integrity of their cytoplasmic domains (Le Borgne and Hoflack, 1997). Many homologues of adaptor subunits AP-1, AP-2 and AP-3 have been found. In one case, clathrin coated vesicles associated with endosomes have been shown to have neither AP-1 nor AP-2, suggesting the presence of another adaptor complex (see Stoorvogel et al., 1996). Some of the adaptors function independently of clathrin (e.g., Stepp et al., 1995; Simpson et al., 1996). These adaptors could function in conjunction with clathrin-like molecules or other unknown coat proteins, possibly interacting with COPI. In summary, adaptors, receptors and clathrin appear to act in concert to produce coated vesicles. The subunits of COPI, coatomer, (β, δ and ζ-COP) have some sequence homology with the β, µ and σ-adaptor subunits of AP-1 and AP-2. Interestingly, the B subcomplex of COPI binds the di-lysine motif (e.g., Fiedler et al., 1996). Therefore, in contrast to clathrin, coatomer subunits have a role similar to the adaptor molecules. Coatomer coat subunits have been shown to interact with KKXX motif (Letourneur et al., 1994) needed for retrieval to the ER. Therefore they do function as receptors for retrograde transport. The interaction of coatomer with a domain of the peptide p23 (a p24 protein thought to act as a receptor for cargo) has been studied in vitro (Reinhard et al., 1999). The binding of the two kinds of protein results in a conformational change and polymerization of the complex in vitro with a stoichiometry of 1:4, COPI:peptide. This conformation is also seen on the surface of isolated COPI vesicles. These results suggest a mechanism by which the induced conformational change of coatomer accompanying its polymerization is responsible for the formation of the bud on the Golgi membrane during biogenesis of a COPI vesicle. In vitro experiments using liposomes reveal the formation of coatomer vesicles requires ARF, GTP and the cytoplasmic tails of the p24 proteins receptors, or cargo proteins with the KKXX retrieval signal (Bremser et al., 1999). http://www.albany.edu/~abio304/text/chapter_11.html (8 of 41) [3/5/2003 7:56:56 PM]
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Knowledge of the possible role of adaptor-like molecules in COPII is still lacking. However, COPIIcoated vesicles are selective, in some cases, as shown by the fact that they concentrate proteins exported from the ER (Balch et al., 1994; Bednarek et al., 1995). We saw that only the COPII proteins are required for vesicle formation (see above) (Matsuoka et al, 1998). However, phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate are needed to bind them to liposomes and the GTP-bound form of Sar1p is needed to recruit the proteins to either liposomes or the ER membranes. Heteromeric complexes of proteins of the p24 family are found in both COPI and COPII vesicles as well as ER and Golgi membranes (e.g., Füllekrug et al., 1999; Marzioch et al., 1999) suggesting that p24 cycles between ER and Golgi as would be expected for receptor proteins. In mammals the p24 proteins are thought to be involved in exit from the ER (Lavoie et al., 1999). In yeast there are eight genes encoding these family members. Mutations of several of these genes exhibit selective protein transport defects or secretion of the ER lumenal protein Kar2p (e.g., Marzioch et al., 1999). For example, deletion of one p24 gene slows down the transport of Gas1p from ER to Golgi (e.g., Marzioch et al., 1999). Gas1p is a GPI-anchored protein. Mutations of the genes coding for p24 proteins do not inhibit anterograde passage from the ER completely and the export of some of the proteins from the ER do not require p24-proteins at all. This suggests that only some proteins require this receptor or any receptor at the ER exit step. In agreement with this notion, many secreted proteins have been shown not to be concentrated in COPII-coated vesicles (Martínez-Menárguez et al., 1999). The incomplete inhibition by some of the mutants that seem to be involved in the p24 system may be explained by the occurrence of bulk flow which would be of significance in the absence of receptors. However, in some cases, the role of p24proteins is clearly that of receptors. The Emp24 complex is needed for for packing Gas1p into vesicles of the ER (Muñiz et al., 2000). In agreement with the notion that Emp23 acts a receptor, Gap1p was shown to become chemically cross-linked to two of the Emp24 proteins in experiments using cross-linking reagents. Cargo proteins have been found to have a role in the assembly of coat proteins (see Springer et al., 1999; Bremser et al., 1999). In addition, in view of the central role of the GTPases in coat assembly, it would seem likely that the GTPases have a role in regulating cargo recognition. A special role of GTPases is suggested by experiments using a cell-free system. During COPI vesicle formation, competing sorting signals were shown to act through a GTPase switch thereby providing specificity for the process (Goldberg, 2000). The signal sequence of hp24a (a p24 protein, supposedly a receptor) inhibits coatomer-dependent GTP hydrolysis. In contrast, the di-lysine retrieval signal (see above) that binds the same coatomer site has no effect on the GTPase. The results suggest that the activity of the GTPase can select or discard a cargo protein. Involvement of phosphatides Several phospholipid modifying enzymes are involved in the formation of vesicles from buds (e.g., endophilin, synaptojanin and phospholipase D). Endophilin is an acyltransferase that binds to the small GTPase, dynamin (see below), and transfers fatty-acyl from arachidonic and palmitic acid to http://www.albany.edu/~abio304/text/chapter_11.html (9 of 41) [3/5/2003 7:56:56 PM]
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lysophosphatidic acid to produce phosphatidic acid (Schmidt et al., 1999). This reaction may induce negative membrane curvature by converting an inverted-cone-shaped lipid into a cone-shaped lipid in the cytoplasmic layer of the bilayer. Phospholipase D produces phosphatidic acid from phospholipids, possibly affecting membrane curvature (Zimmerberg, 2000). Synaptojanin is a polyphosphoinositide phosphatase. See also Chapter 4 for a discussion of membrane curvature. Although their role is not well understood, the phosphoinositides (PIs) have been shown to be part of the machinery responsible for the formation of vesicles, for example, in the the TGN, the plasma membrane and the endosomes (see Martin, 1997; De Camilli, 1996; Ohashi et al., 1995). The phosphoinositides are thought to be part of the discrete membrane sites (see Chapter 4) required for the recruitment of cytoplasmic proteins needed for vesicle formation and budding. An involvement of the lipid system is shown by the dependence of secretion on phosphatitylinositol transfer proteins (PITPs). PITPs are enzymes reponsible for the transfer of phospholipids between membrane structures or serum lipoproteins (see Wirtz et al., 1991). These enzymes have a distinct preference for phosphatidylinositol over phosphatidylcholine. The β isoform, present in the Golgi system, has a high transfer activity in relation to sphingomyelin (see deVries et al., 1995). In yeast, mutations in the SEC14 gene which codes for a PITP, block post-Golgi secretory traffic (e.g., Bankaitis et al., 1990). In agreement with these results, experiments carried out on a cell free system from a neuroendocrine cell line (Ohashi et al., 1995) found that the α and β isoforms of PITPs stimulate formation of vesicles from the TGN. Phosphatidylinositol kinases (PIKs) which phosphorylate PIs have also been implicated. In yeast, the Vps15 protein kinase and the Vps34 PI-3-kinase have been shown to function as a membraneassociated complex which facilitates the delivery of proteins to the yeast vacuole (e.g., Stack and Emr, 1994) which has a similar function than lysosomes in a mammal. Subsequent work has indicated that polyphosphoinosides phosphorylated at the 4' and 5' or alternatively the 3' positions of the inositol ring determine the location of events involved in membrane traffic. It is now generally recognized that phosphorylation-dephosphorylation of the polar heads of phosphoinositides in specific locations coincides with the recruitment or the activation of proteins essential for vesicular transport (see De Camilli et al., 1996). The compartmentation of PPIs is probably the result of synthesis at specific locations. The targeting of PI 4-kinase would permit a segregated synthesis. PI 4-kinase catalyzes the phosphorylation of phosphatidylinositol to form PI 4-phosphate. PI 4-kinases have been found in plasma membranes and intracellular organelles including Golgi, lysosomes, ER, nuclear envelope, coated vesicles, exocytotic vesicles and secretory granules (see De Camilli et al., 1996; Carpenter and Cantley, 1996). The immunoreactivity for this PI 4-kinase molecule was found mostly in close association with the membranes of the Golgi vesicles and vacuoles (Nakagawa et al., 1996). The association between PI-kinases and membranes occurs despite the absence of transmembrane sectors. However, these proteins could bind directly to lipids or indirectly by anchoring to integral proteins. Some of the mammalian kinases have a pleckstrin-homology (PH) domain (Nakagawa et al., 1996) that would allow them to interact with lipids. Alternatively, the kinase could be anchored
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by binding to membrane receptors, as is the case of PI 4-kinase that links to transmembrane proteins and integrins (Berditchevski et al., 1997). A mammalian adaptor protein p150 (homolog of the yeast VPS15) that recruits the PI 3-kinase to the Golgi membrane, has been identified (Panaretou et al., 1997). Recombinant p150, PI 3-kinase and PITP form a complex also present in the cytoplasm of human cells. Not surprisingly, a polyphosphoinositide phosphatase (synatpojanin 1) has been found to be involved in presynaptic endocytotic vesicle recycling in neurons. Synaptojanin 1 was found at high levels at nerve terminals (McPherson et al., 1996), which are involved in the exocytosis of synaptic vesicles. Synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth, phosphatidylinositol 4,5-biphosphate (PIP2) levels are increased and clathrin-coated vesicles accumulate at the nerve endings (Cremona et al., 1999) Undoubtedly, the binding of phosphoinositides to coat components has a role in trafficking. They bind AP-2 (e.g., Gaidarov et al., 1996), AP-3 (AP180) (e.g., Hao et al., 1997) and COPI coatomer (e.g., Chaudhary et al., 1998), dynamin, synaptojanin (Cremona et al., 1999) and in addition arrestins (Gaidarov et al., 1999). Dynamin is involved in vesicle formation (see Chapter 9). Arrestins are involved in the inhibition of Gs proteins (e.g., see Chapter 7) and also act as clathrin adaptors (see Goodman et al., 1996). Mutants of arrestin3 expressed in cells in culture fail to participate in β2-adrenergic receptor internalization and fail to be recruited to the coated pits although they are recruited to the plasma membrane (Gudrov et al., 1999). Similarly, mutation of the α subunit of the AP-2 adaptor protein at high expression levels results in failure to localize at clathrin coated pits (Gaidarov and Keen, 1999). In addition to their separate roles in vesicle formation and secretion, the phosphoinositide system and the GTP-binding proteins are functionally and intimately linked. ARF activates phospholipase D (PLD) (Brown et al., 1993). PLD catalyzes the conversion of phosphatidyl choline to phosphatidic acid. The latter activates PI-4 kinase to produce PIP2 (see Martin, 1997; DeCamilli et al., 1996) which has been shown to be involved in vesicle formation (Tüscher et al., 1997). In addition, ARF has a direct effect in increasing the level of PIP2 (Godi et al., 1998) and PI-4 kinase-β and, by recruitment, another unidentified kinase in the Golgi (Godi et al., 1999). This increases the synthesis of PI-4-phosphate and PI2 levels independently of PLD activation. The PI-4 kinase is required to maintain the integrity of the Golgi. Mutants that lack the kinase exhibit a disorganized Golgi (Godi et al., 1999). Other effects of ARF on the Golgi and vesicle pathway are less clearly understood. The actin binding proteins ankyrin and spectrin, are at the cytoplasmic surface of the Golgi (see Beck et al., 1998; De Matteis et al., 1998). ARF recruits a specific spectrin to Golgi membranes (Godi et al., 1998) in a mechanism involving the PIP2 binding domain of spectrin [the pleckstrin-homology (PH) domain]. Spectrin is needed for maintaining the structural integrity of the Golgi. Agents that block the binding of spectrin inhibit the transport of vesicular stomatitis virus G protein from the ER to the medial compartment of the Golgi complex.
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B. Targeting of Proteins and Vesicles; SNAREs As we saw in Chapter 10, the sorting signals of cargo proteins are responsible for the targeting of proteins. Similarly, retention signals are likely to be required for retention by the appropriate compartment. The sorting signal is recognized directly or indirectly by the proteins that form the vesicle coat to insure packaging of the cargo protein in the corresponding vesicle. Once assembled, specific tethering and docking of the transport vesicle are needed to deliver the vesicle to the acceptor compartment. At the target site, the docking depends on the complementary SNAREs. SNAREs are integral proteins of intracellular membranes with a large domain on the cytoplasmic phase. SNAREs present in a transport vesicle (v-SNARE; e.g. synaptobrevin in neurons) (Söllner et al., 1993a) are recognized by another similar protein on the surface of the target compartment (tSNARE; e.g. syntaxin in neurons) (Rothman, 1994; Søgaard et al., 1994) (see Fig. 1B). The two usually correspond to R-and Q-SNAREs respectively (i.e., named after the glutamine and arginine residues of the their cytoplasmic domains) (see Jahn and Südhof, 1999).The specific interaction is apparently mediated by the extended cytoplasmic domain of the two different kinds of SNAREs (Chapman et al., 1994). The role of SNAREs has been delineated most completely for the shuttles between the ER and the Golgi (see Rothman; 1994, Paek et al., 1997) and from the Golgi to the plasma membrane (Brennwald et al, 1994). A SNARE protein that functions within the Golgi system has been isolated from mammalian cells in culture (Hay et al., 1997; Nagahama et al., 1997). This protein of 28 kDa is associated with Golgi membranes. The transport from the ER to the transGolgi and TGN is blocked by the cytoplasmic domain of this protein or an antibody to it. These agents are thought to block transport between the medial-Golgi and the trans-Golgi and TGN (Hay et al., 1997). A yeast v-SNARE protein has been implicated in retrograde transport to the cis-Golgi (Lupashin et al., 1997) and probably the anterograde transport from late Golgi and a prevacuolar compartment (von Mollard, et al., 1997). The two kinds of SNARES, v-SNARES and t-SNARES, must be capable of binding each other specifically. Complementary pairs have been identified in yeast for the ER-Golgi step (Lian and Ferro-Novick, 1993; Søgaard et al., 1994) and the Golgi-plasma membrane step (Aalto et al., 1993; Propopov et al., 1993; Brennwald et al., 1994). In addition, SNARE-pairs have been identified in regulated exocytosis of neuronal synapses (see Südhof, 1995). A study of Nichols et al. (1997) has shown, in yeast vacuoles, the need for t-SNARE in one membrane and v-SNARE in another for fusion to occur. In a more systematic approach, McNew et al. (2000) tested all of the potential vSNAREs encoded in the yeast genome to examine whether they can partner t-SNAREs of the Golgi, the vacuole and the plasma membrane. Vesicle and target SNAREs were reconstituted into two separate sets of liposomes and tested for fusion (see Weber et al., 1998). In these experiments, the phospholipids in the vesicles reconstiuted with v-SNAREs were labeled with a mixture of probes whose fluorescence is quenched. When fusion to the vesicles containing t-SNAREs occurs, the fluorescent probes are diluted and become fluorescent as the quenching decreases. The SNARE proteins reconstituted in liposomes were found to function as predicted by the SNARE hypothesis by exhibiting the appropriate specificity. However, other factors seem to play an important role as well (see below). These are proteins involved in the localization of vesicles, such as tethering factors, or those involved in the activation of the SNARE complexes (see Waters et al., 1999; http://www.albany.edu/~abio304/text/chapter_11.html (12 of 41) [3/5/2003 7:56:56 PM]
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Mellman and Warren, 2000). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices (Parlati et al., 2000; Fukuda et al., 2000). For the transition between ER and Golgi, one SNARE contains three of the helices (t-SNARE) and the other one (v-SNARE). Fusion does not take place with any other combination. For t-SNAREs on the plasma membrane, the protein syntaxin, supplies one helix and a SNAP-25 protein contributes the other two. The assembly of SNARE complexes involves Rab (see Rothman, 1994; Søgaard et al., 1994) and Sec1. Rab proteins are GTP-binding proteins (Simons and Zerial, 1993, see below). Sec1 proteins (Aalto et al., 1992) bind specifically to t-SNARE subunits (Pevner et al., 1994). The need for separate and complementary v-SNAREs and t-SNARES, one in the cargo carrying vesicle and the other in the target membrane, explains many of the available data. However, in COPII vesicles the situation is more complex. The vesicles originating from the ER must first cluster to form VTCs (see Chapter 10). The large vesicles of the VTC are then targeted to the cisGolgi with which they fuse. Consistent with this view, yeast vesicles can be isolated in clusters (Lian and Ferro-Novick, 1993). In addition, the t-SNARE syntaxin 5 is present in the vesicles, as we might expect if they fuse and not in the Golgi membranes (Rowe et al., 1998) and is essential for the assembly of vesicular-tubular-preGolgi intermediates as well as for the delivery of the cargo to Golgi. We saw that the SNARE hypothesis postulates that the specificity of membrane fusion events resides on the SNARE receptors, SNAREv and SNAREt joining with each other. A good deal of data just reviewed supports this view (e.g., see Weber et al., 1998). However, recent studies suggest that the process is more complex. It has been argued that the specificity of targeting may not depend on SNAREs (e.g., Kaiser and Ferro-Novick, 1998). t-SNAREs are not localized at specific sites of the target membrane (e.g., Garcia et al., 1995) and v-SNARE can bind to more than one t-SNARE (von Mollard, et al., 1997; Holthuis et al., 1998). In some cases the system may be able to bypass SNAREs entirely. The disruption of SNAREs [e.g., caused by microinjection of the cytoplasmic domain of synaptobrevin (Hunt et al., 1994) or cell mutants lacking synaptobrevin or syntaxin (Broadie et al., 1995)] does not prevent vesicle docking. A v-SNARE can reside in both anterograde and retrograde-directed vesicles and a single v-SNARE can bind to several t-SNARES and a single tSNARE can bind to several different v-SNAREs (see Götte and Fischer von Mollard, 1998; Pfeffer et al., 1999). In the case of the ER-to-Golgi transport, another entity, the 800 kDa complex of 8 subunits, the transport protein particle (TRAPP) may account for the specificity. TRAPP, located in the cis-Golgi, has a role in targeting and fusion (Sacher et al., 1998) and may have a role in providing specificity since it is unique to this step, whereas all SNARE molecules are very similar. As reviewed above, t-SNAREs and v-SNAREs present in separate vesicles interact. However, other components are active in this process (Ungermann et al., 1998; Peters and Mayer, 1998). The small GTP-binding protein Ypt7p holds together the vacuoles in a reversible reaction (Ungermann et al., 1998). This stage requires the presence of various factors (see next section) but not SNAREs. A similar SNARE independent attachment was first described in the attachment of the ER vesicles to the Golgi (Cao et al., 1998a). http://www.albany.edu/~abio304/text/chapter_11.html (13 of 41) [3/5/2003 7:56:56 PM]
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In Saccharomyces cerevisiae, Vam7 (a t-SNARE) is targeted to vacuoles in a manner dependent on the presence of phosphatidylinositol 3-phosphate in the vacuolar membrane. This interaction involves the phox homology (PX) domain of Vam7 (Cheever et al., 2001). The PX domain is an 80125 amino acid residue region of proteins involved in binding phosphoinositides (Kanai et al., 2001). C. Fusion The machinery responsible for fusion is composed of three distinct proteins (Söllner et al., 1993): NSF (Sec18p in yeast), SNAP (Sec17p in yeast) family members, and the SNAREs. Once assembled, the complex forms a 20S particle (where 20S refers to the sedimentation coefficient). Models of the structures of the proteins in the complex derived from crystallography give us some understanding of the assembly of the 20S fusion particle (May et al. 1999; Yu et al., 1999; Rice and Brunger, 1999). These studies provide us with a detailed view of the very similar structures seen with the EM of the 20S particles (Hohl et al., 1998). The SNARE complex is rod shaped (2.5 x 15 nm). SNAP binds laterally, whereas NSF binds to one end of the complex to form a particle 22 nm in length. The association of SNAP and NSF with SNARE to form the 20S particle is sequential. The vSNARE-t-SNARE complex binds 3 to 6 SNAP proteins and subsequently NSF (Söllner et al., 1993b; Hayashi et al, 1995). The release of SNAP, which requires NSF, is accomplished before vesicle docking (Mayer et al., 1996). Subsequently, v-SNARE from t-SNARE dissociate (Söllner et al., 1993) and the fusion takes place. The SNAP and NSF proteins are common to most, if not all, different transport steps. It might be expected the SNAREs are specialized since their interaction determines the final fate of the cargo (Fig. 1B) (see Ferro-Novick and Jahn, 1994). However, this was found not to be the case. In vitro testing showed very promiscuous binding of SNAREs derived from different sources (Yang et al., 1999). The SNAP proteins (Clary et al., 1990) and an ATPase, the NSF (Block et al., 1988; Malhotra et al, 1988; Wilson et al, 1989) assemble after docking. The v-SNARE-t-SNARE complex binds 3 to 6 SNAP proteins and subsequently NSF (Söllner et al., 1993b; Hayashi et al, 1995). The hydrolysis of ATP dissociates v-SNARE from t-SNARE and releases the SNAP molecules (Söllner et al., 1993).The fusion process is still not well understood. In one of the current models of fusion, SNAREs have a role in bringing the two bilayers together in a zipper like process (see Chen and Scheller, 2001). When the two bilayers become fused a pore opens and then pore expands producing one continuous bilayer. At least in exocytosis, freeze fracture EM (Chandler and Heuser, 1980) and patch clamping ( Breckenridge and Almers, 1987) confirm the formation of a pore. Present thinking suggests that the pore is in the lipid components with protein scaffolding, possibly SNAREs (see Monck and Fernandez, 1994, 1996;Lee and Lentz, 1997) In mammals, the fusion of early endosomal vesicles and more mature endosomal vesicles requires http://www.albany.edu/~abio304/text/chapter_11.html (14 of 41) [3/5/2003 7:56:56 PM]
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the early endosome-associated protein (EEA1) and Rabaptin-5. EEA1 binds to phosphatidylinositol 3-phosphate in the endosomal membrane through its FYVE domain (e.g., Simonsen et al., 1998) NSF NSF was discovered in isolated systems. In low concentrations, the sulfhydryl alkylating agent NEM, inactivated the acceptor membrane fraction of the intra-Golgi transport system so that it would no longer be able to fuse to the incoming vesicles (Glick and Rothman, 1987). Untreated peripheral proteins of the Golgi membranes restored function. A 76 kDa polypeptide, forming a homotetramer in its native form, NSF, was found to be responsible for the activity (Block et al., 1988). EM examination of the Golgi membranes of blocked cells (Diaz et al., 1989) indicated an accumulation of uncoated intermediate transport vesicles associated with membranes. This observation suggested that NSF acts as part of the complex needed to fuse the vesicle membrane with the acceptor membrane. NSF was subsequently found to be needed for the in vitro fusion of vesicles involved in the transport from ER to Golgi and for the fusion of endocytotic vesicles. NSF shows 48% sequence identity to Sec18p of yeast. Furthermore, Sec18p can replace NSF in the mammalian cell free system. NSF is a hydrophilic molecule not likely to interact with hydrophobic domains such as the hydrocarbon leaflets of the bilayer membrane. However, it has two ATPbinding domains and displays ATPase activity, which determines its attachment to the membrane. In addition to NSF, five other proteins have been found to be necessary in vesicle mediated transport. Many others might also be required. The in vivo requirement for Sec18p was demonstrated using a temperature sensitive mutant (Graham and Emr, 1991). The proteins of the cells were labelled by exposure for a short period to radioactive amino acids at the permissive temperature (20oC), followed by a chase at the nonpermissive temperature (37oC). The fate of two proteins, F and CPY, was examined. These two were selected because the proteins undergo stepwise modifications in the Golgi complex that can be easily recognized using SDS gel electrophoresis and immunoprecipitation. In the case of the wild type cells, the F precursor proteins were rapidly chased to the mature form of the protein (mF). In contrast, in the mutant cells the inactivation of the sec18 protein left the F protein in all intermediate compartments. These results indicate that each sequential step must occur in a separate compartment, as we saw for other systems, and Sec18p is involved in each step. NSF is a member of the ATPases responsible for varied cellular activities, the AAA (ATPases associated with a variety of cellular activities) superfamily (see Patel and Latterich, 1998; Confalonieri and Duguet, 1995, see Frölich on the Web) which also includes proteasomal components (see also Chapter 15). Some members of this family perform chaperone tasks (i.e., folding tasks) (see Chapter 15), others are associated with assembly, remodeling and disassembly. They act in a variety of cellular functions (see Neuwald et al., 1999), including cell-cycle regulation, protein degradation, organelle biogenesis and vesicle-mediated protein transport, and the initiation of transcription. The AAA motif corresponds to a 230-amino-acid domain that contains Walker ATPbinding homology sequences and imparts ATPase activity. NSF is involved in eukaryotic fusion events which also require SNAREs. The SNAREs are associated at their cytoplasmic domains forming stable complexes (Hay and Scheller, 1997; Weber http://www.albany.edu/~abio304/text/chapter_11.html (15 of 41) [3/5/2003 7:56:56 PM]
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et al., 1998). SNAREs (v and t) constitute the minimum requirement for fusion of membranes for one round of fusion. NSF and SNAPs being required for separation of the two SNAREs to allow for the next round of fusions (see Weber et al., 1998). This activity depends on ATP hydrolysis. The dissociation events require the soluble NSF attachment proteins (SNAPs) to bind to SNAREs providing a binding site for NSF (Whiteheart et al., 1992; Wilson et al., 1992). The binding to SNARE and SNAP increases the ATPase activity of NSF and at the same time the complex is disassembled (Morgan et al., 1994, Barnard et al., 1997). NSF is made up of three domains: the amino-terminal domain which is capable of interacting with SNAPs and SNAREs, and two similar ATPase domains (D1 and D2). D1 is thought to be involved in remodeling the 20S particle; D2 is thought to be responsible for the formation of NSF-hexamers (Neuwald, 1999). The complex is thought to function similarly to chaperones in driving the remodeling of effector molecules using the free energy generated by the hydrolysis of ATP (see Patel and Latterich, 1998; Neuwald, 1999) SNAPs SNAPs constitute a family of soluble proteins required for NSF to bind to Golgi membranes (Weidman et al., 1989, Clary et al., 1990). α, β and γ SNAPs are 35, 36 and 39 kDa in molecular weight, respectively. α-SNAP has also been shown to be required in vivo. In yeast, α-SNAP is encoded by the SEC17 gene. In the absence of this protein, vesicles are accumulated (Kaiser and Shekman, 1990). SNAPs bind to specific sites on the membranes the SNAREs) and this binding is required for interaction with NSF. The binding site can be identified by crosslinking with crosslinking reagents (Whiteheart et al., 1992). These show that α-SNAP attaches to a 30-40 kDa protein. The various SNAPs bind at different sites of a receptor complex. D. The GTPases The GTP-binding proteins or GTPases discussed above (also referred to as G-proteins, not to be confused with the VSV-G glycoproteins) have been shown to have a key role in intracellular transport. Mutations in the genes known to code for GTP-binding proteins block several steps in secretion. Furthermore, in mammalian cells, the Rab or Ras proteins which correspond to GTPases are localized in specific membranes associated with the secretory pathway and intracellular transport is blocked by treatments that inhibit small GTP-binding proteins. There are seven major groups of GTPases. They share domains needed for guanine binding and GTP hydrolysis. These domains are so similar that the three-dimensional crystal structure of the proteins are superimposable. They diverge significantly in amino acid sequences in other regions of the molecules. Presumably, these are the regions which determine functional specificity. The role of heterotrimeric GTP-binding proteins in signal transduction was discussed in Chapter 7. In this complex, the α-subunit of 40-50 kDa binds to the guanidine nucleotide. There are also two additional subunits (β and γ of 35-36 and 8 kDa respectively). The present section will discuss both the heterotrimeric and the lower molecular weight GTPases in relation to intracellular trafficking. In contrast to the heterotrimeric GTPase, those of the Ras superfamily (also called Rab when
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derived from cDNA libraries from rat brain) are monomers (Sar1, ARF, Rab/YPT, Rac/CDC42 and Rho families) 20 to 30 kDa in molecular weight. Those of the dynamin family are 60-80 kDa. With the exception of ARF, all form complexes with cytoplasmic or membrane proteins which may have a function similar to the β and γ subunits of the trimeric GTPase. Fatty acid residues are added posttranscriptionally to several GTPases. In some cases, these have been shown to permit direct interaction with membranes (Rossi et al., 1991). Rab, Rho, Rac and the γ-subunit of the heterotrimeric GTP-binding protein have one or two prenyl moieties at the carboxyl terminal or at cysteine residues. Members of the ARF and Gα family are myristylated at an amino-terminal glycine residue. Palmitylation of cysteine residues may occur in the amino or carboxyl terminal of Ras and G. GTPases were originally shown to have a role in all the steps of intracellular transport through the effects of guanosine-5-O-thiotriphosphate (GTPγS) and AlF4-. GTPγS, a non-hydrolyzable analog of GTP blocks all GTPases. It inhibits almost all of the steps in the transport. The heterotrimeric GTPase appears to function as an inhibitor of transport. Two specific α-subunits are associated with the Golgi. Overexpression (by transfection, see Chapter 1) of one of these Gαs leads to slowing of the transport of proteoglycan through the Golgi (Stow et al., 1991). Conversely, inhibition of Gα speeds up transport. A similar role is suspected for ER to Golgi transport at the budding stage (Schwaninger et al., 1992). The well-defined morphology of the mammalian system provides a clear picture of the role of the small GTP-binding proteins. A minimum of 12 mammalian rab genes have been isolated from cDNA libraries using probes recognizing ras sequences. The proteins of six rab genes (rab1 to rab6) have been shown to bind and hydrolyze GTP. Immunofluorescence, immunoeletronmicroscopy and the immunological reactions after cell fractionation have provided information on the localization of these proteins, as summarized in Table 2 (Rothman and Orci, 1992). This association of each Rab protein with a specific structure in the intracellular transport pathway suggests that each GTP protein acts at a different step. However, direct evidence has been provided by genetic approaches in yeast. These findings were extended to other systems using cDNA technology and in vitro reconstitution systems. Numerous additional rab110,111 and ARF65 genes have been cloned and sequenced but the proteins have not yet been localized. In most cases, there is a substantial pool of soluble GTPases in addition to the membrane-bound forms. The transport of VSV G-protein was followed in vitro by measuring the incorporation of [3H]-Nacetylglucosamine (GlcNAc) from donor membranes (from a cell line lacking GlcNAc-transferase) to acceptor membranes isolated from wild type cells (containing GlcNAc transferase) (Balch et al., 1984a,b). The use of synthetic polypeptides representing partial amino acid sequences of specific GTPases, or the use of antibodies to the individual GTPases, identified the individual steps in which they are involved. A polypeptide will block the action of a GTPase when it competes with it, for example, for a receptor site. Similarly, a specific antibody can block individual steps by depleting the cells of the Rab-protein. Table 2 Partial list of GTPases and their location. http://www.albany.edu/~abio304/text/chapter_11.html (17 of 41) [3/5/2003 7:56:56 PM]
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Small GTP-binding protein Rab family
Location(s) when membrane bound
rab1Ap/rab1Bp/ypt
ER and Golgi
rab2p
GCN
rab3Ap
Neurosecretory vesicles
rab4p
Early endosomes, plasma membrane
rab5p
Early endosomes, plasma membrane Golgi stack
rab6p Late endosomes rab7b
ARF family
sec4p
Post-Golgi vesicles, plasma membrane
ARF1p
Golgi stack
SAR1p
ER
Reproduced with permission from Nature (1992) Rothman, J.E. and Orci, L. 355:409-415. Research efforts are now directed toward understanding how the small GTP-binding proteins interact with specific membranes. This is likely to require the identification and study of multiple binding proteins in the various membranes. A summary of some of the functions of GTPases is presented in Table 3. In this table, an important function of the GTPase is listed in the first column, however, some of them have multiple functions. The GTPase responsible is listed in the second column and the evidence available is listed in the third. The precise mechanism of the action of small GTPases is not known. However, their properties provide hints that they can function as switches, initiating or terminating biological processes. We have seen this kind of role in the docking of ribosomes and nascent polypeptide chains in the ER.
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The GTPases change in conformation in response to the phosphorylative state of the bound nucleotide. The active conformation occurs when the bound GDP exchanges with GTP. Hydrolysis of the GTP returns the protein to its inactive conformation. The reactions of the GTPases require the presence of other proteins. Two proteins regulate the conformational changes. The exchange of GDP for GTP is facilitated by the guanine nucleotide dissociation proteins (GEPs) and inhibited by guanine nucleotide dissociation inhibitors (GDIs). In addition, the GTPase activity of the Ras superfamily is slow in the absence of GTPase activating proteins (GAPs). Table 3 Role of GTPases in Intracellular Transport
FUNCTION vesicle budding from ER
GTPase Sar 1
EVIDENCE overcoming mutant block (1) location in ER, trnaslational elements and Golgi (2)
formation of COP vesicles from Golgi
ARF (ADP ribosylation factor)
in vitro assay (3)
endocytotic role
dynamins
accumulation of coated pits in defective mutants (4)
transport from ER and cis to trans Golgi
Rab1
in vitro assay (5)
early endosome fjunction
Rab2, 5 and 7
Rab domains target to endosomes (6)
TGN vesicle budding
Rab6
antibodies to Rab6 block TGN budding (7)
exocytosis: synaptic vesicle
Rab3A
required for transfer from vesicle to cell surface (8)
exocytosis: mast cells
discharge of granules when Rab delivered into cells (9)
Ref. (1) Nakano, A. and Maramatsu, M. (1969), J. Cell Biol. 109:2677-2691. (2) Kuge, O., Dascher, C., Orid, L., Amherdt, M., Plutner, H., Ravazzola, M., Tanigawa, G. Rothman, J.E. and Balch, W.E. (1994) J. Cell Biol.. 125:51-65 http://www.albany.edu/~abio304/text/chapter_11.html (19 of 41) [3/5/2003 7:56:56 PM]
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(3) Orci, L., Palmer, D.J.,, Perrelet, A., Amerdt, M.., Palmer, D.J. and Rothman, J.E.. Nature 362:648-652; 364:732-734. (4) Kosaka, T. and Ikeda, L. (1983) J. Neurobiol.. 14:207-225. (5) Plutner, H., Cox, A.D., Pind, S., Khosravi-Far, R., Bourne, J.R. et al., (1991) J. Cell Biol. 115:3143. (6) Chavier, P., Gorvel, J.P., Steizer, E., Simons, K., Greenberg, J. and Zerila, M. (1991) Nature 353:769-772. (7) Jones, S.M., Crosby, J.R., Salamero, J. and Howell, K.E. (1993) J. Cell Biol . 122:775-788. (8) Matteoli, M., Takel, K., Cameron. R., Hurbult, P., Johnston, P.A. et al. (1991) J. Cell Biol . 115:625-633. (9) Oberhauser, A.F., Monck, J.R., Balch, W.E. and Fernandez, J.M.. (1992) Nature 360:270- 273. Dynamins (see McNiven et al., 2000) constitute a family of large (100-kDa) GTPases that seem to fulfill several roles in membrane trafficking in eukaryotic organisms. Different dynamin isoforms are present in different tissues and even in the same tissue. At least 25 different mRNAs are produced by the 3 dynamin genes by alternative splicing ( Cao et al., 1998b). Dynamins have been shown to play important roles in endocytosis and vesicle formation (see Chapter 9 and above). Dynamin is needed for clathrin-mediated endocytosis (e.g., Herskovits et al., 1993; van der Bliek, 1993) and the formation of vesicles from caveolae (see Chapter 9) (Henley et al, 1998; Oh et al., 1998). Inhibition of dynein function, inhibits the scission of caveolae both in vivo and in vitro. Dynamin has also been implicated in other kinds of endocytosis (see Sandvig and van Deurs, 1996), including the intake of fluid in cultured mammalian cells (Henley et al., 1999) and phagocytosis in macrophages (Gold et al., 1999). Dynamins have a role in vesicle formation in steps of the endocytotic pathway other than vesicle formation from the plasma membrane. In HeLa cells, the expression of a mutant of dynamin does not affect clathrin-independent endocytosis. However, the pathway from endosomes to Golgi is blocked (Llorente et al., 1998). Similarly, disruption of a dynamin-family proteins in Dictyostelium discoideum has a very broad effect (including a defective fluid-phase uptake) (Wienke et al., 1999). In addition, dynamin was found to colocalize with vacuolin, a marker of a postlysosomal compartment. In mammalian cells, confocal imaging (see Chapter 1) showed that dynamin is associated not only with the plasma membrane but also the trans-Golgi network, and a perinuclear cluster of structures containing cation-independent mannose 6-phosphate receptor. Electron microscopy showed that the structures correspond to late endosomes with a localization of dynamin preferentially in tubulo-vesicular processes of these endosomes (Nicoziani et al., 2000). In other studies, dynamin was found associated with the TGN (Henley et al., 1996; Maier et al., 1996). GFPdynamin chimeras (see Chapter 1) expressed in cultured rat hepatocytes appeared in the clathrin coated vesicles at the cell surface and the TGN (Jones et al., 1998). An in vitro system produced dynamin in clathrin coated and non-clathrin coated vesicles. Cells expressing a mutant of dynamin were found to accumulate GFP-protein chimeras in the TGN (Kreizer et al., 2000). In Saccharomyces cerevisiae, a dynamin, dnm1p, has been shown to be involved in transport to the vacuole from the TGN (Gammie et al., 1995).
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The dynamins are capable of interacting with several components. Their pleckstrin homology (PH) domain allows them to bind to phosphoinositides and their proline-rich domain (PRD) allows to bind to the SH3-domains of variety of effector molecules. A coiled-coil region (CC domain) f dynamins is a GTPase effector domain (Sever et al., 1999). In vivo, dynamin interacts with other proteins that may alter the geometry of lipid structures to produce vesicles. An interaction with endophilin 1 (Schmidt et al., 1999) (that transfers arachidonate to lysophosphatidic acid) has been demonstrated. This reaction is likely to favor the formation of highly curved lipid structures. There is at least one example of this mechanism: BARS (Weigert et al, 1999) (that acylates lysophosphatidic acid) was shown to induce the formation of vesicles in Golgi membranes of rat brain. However, in this case the system seems to operate independently of dynamin. Several studies have indicated the dynamin is associated with actin or actin-binding or actindepolymerizing proteins, suggesting a role of these interactions in the formation of vesicles (e.g., Witke et al., 1998; Qualmann et al., 1999) E. ER and Golgi Transport ER to Golgi Transport Present information indicates that COPII vesicles mediate the transfer of cargo from the ER to the Golgi. However, the process may be more complex. We saw that larger vesicular components, formed by vesicle clustering, carry cargo to the cis-Golgi (see Chapter 10, Section III). Therefore, the vesicular tubular clusters (VTC) and not the COPII vesicles are targeted to the cis-Golgi. Consistent with this view, the t-SNARE, syntaxin 5, is present in the vesicles as well as in the cisGolgi (Rowe et al., 1998) and is essential for the assembly of VTCs. What this suggests is that the VTCs are formed by an aggregation of these vesicles. Unfortunately, since retrograde transport also takes place, it is difficult to consider this argument decisive. The role of COPII in anterograde transport from the ER is well established. However, much of the present evidence also supports a role of COPI for anterograde transport between ER and Golgi and between Golgi stacks (Chapter 10, Section 3C; see also Kreis et al., 1995). However, studies with yeast COPI mutants demonstrate ER to Golgi transport in COPI-impaired cells for some proteins but a complete block for others (Gaynor and Emr, 1997). In contrast, the remaining ER to Golgi transport required COPII. A possible explanation for the complex role of COPI in anterograde transport from the ER, might rest on a sequential role of the two coat complexes. In in vitro experiments using isolated ER fragments, showed that vesicles released by the ER-derived system were COPII coated and contained VSV-G protein and p58. p58 is an endogenous recycling protein. However, preparations from ARF1 mutants that prevent COPI recruitment blocked subsequent movement to the isolated Golgi membranes (Rowe et al., 1996). These observations suggest that COPII drives the export from the ER and then COPI replaces COPII in the vesicles.
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In yeast, the transfer of cargo from the ER to the cis-Golgi involves the transport protein particle (TRAPP), a 1100 kDa complex (Sacher et al., 1998). TRAPP mediates vesicle docking and fusion. Proteins analogous to the subunits of TRAPP have been found in mammalian species (see Guo et al., 2000). Subunits of TRAPP have been found in early Golgi membranes, in X-100 insoluble membrane components suggesting a presence in rafts.The protein p115 (also known as TAP) is needed for the transport from the VTC to the cis-Golgi (Nelson et al., 1998). It plays a more general role in intra-Golgi transport (see next section). In yeast, two other factors have been implicated in ER-to-Golgi transfer of cargo. Uso1p acts before SNAREs and is required for tethering (Cao et al., 1998). Sec34p and Sec35p also function in tethering (VanRheenen et al., 1998). Transport from the Golgi stacks Isolated Golgi stacks incubated with cytosol and ATP generate 75 nm vesicles from the cisternae (Balch et al., 1984a) (now recognized as COPI-coated vesicles). They correspond to transport vesicles because they can be shown to contain transported proteins (in these experiments the Gprotein of vesicular stomatatis virus, VSV). The transport between cisternae, also mediated by vesicles, can be followed biochemically through the progression in the glycosylation of proteins. Like other transfers studied so far in vitro, these also require cytosol and ATP (e.g., Braell et al., 1984a). The 75 nm vesicles can be either coated or uncoated (Orci et al., 1986). The relationship between these two kinds of vesicles is revealed by the effect of two inhibitors. GTPγS, a non-hydrolyzable analog of GTP, produces an accumulation of buds and coated vesicles (Melançon et al., 1987). GTP hydrolysis is needed for the fusion of vesicles onto their target compartment and GTPγS blocks this process. N-ethylmaleimide (NEM) block, on the other hand, produces an accumulation of uncoated vesicles (Melançon et al., 1987). As discussed later, NEM blocks the docking of vesicles to the target membrane (see below). Simultaneous treatment with GTPγS and NEM produces an accumulation of coated vesicles (Orci et al., 1989); therefore, the coated vesicles are the precursors of the uncoated vesicles. Both kinds of vesicles should accumulate if the two were produced independently. Alternatively, if the uncoated vesicles were the precursor, only the uncoated vesicles would accumulate. The accumulation of vesicles derived from the Golgi stacks in a cell free system in the presence of GTPγS, permitted the isolation and direct biochemical study of the COPI (coatomer) coated vesicles (Malhotra et al., 1989; Serafini and Rothman et al., 1992). The COPI coatomer is composed of seven proteins (α, β, β', γ, ε and ζ) ranging in molecular weight between 60 and 160 kDa. β-COP has been localized with immunofluorescence and immunoelectron microscopy in the CGN and TGN and has also been found in soluble cytoplasmic complexes (Duden et al., 1991b, Waters et al., 1991). Presumably, the cytosolic β-COP is the source of this subunit of the vesicle's coat. Members of the p24 protein family bind to COPI coatomeres and may have a role in the recruitment
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of COPI to the Golgi membranes (Dominguez et al., 1998). p24 and p23 are cargo receptors found in both COPI and COPII coated vesicles. In COPI coated vesicles, they are present in stoichiometric amounts relative to coatomer and the GTPase, ARF. The cytoplasmic domains of these proteins bind to coatomer (e.g., Sohn et al., 1996) and are needed for cycling in the early secretory pathway (e.g., Nickel et al., 1997). COPI coatomer vesicles also bind to KKXX (di-lysine motif) and KXKXX containing proteins. The KDEL receptor that binds to proteins containing the KDEL motif is also transported in these vesicles. The γ subunit recognized the lysine motifs (Harter et al., 1996). These motifs are retrieval signals (see Table 2, Chapter 10) in line with the role of COPI coated vesicles in retrograde transport. COPI is required for intra-Golgi retrograde transport in vitro (see Lin et al., 1999). COPI vesicles or transport intermediates can be isolated or produced in vitro and have a high level of Golgi-resident enzymes (Lanoix et al., 1999; Love et al., 1998) and KDEL receptors (Sönnichsen et al., 1996) suggesting that they are recycling intermediates. Although there is evidence for the involvement of COPI in anterograde transport (see previous section), the evidence for a role of COPI in retrograde transport is less ambiguous. Subunit of the coatomer bind directly to proteins with the di-lysine retrieval motif (Cosson and Letourneur, 1994). Furthermore, mutation in some of these subunits prevents recovery to the ER of proteins with the dilysine retrieval motif (Cosson et al., 1996). In view of these findings, some investigators are postulating that the role of COPI in anterograde transport is indirect, recycling components needed for COPII transport by retrograde transport. However, results of several experiments continue to support a direct role of COPI in both anterograde and retrograde transport. Immunocytochemistry with the electron microscope using colloidal gold and antibodies (to COPI subunits, KDEL receptors and proinsulin) have demonstrated that both anterograde transport of proinsulin and VSV G protein and retrograde transport of a KDEL receptor occur in COPI vesicles. These vesicles constitute two distinct populations that together account for at least 80% of the vesicles present. The COPI vesicles bud from every level of Golgi cisternae. Similar results were obtained in in vitro experiments (Orci et al., 1997). In addition to a retrograde pathway which involves COPI coat proteins, a transport pathway from Golgi to ER has been found which functions independently from COPI coat proteins (see Storrie et al., 2000). This pathway returns Golgi resident proteins (as well as protein toxins) to the ER and may have a primary role in the recycling of lipids. Microinjection of antibodies to coatomer, block recycling of KDEL receptor and a lectin-like molecule, ERGIC-53, from Golgi to ER. Proteins containing sequences recognized by the KDEL receptor are also inhibited (Girod et al., 1999). In contrast, microinjection of anti-COPI antibodies or an Arf-1 mutant (Arf is required for COPIcoated vesicle assembly) does not interfere with the transport to the ER of Golgi-resident glycosylation enzymes or Shiga toxin/Shiga-like toxin-1. However, overexpression of a Rab6 (a small GTPase, see discussion below) mutant blocks retrieval of Golgi-resident glycosylation enzymes and Shiga toxin/Shiga-like toxin-1. However, it has no effect on KDEL receptor, KDEL containing proteins or ERGIC-53. These observations indicate that there are two pathways for the retrieval of proteins from the Golgi to the ER. The protein p115 (TAP), which has a role in the transport from the VTC to the cis-Golgi (see http://www.albany.edu/~abio304/text/chapter_11.html (23 of 41) [3/5/2003 7:56:56 PM]
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previous section) is required for intra-Golgi transport (Waters et al., 1992) and is likely to function in tethering several steps. It has been implicated in the binding to the plasma membrane during transcytosis (Barroso et al., 1995). II. RECOGNITION OF TARGETS A. Cell Polarity In an organized tissue, many cells have regions of the cytoplasm and the cell surface that differ in composition and function. They are said to be polar. This polarity can be preserved when the cells are cultured on a solid medium. Epithelial and endothelial cells form sheets in which they are held together by junctional complexes which prevent exchange between the two domains and prevent exchanges between compartments. A diagrammatic representation of polar cells in Fig. 2 shows apical and basolateral surfaces. The adherens junction is responsible for adhesion between the cells. In vertebrates the tight junction and in other animals the septate junction, prevent the exchanges. Vertebrate epithelial and endothelial cells, are held together by the tight junctions at contact points, the desmosomes which serve as anchoring points for intermediate filaments. In the polarized cell, the apical and the basolateral surfaces have distinct lipid and protein domains (Simons and Fuller, 1985). This distinct composition could not be maintained unless the two were prevented from exchanging materials and the various components were specifically targeted when newly synthesized. Essentially, tight junctions play a dual role as barriers and fences (see Chapter 4) (e.g., see Gumbiner, 1993; Anderson and Van Itallie, 1995). The barrier function refers to the tight seal that prevents diffusional exchanges between separate compartments The fence function refers to the prevention of exchanges between the basolateral and apical domains of the plasma membrane to maintain specialized functions, such as unidirectional secretion or active transport across the sheets. In addition to their structural role, cell junctions can also provide signals that initiate cascades involved in cell growth and differentiation (e.g., see Clark and Brugge, 1995; Takahashi et al., (1998); Reichert et al., 2000). For example, note that components of the signaling systems are present at junctions (see below). In some cases, the role of the cell junction component may more direct, for example, the adherens junction protein β-catenin (see below) is translocated into the nucleus and binds to a transcription factor (see Nusse, 1997; Eger et al., 2000). Similarly, the junctional component CASK (see Hata et al., 1996) is translocated into the nucleus (Hsueh et al., 2000) and is required for EGF receptor localization and signalling in the nematode Caenorhabditis elegans. CASK is membrane-associated guanylate kinase that is bound to the adhesion protein, syndecan, at epithelial cell junctions (Cohen et al., 1998). In the nucleus, a complex of CASK and Tbr-1 binds to a specific DNA sequence. Tbr-1 is a T-box transcription factor that is involved in forebrain development. The complex of the CASK and Tbr-1 activates several genes containing the T-box. T-box gene family have a binding domain, the T-domain which is important for development.
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The structure and formation of tight junctions as well as the factor that underlie the formation and maintenance of distinct membrane domains have been explored. Maintenance of polarity Some progess has been made in the study of the maintenance of polarity in Drosophila epithelium. Proteins which localize to one of three surfaces and suspected to have a role in organizing the corresponding domain have been called epithelial cell surface organizers (ECSOs) (see Tepass, 1997). These include cadherins (see below) that localize laterally (see Drubin and Nelson, 1996), Crumbs proteins of Drosophila that localize to the apical surface in (e.g., Wodarz et al., 1995) and β1, α integrins (see Chapter 6) located in the basal surface of kidney epithelium (Sorokin et al., 1990) and Madin-Derby canine kidney (MDCK) cells (e.g., Schoenenberger et al., 1994). ECSOs are thought to be recruited and retained to their domain by external signals such as homophilic adhesion in the case of cadherin. The interaction of ECSDOs with cytoplasmic factors permit interactions with the cytoskeletal system. In Drosophila, the mutation in four genes (stardust, sdt, bazooka, baz, crumbs, crb, and discs lost, dlt) has been shown to be lethal by eliminating polarity in epithelial cells (see Tepass, 1997). CRB and DE-cadherin are considered key regulators of polarity. CRB is an integral membrane protein with a single transmembrane domain. It contains thirty EGF-like (for other examples see Chapter 6, Fig. 10 and 11) and four laminin AG domain-like repeats in its extracellular segment and a short cytoplasmic tail of thirty seven amino acids. CRBs is expressed apically in ectodermally derived epithelia. Interestingly the mRNA for CRBs is found in the apical cytoplasm ( Tepass et al., 1990), suggesting that it is the mRNA and not the protein which is targeted to this region. Several examples of proteins targeted via their mRNA are known (see Chapter 10). Overexpression of CRB expands the apical plasma membrane and reduces the basolateral domain. In contrast, BAZ and DLT are cytoplasmic proteins with protein-protein interacting domains such as the PDZ domain. At least in vitro, one of the PDZ domains of DLT binds to the cytoplasmic domains of CRB ( Bhat et al., 1999, Klebes and Knust, 2000) and the laterally localized Neurexin IV (NRXIV). Interference with DLT (mutations or introduction of double-stranded RNA) lead to mistargeted CRB and NRX IV and disruption of epithelial polarity. The apical distribution of DLT depends on the presence of CRB. These and other observations suggest that CRB and DLT together with other proteins define the localization of the zonula adherens ( Klebes and Knust, 2000) and independently the loss of cell polarity (Grawe et al., 1996). An additional protein, Scribble was found to be necessary to maintain the polarization of cells (Bilder and Perrimon, 2000), as development proceeds in Drosophila embryogenesis. Scribble is localized to the epithelial septate junction. Without Scribble, adherens proteins no longer assemble in the apical-basolateral region interface and apical proteins are no longer localized at the apical region. Scribble, a protein of 195 kDa (calculated from the cDNA), was found to contain many protein-protein binding domains and is likely to correspond to a scaffold needed for other proteins to assemble.. The carboxy-end has four PDZ domains (the initials of the first three proteins that were discovered with the motif). The amino-end contains sixteen leucinerich repeats. Similar repeats are known to bind RhoA and Rac-GTPases, known to be associated with polarity and junctions (Jou and Nelson, 1998; see Kaibuchi et al., 1999). Many PDZ proteins, including Scribble localize at septate junctions, although others localize elsewhere. The actual molecular role of Scribble is still unknown. It may have a a role in targeting or it might even be part http://www.albany.edu/~abio304/text/chapter_11.html (25 of 41) [3/5/2003 7:56:56 PM]
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of the fence that prevents exchanges between the two regions. Components of tight junctions EM studies of thin sections (Farquhar and Palade, 1963) revealed discrete sites at which tight junctions produced close contacts between cells ("kissing points"). Freeze-fracture EM showed anastomosing strands in the cytoplasmic leaflet of the plasma membrane with complementary grooves in the external face of the bilayer (see Staehelin, 1974).
Fig. 2. Arrangement of epithelial cells forming a sheet.
Occludin, a 60 kDa integral protein was identified in tight junction strands (Furuse et al., 1993; Ando-Akatsuka et al., 1996). The cloning of the corresponding cDNA (see Chapter 1) and hydrophobicity plots (see Chapter 4) showed that occludin had four possible transmembrane domains and three cytoplasmic domains including the amino- and carboxy-terminals (see AndoAkatsuka et al., 1996). Occludin is involved in forming barriers and fences. The barrier function is shown, for example, by the increase electrical resistance across the epithelium when chicken occludin is overexpressed in under conditions in which they formed tight junctions (McCarthy et al., 1996). Expression of the carboxy-terminally truncated occludin, rather than wild-type occludin (Balda et al., 1996), was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain, indicating that occludin is also involved in providing an apical/basolateral intramembrane diffusion barrier. More recently, claudin-1 and claudin-2, two 23 kDa integral membrane proteins, were found in tight junctions of chick liver or transfected MDCK cells (Furuse et al., 1998a). Hydrophobicity analysis (see Chapter 4) indicated four possible transmembrane domains and cDNA analysis showed no sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy (see Chapter 1) revealed that both claudins were present in the tight junction strands. When introduced in fibroblasts lacking tight junctions, these proteins induced the formation of a network of strands and
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grooves at contact sites (Furuse et al., 1998b). The strands more closely resemble those in native tight junctions, suggesting that the occludins are major components of the junctions. Extensive studies indicate that the claudin family contains many members, as many as fifteen have been found in data-base searches of cDNA (Morita et al., 1999a,b,c; Tsukita and Furuse, 1999). Certain claudins appear to be associated with specific tissues. Claudin-5 and -6 have been found in tight-junctional strands of endothelial, but not of epithelial cells (Morita et al., 1999b). Clostridium perfringes enterotoxin, which binds specifically to claudin-3 and -4, were shown to disrupt tight junction strands in transfected L fibroblast cells and MDCK cells (Sonoda et al., 1999) implicating their presence in those junctions. The possibility of other molecular components forming tight junctions cannot be excluded at this time (see Tsukita and Faruse, 1999) Aside from the proteins forming tight junctions, there must be some structural basis for establishing polarity. In addition to targeting sequences and corresponding receptors there must be an assembly that includes cytoskeletal elements and permits the transfer of cargo to specific locations. The generation of polarity is of fundamental importance in the physiology of a variety of cells. A general model of the development of cell polarity has been presented (Drubin and Nelson, 1996). In this model, clues at the surface lead to localized assemblies of submembrane elements, including the cytoskeleton responsible for the organization of a pathway along an axis of polarity. In the case of epithelial cells, the position of adhesion receptor proteins is determined by adhesion to other cells or the extracellular matrix (ECM) (for cell-cell adhesion, E-cadherin; for adhesion to the ECM, the integrins) (see also Fig. 7C, Chapter 4). This interaction generates localized assembly of cytoskeletal elements. Integrins are bound by α-actinin and talin which recruit actin and actinassociated proteins. In turn, these serve as a scaffold for the assembly of signaling components, such as adhesion kinase and components of the Ras pathway (such as SOS and Grb2 and GTP-binding proteins), and may even lead to regulating gene expression as already discussed. Similarly, cadherin recruits cytoplasmic proteins such as β-catenin, plakiglobin and P120. The binding of these proteins to α-catenin (that has some homology with vinculin) may connect the complex to actin. Cadherincatenin complexes recruit kinases (Src and Yes) and protein-tyrosine phosphatase, components associated with signaling (see Chapter 7). GTP-binding proteins and Ras may associate with the complex. In addition, the region that remains free, the apical region, also seems to respond and assemble a distinct actin network cross-linked with villin, fimbrin and myosin I. Talin, α-actinin and vinculin are involved in the formation of fibers containing actin at focal contacts (see Chapter 23). Catenin, vinculin, α-actinin and plakoglobin have a similar mission but are part of an actin containing belt connecting epithelial cells and are attached to cadherins. Fimbrin and villin are actin bundling proteins (villin is present only in microvilli) (see Chapter 24). Talin, α-actinin and vinculin are involved in the formation of fibers containing actin at focal contacts. The events accompanying cell adhesion also redistribute the microtubules with consequences on the targeting of secretory products. Depletion of kinesin disrupts the basolateral delivery, and depletion of kinesin and dynein disrupts apical delivery (Lafont et al., 1994). Kinesin and dynein are motor molecules that move along microtubules (see Chapter 24). The difference between the microtubules transport to the two faces suggest that the organization of the microtubules is distinct in the two http://www.albany.edu/~abio304/text/chapter_11.html (27 of 41) [3/5/2003 7:56:56 PM]
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systems. The determination of specific targeting of secretory vesicles is clearly closely dependent on these events. In yeast, this is shown by mutations of the genes coding for sec6/sec8 which lead to the accumulation of secretory vesicles in the bud of a daughter cell (Novick et al., 1990; TerBush, 1996). The proteins are found only at the tip of the bud, hence they determine polarity. The sec6/sec8 complex, also known as exocyst, has been implicated in exocytosis and is specifically located at sites of vesicle fusion. In yeast, the complex contains seven subunits of 70 to 155 kDa, whereas in the rat, the complex has eight components ranging from 71 to 110 kDa (see Hsu et al., 1996, 1998). In rat brain, sec6 and sec8 are two components of a 17S complex of 743 kDa homologous to the yeast Sec6/8/15 complex of 834 kDa, which is required for exocytosis. The rat brain complex associated with the plasma membrane has been implicated in exocytosis by its immunoprecipitation with syntaxin, a plasma membrane protein critical for neurotransmission (see above and Chapter 22). In the rat brain, the complex is present at sites of neurosecretion such as the hippocampal synapses (Hsu et al., 1996) and is essential for survival. Mice with a mutation in Sec8 die early during embryogenesis at the primitive streak stage (Friedrich et al., 1997). In the MDCK cell line, the complex is required for calcium dependent cell adhesion (Grindstaff et al., 1998). When the cells are rendered permeable by streptolysin, Sec8 antibodies inhibit delivery of LDL receptor to the basallateral membrane, but not the delivery of the receptor for the nerve growth factor p75NTR to the apical membrane. These findings suggest that the complex is needed to recruit vesicles to specific domains. Similar conclusions were reached in neuronal tissues where the sec6/8 complex seems to specify sites for targeting vesicles at domains of neurite outgrowth and potential active zones during synaptogenesis (Hazuka et al., 1999). B. Targeting of Plasma Membrane Proteins Viral coat proteins with different plasma membrane targets share the transport pathway through the Golgi system (Rindler et al., 1984), as shown by immunological EM methods using colloidal gold markers of different sizes in doubly infected cells. What routes do these glycoproteins follow after leaving the TGN? Are they targeted directly to their surface of residence or do they make a stopover at the other surface? The proteins are sorted out in the TGN (Rodriguez-Boulan and Nelson, 1989). Whether the delivery is direct or indirect can be determined by growing the cells in sheets so that either the apical or basolateral surface is separately accessible to antibodies or proteases. A block in the transport would implicate a stopover in the alternative surface. The answers are not simple. Some of the glycoproteins are sorted out in the TGN so that they are targeted directly to either the apical or the basolateral surface. The delivery of the G-protein of VSV to the basolateral surface (Pfeiffer et al., 1985) and of the hemagglutinin of influenza virus to the apical surface is direct (Matlin et al., 1983; Matlin and Simons, 1984). However, other patterns are possible. In rat hepatocytes, all membrane proteins appear to be delivered to the basolateral surface and eventually, the proteins destined to the
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apical surface are then rerouted to their final destination (Bartles et al., 1987; Schell et al., 1992). In other cells, such as a polarized intestinal epithelium cell line (Caco-2), apical proteins can either proceed directly to the apical surface or arrive first to the basolateral surface (e.g., Matter et al., 1990a, Le Bevic et al., 1990). How are the membrane proteins targeted? The targeting need not differ in principle from other targeting processes. The transported protein may have a targeting domain. Targeting could also have an entirely different mechanism, possibly involving the lipid components of the membrane and acyl chains attached to the targeted protein. As discussed below, there is evidence in some cases, for a targeting mechanism involving glycosphingolipids. The sorting machinery (such as the TGN) must recognize the signal. In addition, the protein must then be targeted to the appropriate membrane site, possibly by the presence of another targeting domain. A separate but related problem is the preservation of the makeup of the target membrane itself. Originally, most investigators assumed that plasma membrane proteins were targeted by a signal to the apical surface, a basolateral targeting occurring by default. However, sorting signals have been demonstrated in the cytoplasmic domain of proteins destined to the basolateral surface (Hunzinker et al., 1991, Casanova et al., 1991, Mostov et al, 1992). One set of signals appears to be contained in the Tyr-containing signals for endocytosis via clathrin-coated pits (Mostov et al., 1992). Mutations of the cytoplasmic tail block basolateral targeting and the proteins are delivered to the apical surface. Internalization signals, required for endocytosis, can substitute for basolateral signals (Collawn et al., 1991). It has been suggested that the signal consists of the presence of a reverse βturn in the protein. Some signals are distinct from the endocytotic signal (e.g., Hunziker et al., 1991, Aroeti et al., 1993) and, in some cases, mutation of the cytoplasmic Tyr that blocks endocytosis has no effect on basolateral sorting (Hunziker et al., 1991). A short amino acid sequence (approximately 14 residues) serves as a signal for basolateral localization of some proteins (Yokode et al., 1992; Mostov et al., 1992). When expressed in livers of transgenic mice, the LDL receptor containing this sequence is targeted to the basolateral surface. Mutant receptors lacking the sequence are delivered to the apical side. The probable sequence of a cytoplasmic domain is Arg Asn X Asp XX Ser/Thr XX Ser, perhaps recognized by an adaptor molecule of the Golgi. In polarized MDCK cells the transferrin receptor (TR) is localized in the basolateral surface. After binding transferrin, ligand and receptor are taken up in coated pits by endocytosis and then returned to the basolateral surface. TR, whether synthesized de novo or recycled from vesicles, depends on its cytoplasmic tail for targeting. The targeting signal for both pathways is contained in residues 19 to 41. However, within this region the targeting sequence for the biosynthetic pathway is distinct from that for the endocytotic pathway (Odorizzi and Trowbridge, 1997) A variety of observations suggest more complexity than that presented in this discussion. For example, integrins and laminin have been shown to be transported from the TGN to the basolateral surface in separate vesicles (Boll et al., 1991), demonstrating the possibility that more than one pathway may be responsible for the same localization.
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The glycosyl phosphatidylinositol (GPI) anchor of certain proteins (see Chapter 4) could serve as an apical signal (see Simons and Ikonen, 1997) by clustering with glycosphingolipids, forming "rafts" that may correspond, at least in part, to caveolin containing elements (see Chapter 4 and Chapter 9). After the separation of lipid-linked proteins in the Golgi, vesicle budding could segregate them from other components. A role of GPI in apical targeting has been demonstrated in Madin-Darby canine kidney (MDCK) cells and intestinal cells by attaching a GPI anchor to proteins not originally destined to the apical surface (e.g., see Soole et al., 1985; Brown et al., 1989; Lisanti et al., 1989). Conversely, replacement of the GPI anchor of placental alkaline phosphatase with the transmembrane and cytoplasmic domains of VSV G, switched its targeting from the apical to the basolateral surface (Brown et al., 1989). The recognition of GPI-anchored proteins could occur in an early step of the cis-Golgi (see Brown and Rose, 1992) because these proteins were found associated with the glycosphingolipid microdomains at stages requiring cis-Golgi reactions. Although many observations are compatible with the raft -GPI model, other significant factors are likely to come into play. Fisher rat thyroid cells (FRT) (Zurzolo et al., 1993) and MDCK Concanavalin A-resistant cells (MDCK-ConAr) (Zurzolo et al., 1994) behave differently from other polarized epithelial cell lines. FRT cells target glycosphingolipids and six out of nine detectable endogenous GPI-anchored proteins to the basolateral surface. In contrast, two other GPI-anchored proteins are apical and one is present at either surface. Transfection (see Chapter 1) of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. The GPI anchored form of Decay accelerating factor (DAF) was targeted to the basolateral domain. Similarly, the Herpes simplex gD-1 protein attached to GPI in the form of fusion protein, gD1-DAF, was targeted basolaterally, where gD1-DAF was delivered directly from the Golgi apparatus to the basolateral surface. Similar discrepancies are exhibited by MDCK-ConAr cells. In most polarized epithelial cell lines (e.g., MDCK), both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane. In contrast, in MDCK-ConAr cells, gD1-DAF was sorted to both surfaces, but GlcCer was still targeted to the apical surface (Zurzolo et al., 1994). In both MDCK and MDCK-ConAr cells, gD1DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In the FRT cell line gD1-DAF and GlcCer were both targeted basolaterally. Although gD1-DAF and glucosyl ceramide distributed to the basolateral surface, gDI-DAF did not associate with membrane clusters. Among several possible alternatives, this surprising finding could be explained by the presence of a small subset of specialized clusters available for basolateral targeting. In addition, in some cases, the role of the GPI anchor is in doubt. In MDCK cells, Thy-1 (a glycoprotein of 25 kDa and unkownn function present in mouse thymocytes, T-cells and neurons) anchored to GPI was delivered apically. However, a truncated form of Thy-1, lacking 22 out of 31 hydrophobic amino acids at the carboxy-terminal, still resulted in apical secretion of Thy-1 despite the fact that the GPI anchor was not attached (Powell et al., 1991). It would therefore seem that Thy1 contains apical targeting information in its protein sector, as well as in the GPI anchor. GPI anchors are synthesized in the ER and added to primary translation products while they are http://www.albany.edu/~abio304/text/chapter_11.html (30 of 41) [3/5/2003 7:56:56 PM]
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being translocated across the ER membrane (see Thomas et al, 1990). In the plasma membrane, the GPI anchors are attached to the external leaflet of the plasma membrane. The motif in the protein directing attachment to the GPI resides in the amino acid sequence. The signal at the carboxy-end of the proteins differ with the protein (see Medof et al., 1996). Characteristically, GPI anchored proteins lack charged amino acids at the carboxy-end of the protein. An additional signal, 15 to 30 amino acids upstream of the terminal hydrophobic stretch of the protein, is needed for GPI anchoring (see Medof et al., 1996). In at least some cases, targeting may be a function of the lipid composition of the membrane. The outer leaflet of the apical membrane of the epithelial Madine-Darby canine kidney (MDCK) cells contains mainly glycosphingolipids held together by H-bonds. These lipids exclude glycerol-based phospholipids (Thompson and Tillack, 1985). Similarly, glycosphingolipid clusters are present in other membranes (see Chapter 4, Section VI). Signals other than GPIs have also been found (e.g. see Weimbs et al., 1997). Saturated acylated proteins (e.g., Src family kinases) appear to be targeted to detergent resistant membrane domains which are thought to correspond to rafts (see Chapter 4). In contrast prenylated proteins (e.g., Ras) are usually not found in these domains (Melkonian et al., 1999). This is likely to be the consequence of the ordered environment of lipid rafts or caveolae (see Brown and London, 1998) which is more likely to favor the incorporation of acyl chains. These chains tend to be present in an extended configuration, whereas the prenyl moieties are branched and bulky. Studies in intact cells confirm these observations (Zacharias et al., 2002). This study used FRET (see Chapter 1) a technique which allows the study the interaction between two chromatophores separated by 10 nm or less and therefore the proximity of the protein pairs. GFP variant pairs (donor-acceptor) were used. They were combined to peptides with consensus sequences for either acylation or prenylation or with caveolin, a protein components of rafts. The fluorescent GFP variants had to be modified to prevent their dimerization. Again, the acyl proteins but not the prenylated proteins were found in rafts. Furthermore, in contrast to the acylated proteins, the prenylated proteins were found insensitive to cholesterol depletion. The finding of the location of proteins in microdomains or caveolae is significant in relation to regulatory cascades. The lipid microdomains contain signaling kinases so that when receptors are bound to their ligand they move to these microdomains initiating a signaling cascade (see Pierce, 2002). N-glycans of secretory (Scheiffele et al., 1995) or in N- and O-glycans of membrane proteins act as apical signals (Yeaman et al., 1997; Gut et al., 1998). The hypothesis that N-linked carbohydrates are responsible for apical targeting was tested with three membrane proteins (Gut et al., 1998). Two are normally not glycosylated and another is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting. However, the presence of cytoplasmic basolateral targeting motifs remained basolateral even when attached to N-linked sugars. To compare the role of GPI and N-glycans, GPI was attached to the rat growth hormone (rGH) which is normally secreted in non-polarized manner (Benting et al., 1999). This modification did not lead to an apical delivery. However, the addition of N-glycans to the GPI-anchored rGH did target mostly to the apical surface. A transmembrane form of rGH accumulated intracellularly unless attached to http://www.albany.edu/~abio304/text/chapter_11.html (31 of 41) [3/5/2003 7:56:56 PM]
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N-glycans that delivered them to the apical surface. The N-or O-linked oligosaccharide could possibly attach to lectins present in the rafts (such as VIP36) (see Fiedler and Simons, 1995). As with other transport systems, it has been possible to partially reconstitute the basolateral targeting system (Gravotta et al., 1990). The fusion of the vesicles to the surface requires energy and cytoplasmic extracts. A GTP-binding protein is presumed to be involved because the fusion is inhibited by GTPγS. As already discussed, GTP-binding proteins of the Rab family are thought to be involved in fusion of vesicles in the intracellular transport system. There are some indications that Rab8 is localized in the basolateral transport vesicles in MDCK cells. Once incorporated into one of the cell membrane faces, the diffusibility of the proteins in the plane of the membrane appears to be constrained by binding to the cortical cytoskeleton (Vega-Salas, 1987). Na+-K+, ATPase, the Na+-channel, and the anion exchange protein bind to ankyrin/fodrin complexes (see Nelson and Hammerton, 1989). The probable interactions involved in establishing and maintaining cell polarity are summarized in Fig. 3 (Nelson, 1992). C. Targeting in Secretion and Transcytosis Secretion may also require targeting to a specific cell surface. This may be a consequence of the common mechanism accounting for plasma membrane transport and secretion. Although some cells release secretory products around their perimeter, many polar cells release them only in specific regions of the membranes. Thus, the same kind of targeting has to be considered in secretion. In MDCK cells, endogenous constitutively secreted proteins are released in the apical domain (KondorKoch et al., 1985; Gottlieb et al., 1986), whereas exogenous proteins (produced after transfection) release at both the apical and basolateral cell surfaces.
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Fig. 3 Mammalian polarized epithelial cell organization of protein trafficking pathways. Protein sorting and transport between the trans Golgi network (TGN) and different cell surface domains are regulated. In the TGN, proteins can be sorted by signal-mediated or bulk flow pathways into different vesicles. Signal-mediated sorting of proteins to the apical plasma membrane (Ap-PM) is regulated by glycosphingolipid patching, whereas signal-mediated sorting of protein to the basolateral plasma membrane (BL-PM) is regulated by protein clustering of adaptor proteins; other signal-mediated pathways may also exist. Docking with targeting patches in each membrane is regulated by domain-specific GTPase cycles (Ap-GTPase, apical membrane GTPase; BL-GTPase, basal-lateral membrane GTPase) (from Nelson 1992). Reproduced by permission.
Most cells carry out receptor-mediated endocytosis (Chapter 9), in which the ligand is generally degraded in the lysosomes and the receptor is either degraded or recycled to the surface. However, in transcytosis, which is ubiquitous in epithelial cells, the endosomes transfer receptor and ligand to the surface of opposite polarity, so that the material traverses the cell. The transcytosis of polymeric immunoglobulin (poly-Ig) is probably the best understood of the various known cases. Poly-Ig is produced by plasma cells and transported through epithelial cells by transcytosis. In this process, the epithelial cell adds a polypeptide, the secretory component (SC) or secretory piece, part of the receptor molecule, to the poly-Ig. The poly-Ig receptor is originally incorporated in the basolateral surface, where it binds poly-Ig. After transcytosis, the endocytotic vesicle discharges its contents at the apical surface by exocytosis and the receptor is cleaved. So far, only one signal has been identified in transcytosis, the phosphorylation of a Ser in the cytoplasmic domain of the receptor (Casanova et al., 1990).
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Despite the flow of membranes from one pole of the cell to the other in transcytosis, the two surface domains remain distinct. This is illustrated by VSV G-protein, which is normally in the basolateral surface of the infected MDCK cells. When artificially inserted into the apical surface by fusing it to viral coats at low pH, the VSV-G protein is taken up by endocytosis and delivered to the basolateral surface (Matlin et al., 1983). Therefore, it would seem possible that portions of the membranes involved in transcytosis can be recycled to their original location by a process akin to transcytosis in reverse. D. Transport of the Vesicles What is responsible for the movement of vesicles? The information presently available suggests a varied pattern (see Bloom and Goldstein, 1998). The diffusion coefficient of granules in cells is approximately 2.5 x 10-10 cm2/s (Felder and Kam, 1994). It has been calculated that a vesicle 160 nm in diameter can diffuse for a distance of 10 µm in 10 minutes (Bloom and Goldstein, 1998). Therefore, no special mechanism is required in small cells for a variety of vesicular transport events. For a neuron, the distances from cell body to periphery, however, is prohibitive (the vesicles may have to travel 1 m or more from the cell body to the neuron terminal) and microtubular transport is essential. Normally, the transport from the compartments intermediate between ER and Golgi (ICs) to the Golgi, occurs on microtubules (Presley et al., 1997; Scales et al., 1997) as shown by direct observation using conjugates of VSV-G glycoprotein and green fluorescent protein (see Chapter 1, Chapter 10, Section III). However, in some cases, the absence of MTs does not preclude secretion at close to normal rates (e.g., van de Moortele et al., 1993). Apparently, in these cases, the Golgi cisternae segment in ministacks. These ministacks distribute throughout the cytoplasm (Rogalski and Singer, 1984) adjacent to IC sites and the ER (Cole et al., 1996). Presumably in these cases the transport can proceed efficiently by diffusion. These considerations and other presently available information suggest that in anterograde transport microtubules have no role in transport from ER to IC or in intra-Golgi transport. However, they are needed for transport from IC to Golgi or from TGN to the cell surface (see Lippincott-Schwartz, 1998). In the retrograde pathway, microtubules are also involved in many steps. Golgi to ER transport is driven by kinesin (Lippincott-Schwartz et al., 1995), a microtubular motor, and microtubules are involved in the transport of endosomes and lysosomes toward the centrosome, as shown by direct observation and the use of microtubular inhibitors (Matteoni and Kreis, 1987). In polarized epithelial cells, microtubules seem to be involved in the movement of vesicles to the apical surface (Nelson, 1991, 1992). Depolymerization of microtubules interferes with trafficking between TGN and the apical membrane (e.g., Rindler et al.,1987; Van Zeijl and Matlin, 1990) and decreases transcytosis from the basolateral to the apical surface (e.g., Matter et al., 1990b, Breitfeld et al., 1990). The presence of colchicine, vinblastine or nocodazole (all drugs which interfere with http://www.albany.edu/~abio304/text/chapter_11.html (34 of 41) [3/5/2003 7:56:56 PM]
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the microtubules) redirects the vesicles from the apical to the basolateral surface. In contrast, the basolateral traffic is not affected. However, in budding yeast, microtubules do not have a role in polarized secretion (e.g., Huffaker et al., 1988; Jacobs et al., 1988). In contrast, in these cells, actin (e.g., see Novick and Botstein, 1985) has been found to be involved in conjunction with Myo2p, a myosin V (see Chapter 24) (e.g., Santos and Snyder, 1997; Catlett and Weisman, 1998; Schott et al., 2000). The experiments carried out with mammalian cells indicate a primary role of microtubules in apical vesicular transport. The presence of myosin I in Golgi derived vesicles (Fath and Burgess, 1993) and in apical membranes (Mooseker and Coleman, 1989) also suggests a role of the actomyosin system in at least some of the processes of intracellular transport. Myosin I is found in vesicles in intestinal brush border cells where they are linked to actin filaments (Drenckhahn and Dermietzel, 1988). Similarly, myosin I was localized by immunoblotting and immunolabel negative staining of the isolated vesicles during the assembly of these cells (Fath and Burgess, 1993). This protein was found at the outer surface of Golgi associated vesicles during the assembly of these cells. The vesicles contained galactosyl transferase, a trans-Golgi enzyme, as well as alkaline phosphatase, an apical membrane targeted enzyme. The vesicles were also shown to bundle actin, suggesting that the actomyosin system functions in the peripheral translocation of vesicles. The results suggest an involvement of both microtubules and the actomyosin system, the latter perhaps only in the final step of the delivery in the apical pathway. However, the details are still not clear. How are the vesicles connected to the transporting systems? Present evidence implicates dynactin. Dynactin is a 1.2 MDa complex of ten peptides (and includes actin) that is required for cytoplasmic dynein motility and in vitro vesicle movement (Schroer and Sheetz, 1991). Electron microscopy of this complex shows an actin-like filament, 37 nm long, with laterally projecting sidearms (Schafer et al., 1994). A model of dynactin is shown in Fig. 4 (Schroer et al., 1996). The filamentous part of the molecule is made up of the actin related protein (Arp1) and probably one molecule of actin. The interaction between dynactin and the MT-dynein system probably involves the sidearms that contain a microtubule binding site (Waterman-Storer et al., 1995) and also bind dynein (Collins and Vallee, 1989). The most likely role of the Arp1 portion of the complex is to provide a connection to the cargo vesicle. Many myosins of the myosin I family bind directly to phospholipid. Myosin I isoforms associate with specific membranes (Baines et al., 1995). It is entirely possible then that dynactin binding to the vesicles is mediated by myosin, although other possibilities are also likely (e.g., ponticulin or an annexin, see Schroer et al., 1996).
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Fig. 4 Current model of dynactin structure. The localization of Arp1, actin-capping protein, p62 and p150Glued/P135Glued are based on ultrastructural analysis of antibody decorated molecules. The location of actin, p50, p24 and p27 is uncertain. Because of their similar properties, actin is likely to be in the Arp1 filament. The overexpression of p50 causes p150Glued to dissociate suggesting the location in the diagram. From Schroer et al., 1996, reproduced by permission.
E. Recycling of the Plasma Membrane The process of exocytosis adds a considerable amount of material to the plasma membrane. Much of this material is recycled through endocytosis. Because of this continuous recycling, the turnover time of the membranes of granules is relatively long compared to that of ordinary cytoplasmic proteins (Meldolesi, 1974). The availability of antibodies to membrane proteins of the luminal side of the secretory vesicle membrane has permitted following the fate of the secretory vesicles in the regulated secretion of catecholamine granules (Patzak and Winkler, 1986). Glycoprotein III (gpIII) is exposed to its fluorescently labelled antibody at exocytosis. The protein appears in coated pits and vesicles in the first 5 min after exocytosis. Then it passes through the smooth ER and reappears in the trans Golgi network and in dense-core secretory granules within 30 to 45 min. The protein was never found in the cisternal lumen, indicating that the membrane itself is being recycled. Similar results were found for the transferrin receptors (Woods et al. 1986). Their presence was demonstrated in several Golgi cisternae; therefore, recycling must involve some of the same steps followed by the transport of newly synthesized protein. These observations imply that some steps in the recycling process must involve transport in the opposite direction from that discussed in most of this chapter, that is retrograde transport. Retrograde transport is beginning to be studied by taking advantage of the effect of the antibiotic Brefeldin A (Tan et al., 1992). Brefeldin, a macrocyclic lactone synthesized by fungi, prevents the assembly of nonclathrin coated vesicles and blocks the transport from ER to Golgi (e.g., Klausner et al., 1992). The retrograde transport, however, is not inhibited, redistributing material such as enzymes from the Golgi back to the ER. This transport is also blocked by GTPγS, suggesting that http://www.albany.edu/~abio304/text/chapter_11.html (36 of 41) [3/5/2003 7:56:56 PM]
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GTP-binding proteins are involved in both forward and backward transport. F. Formation of Lysosomes and Secretory Storage Vesicles Lysosomes The study of the transport system in the formation of lysosomes received great impetus from the recognition of at least 30 human lysosomal storage disorders. I-cell disease, a deficiency in lysosomal enzymes, results from a failure in the recognition marker needed for targeting. Study of Icell mutants has permitted the identification of the recognition marker, the mannose-6-phosphate (M6P) residue, and the M6P receptor, MPR (Sahagian et al., 1981). The receptor spans the membrane and 10 kDa of its carboxy-terminal protrudes into the cytosol (Sahagian and Steer, 1985). Two distinct but related MPRs are known (see Kornfeld, 1992). One is a type I (amino group external to the cell) transmembrane glycoprotein of 275 kDa which does not require divalent cations. The other receptor is also a type I glycoprotein of 46 kDa. The bovine and murine forms of the latter, but not the human or porcine forms, require divalent cations for optimal binding. M6PRs bind to lysosomal hydrolases while they are transported from the trans-Golgi to the lysosomes. In turn, targeting signals in the M6PRs are needed to arrive at their final destination. The Golgi-localized γ-ear containing, ARF-binding (GGAs) adaptors have been found to bind to the lysosomal targeting signals of the cation-independent M6PRs, the acidic cluster-dileucine motif of their cytoplasmic tails. The GGAs, already implicated in protein trafficking between the Golgi and the endosomes, have all the properties expected for an adaptor mediating the binding of the receptors to the components needed for transport. They contain a Vps27p/Hrs/STAM (VHS) domain, binding sites for clathrin, a GTP-ARF binding domain and a domain that binds to proteins involved in coat assembly such as γ-synergin. The VHS domain of 153 residues is present in various proteins involved in endocytic trafficking (Lohi and Lehto, 1998). The M6PRs bind to the VHS domain of the GGAs. The GGAs were found to be present in the TGN, tubules and vesicles which bud from the TGN and the cell surface as expected from its presumed function (Zhu et al., 2001; Puertollano et al., 2001). Lysosomal enzyme precursors are transported through the common pathway, as indicated by immunocytochemical experiments (Geutze et al., 1984). However, processing continues on arrival in the cis-Golgi (Fig. 5, Kornfeld, 1987), where lysosomal hydrolases are recognized by GlcNAcphosphotransferase, which adds GlcNAc-phosphate to α-1,2-mannose residues of the hydrolases. The phosphotransferase probably recognizes a signal patch, because recognition is very sensitive to conformation changes. After this modification, M6P residues are exposed by removal of the Nacetylglucosamine and are recognized by MPR. The interaction between receptors and M6P residues is responsible for the lysosomal enzyme segregation (see von Figura and Hasilik, 1986; Kornfeld, 1987). The two can be detected together in buds and coated vesicles in the TGN, which eventually form lysosomes (Geutze et al., 1985, Griffiths et al., 1985). In addition to the processing of the oligosaccharides, lysosomal enzymes are proteolytically cleaved to their mature form (Gieselman et al., 1983).
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Fig. 5 Schematic pathway of lysosomal enzyme targeting to lysosomes. Lysosomal enzymes and secretory proteins are synthesized in the rough endoplasmic reticulum (RER) and glycosylated by the transfer of a performed oligosaccharide from dolichol-P-P-oligosaccharide (DOL). In the RER, the signal peptides (hatching) are excised. The proteins are translocated to the Golgi, where the oligosaccharides of secretory proteins are processedto complex-type units and the oligosaccharides of lysosomal enzymes are phosphorylated. Most of the lysosomal enzymes bind to mannose6) and are translocated to an acidified prelysosomal phosphate receptors (MPRs) ( compartment where the ligand dissociates. The receptors recycle back to the Golgi or to the cell surface, and the enzymes are packaged into lysosomes where cleavage of their propieces is ). The Pi may also be cleaved from the mannose residues. A small number of the completed ( lysosomal enzymes fail to bind to the receptors and are secreted along with secretory proteins ( ). These enzymes may bind to surface MPRs in coated pits ( ) and be internalized into the prelysosomal compartment. (
) N-Acetylglucosamine; (
) mannose; (
) glucose; (
)
) sialic acid. Reprinted with permission from S. Kornfield, Federation of American galactose; ( Societies for Experimental Biology Journal, Vol.1, No.6: 463, 1987.
The MPRs are recycled. Before the primary lysosomes are formed, the MPRs are sent back to the trans Golgi. Recovery of receptor probably follows its dissociation from the ligands brought about by lowering the pH, as in the case of endocytotic vesicles discussed in Chapter 9. Although most lysosomal enzymes remain segregated in vesicles, a small portion of the lysosomal enzymes are secreted and are thought to be recovered by endocytotic uptake after binding MPRs present at the surface (Willingham et al., 1981).
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Secretory storage granules Regulated secretion differs from constitutive secretion in the need to store the secreted products in the secretory vesicles until a physiological signal permits their discharge. Therefore, they must be separated from the other products destined to the cell surface. This separation occurs in the transGolgi, but the selection mechanism is still unknown. The process can be very selective. However, it also allows for packaging very different proteins in the same vesicle. The possibility that a receptor is involved in targeting storage secretory products is also supported by the observation that the precursor of insulin, proinsulin, is bound to Golgi membranes (Munro and Pelham, 1986), indicating the likelihood that a receptor is present. G. Synaptic Vesicles Presynaptic cells discharge neurotransmitters by exocytosis of their synaptic vesicles. Postsynaptic membrane receptors bind the neurotransmitters. These receptors are channels that open more frequently after binding the neurotransmitters. The increased conductance of the membrane initiates the depolarization that can culminate in an action potential (see Chapter 22). The presynaptic neurotransmitter vesicles are continuously discharged and continuously reformed. Most of the recycling involves the recovery of synaptic vesicles by endocytosis and their reloading with neurotransmitter (See Chapter 22). However, there is also a turnover of the synaptic vesicles themselves; their components are degraded and resynthesized in the cell body. Eventually, the newly formed vesicles must be targeted to the nerve terminals through axonal transport. The integral proteins follow the usual path from RER to TGN. These proteins obviously can be found at synaptic sites, but also (with few exceptions) throughout the Golgi system. All indications are that the forward or anterograde transport of newly synthesized synaptic vesicle components is microtubular and is powered by the motor protein of the kinesin family (Chapter 24). Kinesin is implicated by its association with vesicles (e.g., Morin et al., 1993) and by its accumulation when the axonal flow is blocked by ligation (Hirokawa et al., 1991). The vesicles and the kinesin (indentified by immunocytochemistry) accumulate on the cell body side of the ligature. Evidence from genetic studies also implicates a kinesin-like molecule. The unc-104 gene codes for a kinesin-like motor, thought to be neuron specific in the nematode Caenorhabditis elegans. This protein has a kinesin-like motor domain at the amino terminal, but otherwise it has little homology to other kinesins. Mutant alleles of this gene block the accumulation of synaptic vesicles in axons (Hall and Hedgecock, 1991). The movement of other vesicles is not affected, suggesting a unique targeting role for the protein coded by the unc-104 gene. The study of the biogenesis of synaptic vesicle proteins can be carried out most readily in neuroendocrine cells and neurons in culture. PC12 cells, derived from pheochromocytoma of the rat adrenal medulla, have characteristics of both neural and endocrine cells. They synthesize, store and release the neurotransmitter acetylcholine (Greene and Rein, 1977) and have both regulated and constitutive secretory pathways. In these cells, regulated secretion involves large dense-core granules related to chromaffin granules. Small electron-translucent vesicles are also present and are thought to be related to cholinergic synaptic vesicles. Dense granules were purified and shown to http://www.albany.edu/~abio304/text/chapter_11.html (39 of 41) [3/5/2003 7:56:56 PM]
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contain the regulated secretory protein secretogranin II. The synaptic protein synaptophysin was used as a marker for the smaller vesicles (Cutler and Cramer, 1990). Synaptophysin is a major integral protein of synaptic vesicle membranes. Pulse-chase experiments using immunoprecipitation, demonstrated that synaptophysin is associated with the smaller vesicles and does not occupy the dense granules at any time. These findings indicate two separately regulated secretion routes. In another study, synaptophysin was also traced by pulse-chase (Régnier-Vigouroux et al., 1991). It was found to follow a route involving the trans Golgi network (TGN). The protein was found to reach the cell surface from the TGN with a half time of 10 min and was found to cycle between cell surface and vesicles. The endosomal fraction was identified by exposing the terminals to peroxidases and tracing this enzyme. Peroxidase is taken up by endocytosis and represents the material dissolved in the external medium. The molecular mechanism by which the synaptic vesicle proteins are sorted out is not clear. A common sequence motif that could be recognized by receptors used in targeting has not been found in examining the various proteins of the secretory vesicles, and signals involving secondary or tertiary folding are suspected. A role of specific complexes of synaptic proteins in targeting is also possible because multimeric complexes can be recovered from synaptic vesicles after detergent treatment (Bennett et al., 1992). SUGGESTED READING Bloom, G.S. and Goldstein, L.S.B. (1998) Cruising along microtubule highways: how membranes move through the secretory pathway, J. Cell Biol. 140:1277-1280. (MedLine) Kirchhausen, T. (2000) Clathrin, Annu. Rev. Biochem. 69:699-727. (MedLine) Lippincott-Schwartz, J. (1998) Cytoskeletal proteins and Golgi dynamics, Curr. Opin. Cell Biol. 10:52-59. McNiven, M.A., Cao, I., Pitts, K.R. and Yoon, I. (2000) The dynamin family of mechanoenzymes: pinching in new places, Trends Biochem Sci 25:115-120.(MedLine) Mellman, I. and Warren, G. (2000) The road taken: past and future foundations of membrane traffic, Cell 100:99-112. (MedLine) Morgan, A. (1995) Exocytosis, Essays in Biochemistry 30:77-95. Nelson, W. J. (1992) Regulation of cell surface polarity from bacteria to mammals, Science 258:948955. (MedLine) Robinson, M.S. (1997) Coats and vesicle budding, Trends in Cell Biol. 7:99-102. Schekman, R. and Orci, L. (1996) Coat proteins and vesicle budding, Science 271:1526-1533. (MedLine)
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Simons, K. and Ikonen, E. (1997) Functional rafts in cell membranes, Nature 387:569-572. (MedLine) Springer, S., Spang, A. and Schekman, R. (1999) A primer on vesicle budding, Cell 97:145-148. (MedLine) Weimbs, T., Low, S.H., Chapin, S.J. and Mostov, K.E. (1997) Apical targeting in polarized epithelial cells: there's more afloat than rafts, Trends in Cell Biol. 7:393-399. Wieland, F. and Harter, C. (1999) Mechanisms of vesicle formation: insights from the COP system, Curr. Opin. Cell Biol. 11:440-446. (MedLine) WEB RESOURCES Kirchhausen, T. and Bruce, A. Clathrin-coat formation in time and space: modelling. http://www.hms.harvard.edu/news/clathrin REFERENCES Search the textbook
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Chapter 11: References
Back to Chapter 11
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Balch, W. E., Glick, B. S. and Rothman, J. E. (1984b) Sequential intermediates in the pathway of intercompartmental transport in a cell free system, Cell 39:525-536. (Medline) Balch, W. E., Wagner, R. R. and Keller, D. S. (1987) Reconstitution of transport vesicular stomatitis virus G protein from the endoplasmic reticulum to the Golgi complex using a cell free system, J. Cell Biol. 104:749-760. (Medline) Balch, W.E., McCaffery, J.M., Pluttner, H. and Farquhar, M.G. (1994) Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum, Cell 76:841-852. (Medline) Balda, M.S., Whitney, J.A., Flores, C., Gonzalez, S., Cereijido, M. and Matter, K. (1996) Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein, J. Cell Biol. 134:1031-1049. (Medline) Bankaitis, V.A., Aitken, J.R., Cleves, A.E. and Dowhan, W. (1990) An essential role for a phospholipid transfer protein in yeast Golgi function, Nature 347:561-562. (Medline) Bannykh, S.I. and Balch, E.E. (1997) Membrane dynamics at the endoplasmic reticulum-Golgi interface, J. Cell Biol.138:1-4. (Medline) Barlowe, C. (1998) COPII and selective export from the endoplasmic reticulum, Biochim. Biophys. Acta 1404:67-76. (MedLine) Barlowe, C. and Shekman, R. (1993) SEC12 encodes a guanine-nucleotide-exchange factor essential for transport vesicle budding from the ER, Nature 365:347-349. (Medline) Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Ravazzola, M. Amherdt, M. and Shekman, P. (1994) COPII: a membrane coat formed by SEC proteins that drive vesicle budding from the endoplasmic reticulum, Cell 77: 895-907. (Medline) Barnard, R.J., Morgan, A. and Burgoyne, R.D. (1997) Stimulation of NSF ATPase activity by α-SNAP is required for SNARE complex disassembly and exocytosis, J. Cell Biol. 139:875-883. (Medline) Barroso, M., Nelson, D.S. and Sztul, E.(1995) Transcytosis-associated protein (TAP)/p115 is a general fusion factor required for binding of vesicles to acceptor membranes, Proc. Natl. Acad. Sci. USA 92:527531. (MedLine) Bartles, J. R., Feracci, H. M., Stieger, B. and Hubbard, A. L. (1987) Biogenesis of the rat hepatocyte
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12. Energy and Biological Systems
12. Energy and Biological Systems. I. II. III. IV. V. VI. VII.
Free Energy Coupled Reactions Redox Potentials ∆G as a Function of the Concentration of Reactants ∆Go Energy Cost of Transport Muscle Contraction Suggested Reading Web Resources References Back to List of Chapters
Energy is central to cell function. Energy is needed for carrying out the tasks of the cell, the displacement of mass in biological movement, the fluxes of solutes through membranes against electrochemical gradients (concentration and electrical potential gradients), the emission of light (bioluminescence), and the synthesis of macromolecules to replace cell components that are broken down. Ultimately, the energy must come from the environment: from the light absorbed by photosynthetic pigments and from the substances that serve as substrates in the cell's metabolism. In a sense, cells or cell organelles may be regarded as transducer systems, i.e., devices that convert one form of energy into another. The present chapter reviews very briefly some of the principles of bioenergetics and is intended to serve as a framework for the discussion of later chapters. I. FREE ENERGY The Gibbs free energy change, ∆G, expresses the maximal amount of work that can be performed by a system or, conversely, the minimal amount of energy input required for work. The definition of ∆ G is such that net work can be performed by a reaction when ∆G is less than zero; i.e., the reaction is exergonic. Unless coupled to exergonic reactions, endergonic reactions (∆G > 0) take place to a very limited extent. When ∆G = 0, the system is at equilibrium. For reactions taking place at constant temperature, ∆ G can be expressed as shown in Eq.(1). ∆H is the heat transferred between the system under discussion and the surrounding at constant temperature. In an exothermic reaction, heat is released by the system (∆H < 0), whereas in an endothermic reaction, heat is absorbed by the system (∆H > 0). The entropy of the system, ∆ S, is defined as shown in Eq. (2), where dQ is the chance in the heat of the system.
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∆G = ∆H - T∆S (1) dS = -dQ/T (2) In practical terms, the entropy is related to the organization of the system. In an isolated system (i.e., one that does not exchange heat or matter with its environment) ∆ S must be positive. Two examples of exergonic reactions are shown below in Eqs. (3) and (4). The superscripto (e.g., in ∆Go, ∆Ho) means that the quantity has been determined under standard temperature (usually 298 K), pressure (1 atmosphere), and concentration (1 molal). Since biological reactions do not take place under these standard conditions, we should be concerned with ∆ G, but for simplicity the system will be assumed to be at standard conditions. The difference between ∆G and ∆Go and the meaning of these two parameters are discussed in Sections IV and V. The oversimplification of equating ∆G to ∆Go has been vigorously challenged (Banks and Vernon, 1970). The ∆G value changes as a function of concentration and, in the case of active transport of ions, ∆G is also a function of membrane potential. This aspect will be taken up in Sections IV and VI. ATP →ADP + Pi (3) ∆Ho = -7.4 kcal; -T∆ So = -2.6 kcal ∆Go = -10.0 kcal Equation (3) represents the hydrolysis of the terminal phosphate of adenosine triphosphate (ATP) to form adenosine diphosphate (ADP) and inorganic orthophosphate (Pi). In this reaction ∆Ho and ∆ Go are both less than zero. However, ∆H need not always be less than zero for a reaction to take place, as shown for the activation of NADH dehydrogenase, represented by E in Eq. (4). E + AMP →E-AMP (4) ∆Ho = 12.5 kcal -T∆So = -17.3 kcal ∆Go = -4.8 kcal The units used for energy vary in part for convenience, in part for historical reasons. The calorie (or kilocalorie = 1000 cal) has been frequently used, and kilocalories can easily be converted to electrical http://www.albany.edu/~abio304/text/chapter_12.html (2 of 22) [8/10/2001 1:30:57 PM]
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potential units by dividing by the Faraday constant, F (23 kcal/V). For a direct comparison of ∆G values to the experimentally obtained redox potentials, see Section III. In recent years, the joule (1 X l07 ergs, 0.24 cal) or kjoule have been frequently used. II. COUPLED REACTIONS Chemical reactions with ∆ G > 0 can take place to a significant extent, only if coupled to another reaction in which ∆G < 0. At constant pressure and temperature, the coupling requires the product of the first reaction to be the reactant in the subsequent reaction. If the reactions were not linked in this manner, they would occur independently of each other and therefore no energy could be transferred between these molecules by ordinary means. An hypothetical set of coupled reactions is shown in Eq. (5).
The hypothetical product of reaction (5a), C, is a reactant in the second reaction (5b). Consequently, in the overall reaction (5c), C does not appear at all, since no net change in C occurs. The G of the overall reaction (5c) is less than zero since the G of the coupled reactions is additive. Equation (6a) gives the formal description of an actual biochemical reaction that requires an energy input in order to proceed to a significant extent. The energy-yielding reaction is represented by Eq. (6b). Equations (6a) and (6b) ignore the reaction needed to link the two. In fact, the molecular details need not be known; only knowledge of the overall reaction is necessary.
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The reaction actually takes place by the mechanism shown in Eqs. (6d) and (6e). An intermediate glutamylhttp://www.albany.edu/~abio304/text/chapter_12.html (3 of 22) [8/10/2001 1:30:57 PM]
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P-E in the first reaction is a participant in the second reaction.
In Eqs. (6d) and (6e), E represents an enzyme molecule. The energy for reaction (6a) is said to be coupled to the hydrolysis of ATP, (reaction 6b), and the actual common intermediate (glutamyl-P-E) need not be known to carry out calculations involving the energy balance of the reactions, i.e., ∆G. The synthesis of ATP from ADP and Pi in so-called substrate-level phosphorylation, takes place by a coupling similar to that discussed in these reactions. They involve as an intermediate a phosphorylated substrate. In contrast, in most cells, the reactions responsible for most of the synthesis of ATP involve oxidation-reduction reactions of the cytochrome system in a process referred to collectively as oxidative phosphorylation. These reactions are likely to occur by a distinct mechanism (see later discussion). In eukaryotic cells, the cytochrome system is in the mitochondria. An example of substrate-level phosphorylation is the oxidation of glyceraldehyde 3-phosphate to form 3phosphoglycerate in one of the reactions of glycolysis. In this step, ADP is concomitantly phosphorylated to form ATP. Equations (7a) and (7b) represent the process schematically.
The oxidation of glyceraldehyde 3-phosphate has a ∆G of less than -10 kcal, whereas the phosphorylation of ADP is endergonic and has a ∆G of about 10 kcal. In effect, the energy yielded by one reaction is trapped by the synthesis of ATP. A later event, hydrolysis of the ATP, can yield the energy necessary for other processes. The details of the reactions show that in one reaction, Eq. (8a), a product, 1,3-phosphoglycerate, is formed that is used in the subsequent reaction depicted by Eq. (8b). The coupling scheme shown by Eqs. (8a) and (8b) is somewhat oversimplified but sufficiently detailed to illustrate this mechanism.
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In the past, couplings similar to those represented in Eqs. (8a) and (8b) have also been postulated for the phosphorylation of ADP in the oxidative phosphorylation reactions of the cytochrome chain. The mechanism of phosphorylation involving the cytochrome chain is still not completely understood; however, it does not involve a phosphorylated intermediate. Not all the energy derived from a reaction can be used for the synthesis of ATP. Some of the energy is released as heat (Poe and Estabrook, 1969). This release of heat is of great physiological importance in mammals. The nonshivering thermogenesis that occurs in brown fat apparently results from an increase in the energy dissipated as heat in the oxidative reactions in mitochondria (Nicholls and Locke, 1984). This form of thermogenesis plays a fundamental role in cold adaptation and arousal from hibernation. The idea of energy coupling and in particular the concept that ATP can be used to power in vivo reactions, has been questioned at various times (e.g., see Banks and Vernon, 1970). Although the concepts are sometimes misunderstood and should be used with caution (e.g., see McClare, 1972), they are nevertheless valid. III. REDOX POTENTIALS In some reactions, a reactant serves as an electron donor and another as an electron acceptor. In these cases, the ability to exchange electrons can be expressed as an oxidation-reduction potential (redox potential). Removal of electrons from the donor and acceptance of electrons by the acceptor can be formulated as separate reactions, as shown in Eqs. (9a) and (9b). In these equations, e represents an electron that is being exchanged. Equation (9c) represents the overall reaction. "Red" and "ox" indicate, respectively, the reduced and oxidized species of the compound. Oxidation-reduction reactions must be coupled to each other in order to take place, but they can be separated out, as done in Eqs. (9a) and (9b), for calculations involving the energetics of the system.
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If an electrical connection is made between two containers, these reactions can actually take place separately. Each container is called a "half-cell." The system made up of two half-cells is represented in Fig. 1, in which the electrode of half-cell A receives an electron that serves to reduce the component of half-cell B.
The potential of a half-cell is conventionally defined by comparison to that of an H2 half-cell that operates as shown in Eq. (10). When electrons are given to the H electrode, the potential is considered to be less than zero (i.e., Eh is negative). When the hydrogen half-cell is the electron donor, the potential is considered greater than zero (Eh is positive). In comparing the tendency of two reactions to take place, we are actually concerned with the difference in the redox potential between the two reactions (Eh1 - Eh2) which is expressed as the ∆E of the two half-cells, and the ∆G of the overall reaction is represented by Eq. (11). ∆G = -nF ∆E (11) Here, ∆E is the redox potential difference (in volts), n refers to the number of electrons and F is the Faraday constant (23 kcal/V). The use of E and its interconvertibility with ∆G can best be illustrated by an example. A hypothetical redox scheme is shown in Eq. (12a). The two half-reactions (i.e., the portions of the reactions occurring in the two half-cells) are shown in Eqs. (l2b) and (l2c).
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Since reaction (12c) is driven by reaction (6.12b), ∆Eo = 0.30 V - (-0.25 V) = 0.55 V or ∆Go = -2 X 0.23 kcal (V mol)-1 x 0.55 V = -25.3 kcal The reaction depicted in Eq. (l2c) involves H+ and, therefore, ∆E will depend on the pH of the mixture (pH = -log[H+]). Usually the redox potentials have been evaluated from values determined at some standard pH (e.g., pH 7).
Fig. 1 Two half-cells.
IV. ∆G AS A FUNCTION OF THE CONCENTRATION OF REACTANTS As already mentioned, the actual parameter that is pertinent to the energy available to perform work is ∆G rather than ∆Go and in this section, we will focus on this parameter. The total capacity of a substance to perform useful work actually depends on its chemical potential, µ. The change in µis as a function of concentration as shown in Eq. (13a), which follows directly from the gas laws and is integrated in Eq. (13b) dµ = RT dC / C (13a) µ = µo + RT loge C (13b) In these equations, C is the concentration of the substance in question, and µ is the chemical potential under standard conditions. In a chemical reaction such as AB, the ∆G will correspond to the difference in chemical potential between the two components as shown in (Eq. 14). http://www.albany.edu/~abio304/text/chapter_12.html (7 of 22) [8/10/2001 1:30:57 PM]
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∆G = µB - µA = µBo- µAo + RT ln CB - RT ln CA (14) = ∆Go + RT ln CB - RT ln CA = ∆Go + RT ln(CB/CA) In Eq. (14), ∆Go = µBo - µAo. Following Eq. (14), the ∆ G for the synthesis of 1 mol of ATP would be
To convert loge (ln) to log10 and to simplify the calculation, 2.3RT can be considered to be 1.4 kcal/mol (i.e., 2.3 x 2 cal mol-1 deg-1 X 300 K). Equation (15a) can be represented by Eq. (15b) if ∆G is assumed to be 10 kcal/mol. ∆G = 10 kcal/mol - 1.4 kcal/mol (log[Pi]) + 1.4 kcal/mol (log10[ATP]/[ADP]) (15b) Equations (15a) and (15b) show that the ∆G for the synthesis or, inversely, the ∆G available from the hydrolysis of ATP, depend on the concentrations of the components. This fact has important consequences. For example, in muscle the ATP concentration is maintained maximally by the transfer of the phosphate from phosphocreatine. Creatine phosphokinase replenishes any ATP hydrolyzed by muscle contraction and other energy-requiring reactions. It is only when 90% of the phosphocreatine is used up that ATP begins to fall significantly, to about 10% of its resting state. The high [ATP]/[ADP] ratio permits a ∆G of -12.5 kcal/mol, a much greater magnitude than the ∆G, which may be as low as -7.6 kcal/mol. Generally, the hydrolysis of ATP or the reverse reaction, the synthesis of ATP, is written in a simplified form that involves only ATP, ADP, and Pi, as in (Eq. 3). In actuality, the reaction involves H+, as shown in (Eq. 16). ATP ---> ADP + Pi + H+ (16) The calculation of the ∆G for ATP synthesis therefore would follow Eq. (17a).
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Since pH = -log10 [H+], Eq. (a) can be represented as shown in Eq. (17b).
When the simpler Eq. (15a) is used, the ∆ G is assumed to be at a standard pH (e.g., 7.0 or 7.4). As noted previously, ∆G is significantly sensitive to pH, and a difference of one pH unit lowers the ∆G for the formation of ATP by 1.4 kcal/mol. Since the synthesis of ATP during oxidative phosphorylation (Chapters 16 and 18) or photophosphorylation (Chapters 17 and 18) is associated with membranes, the actual pH values are not known. There have been proposals that during oxidative phosphorylation or photosynthetic phosphorylation the pH of the phosphorylation sites is changed. A decrease of pH from 7 to 5, for example, would lower the ∆G as much as 2.8 kcal/ mo, driving the reaction of Eq. (6.16) toward ATP synthesis. Actually, the ∆G depends on many other factors, although in the present discussion, we have assumed that the ∆G for the synthesis of ATP (i.e., -∆G of its hydrolysis) is in the neighborhood of 10 kcal/mol. Published estimates of ∆G for the hydrolysis of ATP (pH range 6-9, at several Mg2+ concentrations and at 25o-35oC ) was calculated to be between -6.1 to -10.9 kcal/mol (see Bridger and Henderson, 1983). The energy required for the synthesis of 1 mol of ATP is frequently referred to as the phosphate potential and is expressed in either kilocalories or units of electrical potential (millivolts). Electrical potential units make it possible to compare the energy needed for ATP synthesis directly with the redox potentials for the cytochromes. Calculations of phosphate potentials for metabolizing isolated mitochondria have been published (Slater et al., 1973). V. ∆Go As mentioned in Section 1, ∆Go is the ∆G under standard conditions of temperature, pressure, and concentration. ∆Go is related to the equilibrium constant (K) of the reaction. In Eq. (14), ∆G has been shown to correspond to ∆G o + RT ln(CA/CB). Since ∆G = 0 at equilibrium, ∆Go can be expressed as shown in Eq. (18).
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VI. ENERGY COST OF TRANSPORT Biological systems are made up of many compartments, such as kidney tubules, cells, or subcellular organelles, enclosed by selective membranes of varying complexity. The movement of molecules from one biological compartment to another is known as transport. Transport requires an expenditure of energy whenever it occurs against an electrochemical gradient (i.e., the transport is in an uphill direction). In these cases, it is referred to as active transport. For a nonelectrolyte, the chemical potential µ of a compartment in relation to a single solute is represented by Eq. (19a), where n represents the number of moles and µ1, the chemical potential per mole of the solute. µ = nµ1 (19 a) The total chemical potential is the summation of µ's for all solutes as in Eq. (19b). Each subscript of µ or n indicates a different solute. Here the discussion will be restricted to a single solute. µ = n1µ1 + n2µ2 + n3µ3 + ... + niµi (19b) In a system made up of two compartments separated by a biological membrane, the ∆G for the transfer of 1 mole of solute from one compartment to another will correspond to the difference in chemical potential of the solute between the two compartments. If µA represents the chemical potential of the solute in compartment A and µB the chemical potential in compartment B, the ∆G can be shown to correspond to Eq. (20) by subtracting the individual chemical potentials as expressed by Eq. (13). Since the standard chemical potential is the same in both compartments, µB - µA = 0. ∆G = µB - µA = µBo - µAo + RT ln CB - RT ln CA = RT ln (CB/CA) (20) In the case of ions, the electrical potential across the membrane and the charges of the solute molecules also have to be taken into account. We are then concerned with the electrochemical potential of the solute. The electrochemical potential (µ) for a given solute corresponds to Eq. (21). µ = µ1 + zΨ F (21) Here Ψ is the electrical potential, z is the valence, and F is the Faraday constant. Consequently, the ∆G for the transfer of 1 mole of the ion is represented by Eq. (22), where ∆Ψ m is the membrane potential. ∆G = RT ln(CB/CA) + z∆ΨmF (22) Eq. (22) was obtained by subtracting the µ of phase B from that of phase A.
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One of the compartments may contain a nondiffusible charged component such a macromolecule or a colloid. This can give rise to a special kind of equilibrium, called a Donnan equilibrium, in which the diffusible ions distribute unequally between the two phases. Ions opposite in charge to the nondiffusible component will have a higher concentration in its compartment. The reverse is true for ions with charges of the same sign as that of the nondiffusible component. The exact proportion can be calculated from Eq. (22). When ∆G = 0, RT ln(CB/CA) = -zF∆Ψm or CB/CA = [exp(-z∆ΨmF)]/RT. For this special case, the electrical potential is known as the Donnan potential. The ∆Ψ m is related to the charge of the nondiffusible component. A Donnan distribution does not require the presence of a membrane. The nondiffusible component may be fixed to a structure rather than restrained by a limiting membrane. The energy required for the transport of ions can be calculated from Eq. (22). For instance, consider the transport of Na+ and K+. In most cells, the two transports are coupled and they take place in opposite directions. The Na+ is transported outward, whereas K+ is transported inward. The ion concentrations used in the calculation, which are shown in Table 1, approximately correspond to those of the squid giant axon. Columns 2 and 3 give the internal and external concentrations of Na+ and K+. The potential across the membrane is shown in column 4. The inside of the axon is negative in relation to the outside. The electrical potential across the cell membrane would therefore favor the entry of K+ and oppose the exit of Na+, as shown in column 5, which represents the electrical potential component of ∆G. The chemical potential component of ∆ G is represented in column 6, and column 7 shows the ∆G for each transfer. The energy expenditure is due primarily to the transport of the Na+. If we assume that the transport is coupled to ATP hydrolysis, the breakdown of 1 mol of ATP would suffice for more than the simultaneous transfer of 1 mol of Na+ and K+, since the ∆G of hydrolysis of ATP is approximately -10 kcal/mol. More realistic calculations (Tedeschi and Kinnally, 1992) indicate the cost for 3 Na+ and 2 K+ transported per ATP hydrolyzed to be approximately 10 kcal under conditions in which ATP hydrolysis provides -13.2 kcal/mole. So far, the discussion has concerned the expenditure of energy necessary for active transport and the minimal energy cost (i.e., assuming 100% efficiency) has been calculated for one example. We may well want to ask the opposite question. Can the energy available from the passage of solute in the direction of the electrochemical gradient be harnessed and used for some other process? As shown by substituting the appropriate values in Eq. (20) or (22), the transfer in the direction of the gradient provides a ∆G < 0, for example, in the transfer of a nonelectrolyte from phase A to phase B where CA/CB. Such coupling should be feasible at least as far as the energy available for the process is concerned. The flow of one solute in the direction of its gradient can be coupled to the flow of another solute against the electrochemical gradient and in the opposite direction. This is shown, for example, in the experiment depicted in Fig. 2 (Rosenberg and Wilbrandt, 1958). In this experiment, a suspension of human red blood cells has been equilibrated with [14C]glucose. The radioactivity of the external medium is represented on the ordinate and time is shown on the abscissa. Although it has been recognized that a special mechanism is needed for the transfer of the sugars across the plasma membrane, these cells normally do not transport against a concentration gradient. However, on addition of mannose or unlabeled glucose to the medium, the cells begin to transport labeled glucose outward against the concentration gradient, as shown in curves 1 and 2. Curve 3 represents a http://www.albany.edu/~abio304/text/chapter_12.html (11 of 22) [8/10/2001 1:30:57 PM]
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control in which only a salt solution was used; the radioactivity of the external medium corresponds to the appropriate dilution. The passage against the concentration gradient is maintained only for a limited period, and eventually the system equilibrates again. This equilibration is to be expected, since the gradient for the mannose or the unlabeled glucose is dissipated. This experiment demonstrates that the energy from the concentration gradient of the solute added to the medium can be harnessed to provide an outward flow of another solute against a concentration gradient. Outflow of one solute in response to the inflow of another has been called counterflow, and it will be discussed later. Table 1 Energy Requirement for the Transport of Na+ and K+ in an axon of a Marine Invertebratea
(1)
(2)
(3)
Ions
Internal Concentration (M)
External Concentration (M)
K+
0.40
Na+
0.05
a2.3
(4)
(5)
(6)
(7)
(8)
(V)
zFm kcal/mole
2.3RT log (C2/C1) kcal/mole
∆G kcal/mole
∆G kcal/mol
0.010
0.06
-1.4
2.2
0.8
3.5
0.460
0.06
+1.4
1.3
2.7
m
RT was assumed to be 1.4 kcal/mole
Fig. 2 Experiment showing uphill transport of labeled glucose across the human red cell membrane induced by counterflow of ( ) mannose or ( ) unlabeled glucose. Ordinate, activity in 10 µl of the external medium. Circle with x in center, activity before addition of 0.16 volume of unlabeled sugar (0.72 M in
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saline). Circle with bar, activity after addition of 0.16 volume of saline. Temperature 0oC until second arrow, then 37oC. (The temperature was raised to accelerate the penetration, which, however, proved to be unnecessary.) The calculated maximal concentration ratio for labeled glucose is approximately 4 (Rosenberg and Wilbrandt, 1958). Reproduced from Journal of Gen. Physiol., ©1958, vol. 41, pp. 289-296 by copyright permission of The Rockefeller University Press.
The transport of one solute may also proceed against its concentration gradient when it is coupled to the flow of another solute in the direction of its own concentration gradient, providing the necessary energy. This type of translocation has been called cotransport. A study of pigeon red cells (Vidaver, 1964) has shown inward cotranspont of Na+ and glycine, with two Na+ transferred per glycine molecule. The dependence of the glycine transport on the Na+ gradient has been shown in experiments where the internal concentration of ions has been varied. Red blood cells can be made leaky, exposed to a medium of the desired composition, and then resealed. Table 2 shows how the internal composition of human red blood cells can be varied over a wide range of concentrations. In this example, both Na+ and K+ levels were varied. A similar procedure was used with pigeon red cells to vary the internal concentration of Na+. The flow of glycine in response to changes in the Na+ gradient is shown in Table 3. The initial internal concentration of Na+ is represented in column 4; that in the medium, in column 5. The initial concentration ratios of glycine (internal/external) are shown in column 6. Column 7 represents the final ratio after a period of incubation. A comparison of these ratios shows that a higher external concentration of Na+ results in accumulation of internal glycine. When the internal concentration of Na+ is higher than that outside, the glycine is actively transported outward (results marked with arrows). In the intact cell, since the Na+ concentration inside the cell is low, the flow of glycine is invariably inward. In the intact cell, the internal Na+ concentration is kept low by the net outward transport of Na+, the so-called sodium pump, which expends energy from the hydrolysis of ATP. Table 1 shows the calculation of the energy requirement for the inward transport of 1 mole of Na+ in the squid axon. In Chapters 21 and 20, we will examine evidence for the coupled outward transport of Na+ and inward transport of K+ powered by the hydrolysis of ATP, and the possible mechanism of this pump. The converse process, transfer of ions in the direction of the electrochemical gradient coupled to the synthesis of ATP from ADP and Pi, is also possible. An experiment to test this was carried out with human red blood cells in a medium with a high external concentration of Na+ and low external concentration of K+ (Glynn and Lew, 1970). The synthesis of ATP from metabolic sources was inhibited with iodoacetate (which inhibits glycolysis, the major metabolic pathway in these cells). Results of the experiment are shown in Fig. 3. Curve 1 represents the incorporation of 32P into ATP as a function of external Na+ concentration. The concentration of Na+ was varied without changing the osmotic pressure or the ionic strength of the medium by replacing the Na+ with choline whenever necessary. Placing the red blood cells in a medium with a higher Na+ concentration Table 2 Variations of Erythrocyte Cation Composition after Special Treatment
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Concentration of cation in loading medium (mM)
Final cation content of cells (µEq/ml of cells)
Na+
Na+
K+
K+
Na++K+
40
260
12
108
120
50
250
16
101
117
75
225
24
89
113
100
200
31
78
109
150
150
50
65
115
300
0
101
12
113
Whittam and Ager (1965) Reproduced by permission from Biochemistry Journal , 97:214-227, copyrights ©1965 The Biochemical Society, London.
increases the gradient for Na+, since the low initial internal Na+ level remained the same. Increased external Na+ led to increased ATP synthesis. Curve 2 represents the effects of ouabain, a drug that inhibits the Na+ pump. Since the incorporation into ATP was prevented by the inhibitor, it is most likely that the reaction does correspond to the reverse of transport against the electrochemical gradient.
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Fig. 3 ATP synthesis as a function of external sodium concentration. Choline was used to replace Na+ to maintain constant osmotic pressure and ionic strength. Reproduced with permission from I.M. Glynn and V.L. Lew, Journal of Physiology, 207:393-402. Copyright ©1970 The Physiological Society, Oxford, England.
The incorporation of 32Pi into ATP can be related to the flow in the direction of the gradient by following either the penetration of Na+ or the exit of K+, since the transport of the two in opposite directions is coupled. In these experiments, the exit of K+ was estimated by the appearance of 42K in the medium after suitable loading of the cells with the labeled ion. A correction for the hydrolysis of [32Pi]ATP, which takes place presumably by independent processes, makes it possible to relate the K+ exit with the ATP synthesis. The exit of 2 to 3 moles of K+ (accompanied by the Na+ influx) produces 1 mole of ATP. Similar results have been obtained in experiments with other membrane-bound systems that can carry out active transport of ions. In isolated sarcoplasmic vesicles, the release of Ca2+ in the direction of the electrochemical gradient results in phosphorylation of ADP (Makinose and Hasselbach, 1971a, 1971b). The sarcoplasmic reticulum is a system of tubes and vesicles of striated muscle. Elements of the reticulum liberate Ca2+ as a signal for muscle contraction and subsequently sequester it during relaxation by an active transport. The active transport depends on the hydrolysis of ATP and is thought to resemble the Na+ pump. In isolated mitochondria, the antibiotic valinomycin induces active transport of K+ inward. The energy for http://www.albany.edu/~abio304/text/chapter_12.html (15 of 22) [8/10/2001 1:30:57 PM]
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this transport can be Table 3 Glycine Accumulation and Expulsion by Intact or Lysed and Restored Cells
(1)
(2)
(3)
Experiment
Sample
Preparation
Initial cell Na+(mM)
Na+ in medium (mM)
Initial ratio glycinein/ glycineo
Final ratio glycinein/ glycineo
1
a b
lysed and restored
24 24
140 140
1.45 1.45
2.76 3.02
c d
lysed and restored Intact
24 17.5
140 140
1.17 6.06
2.31 8.22
3
e f g h
lysed and restored " "
24 24 115 126
140 0 0 0
1.59 1.57 1.43 1.24
2.39 1/1.08 1/2.06 1/2.34
4
i j
lysed and restored
24 126
140 0
1.12 1.07
1.95 1/1.94
5
k l m
lysed and restored "
115 84 24
140 134 125
1.63 1.67 1.68
1.73 1.91 2.57
2
(4)
(5)
(6)
(7)
From: Vidaver (1964). Reproduced with permission from Biochemistry 3:795-799. Copyright ©1964 American Chemical Society.
supplied by either hydrolysis of ATP or respiratory reactions. Conversely, the efflux of K+ in response to the addition of valinomycin results in net synthesis of ATP from Pi and ADP (Cockrell et al., 1967). Valinomycin is one of the compounds, referred to as ionophores, that are capable of ligating ions. Because of their high solubility in the membrane lipid, they are involved in the transport of ions. The mechanism by which valinomycin affects mitochondrial transport is still a matter of debate. These experiments show that the transport mechanism can operate in either direction. In one direction, an http://www.albany.edu/~abio304/text/chapter_12.html (16 of 22) [8/10/2001 1:30:57 PM]
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exergonic chemical reaction powers the transport against an electrochemical gradient. In the other direction, the passage of ions along the electrochemical gradient can be harnessed to drive the synthesis of a chemical bond. The minimal energy expenditure necessary to transport solute is given by Eq. (20) or (22). According to these relationships, the minimal expenditure necessary to transport 1 mole of solute (i.e., the ∆G per mole transported) increases with the steepness of the gradient. Experimental examination of transport at different gradients leads to a number of conclusions that bear on the mechanism of transport. As already discussed, it is possible to vary either the internal or external ionic composition of red blood cells. The internal composition of the red blood cell can be changed by several experimental manipulations. The result of one such procedure has been shown in Table 2. In other work discussed above (see Table 1), the transport of glycine was studied as a function of the Na+ gradient. Other studies concerned the influx of K+ as a function of external K+ concentration and the efflux of Na+ as a function of internal Na+ concentration. The ATP hydrolyzed can be calculated from the Pi liberated by the reaction. It is necessary to correct this value by subtracting the amount of ATP produced by the red blood cell's metabolism. The latter can be calculated from the lactate produced by the glycolytic reactions that represent the major metabolic pathway of the red blood cell. In Fig. 4, the influx of K+ is shown on the ordinate (Whittam and Ager, 1965). Each point represents an experimental determination at a different external K+ concentration. The hydrolysis of ATP inhibited by ouabain is shown on the abscissa. Ouabain has little or no effect on reactions other than the active transport. The slope of the line indicates the K+ transported per ATP hydrolyzed and remains constant (mean of 2.4 ± 0.1 K ATP) over a wide range of K+ concentrations in many independent experiments. In experiments in which the Na+ efflux was estimated at various internal Na+ concentrations, the Na+ transported per ATP hydrolyzed was found to correspond to 1.1 ± 0.2. The results indicate that the energy cost of transporting one Na+ or one K+ by the Na+/K+ pump is the same regardless of the magnitude of the gradient. This suggests that the transport mechanism functions in precise stoichiometry, in a manner analogous to any other biochemical reaction. The implication of this observation can best be seen by calculating transport in specific cases.
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Fig. 4 Relationship between active K+ influx and ouabain sensitive ATPase activity in media with different K+ concentrations (mM, shown by the numbers next to the data points). From Whittam and Ager (1965). Reproduced by permission from Biochemistry Journal, 97:214-227, copyright ©1965 The Biochemical Society, London.
Table 4 Calculation for ∆G of Transport Corresponding to the hydrolysis of 1 ATP
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
Experiment
Nao
Nai
Ko
Ki
Nao/Nai
Ki/Ko
n2.3RT x log10 Na0/Nai (kcal)
m2.3RT xlog10 Ki/Ko (kcal)
1
150
10
10
96
15
9.6
5.3
2.8
2
150
101
10
12
1.48
1.2
0.8
0.3
Table 4 shows some of the concentrations reported by Whittam and Ager (1965) and gives the calculation of G for these two cases (as in Table 1). The external concentrations of Na+ and K+ are shown in columns 2 and 4 and the internal concentrations in columns 3 and 5. Columns 8 and 9 show the calculations of G for the transport of Na+ and K+. In these calculations, the membrane potential of the red blood cell has been neglected because its contribution to the energy is small. The values in columns 8 and 9 were obtained by multiplying the appropriate equation by n or m, the equivalents of Na+ or K+ transported per ATP hydrolyzed. The total energy required for transport under the conditions of experiment 1 is about 8.1 kcal (columns 8 and 9), and under the conditions of experiment 2 it is only 1.1 kcal. Nevertheless, as in Fig. 4, the ATP hydrolyzed per Na+ or K+ transported remains invariant. Under these conditions, the ∆G for the hydrolysis of one mole of ATP is about -13 kcal, sufficient for the transport under either of the conditions. Therefore, a constant stoichiometry is maintained by varying the efficiency. When the gradient becomes smaller the efficiency drops, even if theoretically there is sufficient energy in each ATP hydrolyzed to support a much larger cation/ATP ratio. VII. MUSCLE CONTRACTION Biological systems expend energy in a number of processes. We examined in some detail the transport of ions. Cells also expend energy in moving by processes involved in contraction. The contraction of striated vertebrate muscle has been studied intensively. The work performed by muscle can be calculated readily, since it corresponds to the mass of the object lifted (or, alternatively, the tension τ exerted) times the displacement (∆L). The energy expended that is not used to perform work is liberated as heat. The heat liberated can be calculated from the change in temperature of an insulated system whose heat capacity (∆H http://www.albany.edu/~abio304/text/chapter_12.html (18 of 22) [8/10/2001 1:30:57 PM]
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= µCH ∆T) is known. Note that if no work has been performed (the muscle has been held stationary in a socalled isometric contraction) or a contraction-relaxation cycle has been completed, all energy expended must appear as heat. The heat released by muscle is of great physiological importance in animals (even in some fish, which are considered ectothermic, or cold blooded) and in the physiological control of body temperature. In mammals, the heat released by contraction-relaxation cycles is expended in the basic function of shivering. Fig. 5 shows the work performed (lower curve) and the heat liberated (upper curve) as a function of tension (τ) relative to peak tension τ0. An apparatus kept tension constant during the contractile event. The amount of work is expressed solely by the shortening. The results show that the work performed and heat liberated undergo parallel changes. This is expressed most simply in Fig. 6, where the heat liberated is presented as a function of work. It follows from these results that the energy expenditure is proportional to the work performed. However, some energy is expended whether work is performed or not. In Fig. 5, at zero work, 2.95 mcal/g was expended; this minimal expenditure is known as the activation energy. The chemical events underlying contraction would, therefore, be graded with the amount of work performed. The efficiency of the system does not vary with increases in the amount of work performed. It has been shown (discussed in Chapter 23) that the amount of ATP (or phosphocreatine) hydrolyzed is proportional to the work, as we would expect from these considerations (Cain et al., 1962). Phosphocreatine is one of the energy reservoirs of muscle and can transfer its terminal phosphate to ADP to regenerate ATP. We saw that in the transport of Na+ and K+, the energetic cost of transferring 1 mol of Na+ is the same, regardless of the steepness of the gradient. Therefore, the case of ion active transport seems to be quite different in principle from that of contraction of striated muscle. The results suggest that muscle contraction corresponds to a graded process, as if contraction were the result of small finite steps. The molecular mechanisms of contraction are likely to involve such small finite steps (see Chapter 22 and 23). As noted above, some heat is evolved by muscle whether work is performed or not. This heat evolution seems inherent to the active state. When contracting muscle is slowly stretched, the total heat evolved is less than the sum of the heat of activation and the heat evolved from the work performed on the muscle by the stretching process (Hill, 1960). The difference corresponds rather closely to the work being performed on the muscle.
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Fig. 5 Variation of energy output with load in after load isotonic twitches; the load was allowed to fall during relaxation. Abscissa, load as a fraction of the peak isometric twitch tension 0. Ordinate, energy output in millicalories per gram and twitch (mean of 100 twitches, 20 by each of five muscles, ± 1 standard error plotted as a vertical bar). Upper curve, total heat; lower curves, external and internal work. Crosses, total heat minus total work. Reproduced with permission from F.D. Carlson, et al., Journal of General Physiology, 40:851-882. Copyright ©1963 Rockefeller University Press.
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Fig. 6 Ordinate, total heat, millicalories per gram and twitch. Abscissa, total work, millicalories per gram and twitch. Reproduced with permission from F.D. Carlson, et al., Journal of General Physiology, 40:851882. Copyright ©1963 Rockefeller University Press.
The results lend themselves to two possible interpretations: either the stretching has arrested the active state, or the energy input (so-called negative work) has been absorbed by reversing the contractility process. The latter alternative, which is supported by some investigators, would have rather far-reaching repercussions. The energy of contraction originates from the hydrolysis of high-energy phosphates. It has been found that the stretching of striated vertebrate muscle does not result in the synthesis of the main highenergy compound, phosphocreatine or ATP. These experiments have been interpreted as being consistent with the idea that the primary event in contraction does not directly involve the hydrolysis of ATP or phosphocreatine. However, in insect flight muscle preparations, stretching does increase the incorporation of 32Pi into ATP (Ulbrich and Ruegg, 1971). In addition, the interpretation involving the "disappearance" of work may well be incorrect. A more likely explanation is that the events of contraction are arrested and that the heat evolved is simply a quantitative conversion of the work performed on the system into heat. In Chapters 13 to 18 we will examine primarily the processes involved in capturing energy in a biologically utilizable form. The mechanisms involved in dissipating this energy will be taken up primarily in Chapters 19 to 24. SUGGESTED READING Becker, W.M. (1977) Energy and the Living Cell, Lippincott, Philadelphia. Christensen, H.N. (1975) Thermodynamic aspects of transport. In Biological Transport, 2d ed. Benjamin, http://www.albany.edu/~abio304/text/chapter_12.html (21 of 22) [8/10/2001 1:30:57 PM]
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Reading, Mass. Cramer, W.A. and Knaff, D.B. (1990) Energy Transduction in Biological Membranes, Chapters 1 and 2. Springer-Verlag, New York. Dutton, P.L. (1978) Redox potentiality: determination of midpoint potential of oxidation reduction components of biological electron transfer systems, Methods Enzymol. 54:411-425. (Medline) Morris, J.G. (1968) The Biologist's Physical Chemistry, Addison-Wesley, Reading Mass. Woledge, R.C., Curtin, N.A. and Homsher, E. (1985) Energetic Aspects of Muscle Contraction, Academic Press, New York (see Chapters 1, 2, and 4). (Medline) Alternative References Harold, F.M. (1986) The Vital Force: A Study of Bioenergetics, Chapter 1 to 3. W.H. Freeman, New York. Tedeschi, H. and Kinnally, K.W. (1992) Bioenergetics in Fundamentals of Medical Cell Biology, Bittar, E.E. ed., JAI Press Inc., Greenwich, CT., vol. 3B pp. 609-642. WEB RESOURCES Diwan, J.J., Bioenergetics, http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/bioener.htm REFERENCES Search the textbook
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Chapter 12: References
Back to Chapter 12
REFERENCES Banks, E.C. and Vernon, C.A. (1970) Reassessment of the role of ATP in vivo, J. Theor. Biol. 29:301306. (Medline) Bridger, W.A. and Henderson J.H. (1983) Cell ATP, Chapter 2. Wiley, New York. Cain, D.F., Infante, A.A. and Davies, R.E. (1962) Chemistry of muscle contraction. Adenosine triphosphate and phophoryl creatine as energy supplies for single contractions of working muscle, Nature 196:214-217. Carlson, F.D., Hardy, D.J. and Wilkie, D.R. (1963) Total energy and phosphocreatine hydrolysis in the isotonic twitch, J. Gen. Physiol. 46:851-882. Cockrell, R.S., Harris, E.J. and Pressman, B.C. (1967) Synthesis of ATP driven by potassium gradient in mitochondria, Nature 215:1487-1488. (Medline) Glynn, I.M. and Lew, V.L. (1970) Synthesis of adenosine triphosphate at the expense of downhill cation movements in intact red cell, J. Physiol. (London) 207:393-402. (Medline) Hill, A.V. (1960) Production and absorption of work by muscle, Science 131:897-903. McClare, C.W.F. (1972) In defense of the high energy phosphate bond, J. Theor. Biol. 35:233-246. (Medline) Makinose, M. and Hasselbach, W. (1971a) Calcium efflux dependent formation of ATP from ADP and orthophosphate by membranes of the sarcoplasmic vesicles, FEBS Lett. 12:269-270. Makinose, M. and Hasselbach, W. (1971b) ATP synthesis by the reverse of the sarcoplasmic pump, FEBS Lett. 12:271-272. Nicholls, D.G. and Locke, R.M. (1984) Thermogenic mechanism in brown fat, Physiol. Rev. 64:1-64. (Medline) Poe, M. and Estabrook, R.W. (1969) Kinetic studies of temperature changes and oxygen uptake concomitant with substrate oxidation by mitochondria: the enthalpy of succinate oxidation during ATP formation in Mitochondria, Arch. Biochem. Biophys. 126:320-330. (Medline) Rosenberg, T. and Wilbrandt, W. (1958) Uphill transport induced by counterflow, J. Gen. Physiol. 41:289-296. http://www.albany.edu/~abio304/ref/ref12.html (1 of 2) [8/10/2001 1:31:21 PM]
Chapter 12: References
Rosing, J. and Slater, E.C. (1972) The value of ∆G for the hydrolysis of ATP, Biochim. Biophys. Acta 267:275-290. (Medline) Slater, E.C., Rosing, J. and Mol, A. (1973) The phosphorylation potential generated by respiring mitochondria, Biochim. Biophys. Acta 292:534-553. (Medline) Tedeschi, H. and Kinnally, K.W. (1992) Bioenergetics, in Fundamentals of Medical Cell Biology, JAI Press Inc., Greenwich, CT, vol 3B, pp. 609-642. Ulbrich, M. and Ruegg, J.C. (1971) Stretch induced formation of [P]ATP in glycerinated fibers of insect flight muscle, Experientia 27:45-46. (Medline) Vidaver, G.A. (1964) Glycine Transport by hemolyzed and restored pigeon red cells, Biochemistry 3:795799. (Medline) Whittam, R. and Ager, M.E. (1965) The connexion between active cation transport and metabolism in erythrocytes, Biochem. J. 97:214-227.
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13. Enzymes and Enzyme Complexes
13. Enzymes and Enzyme Complexes I. Chemical Reactions II. The Role of Enzymes III. Kinetics of Enzyme Reactions A. Michaelis-Menten Kinetics B. Sigmoidal Kinetics C. Regulatory Mechanisms Allosteric interactions Intrasteric interactions D. Transitions from Inactive to Active Forms E. Regulation of Phosphorylation and Dephosphorylation A. Protein Kinases Interactions with substrate docking sites cAMP-dependent protein kinases Cyclin-dependent protein kinases B. Protein Phosphatases F. Multienzyme Complexes A. The Pyruvate Dehydrogenase Complex B. Other Multienzyme Complexes Mitochondrial enzymes Binding of glycolytic enzymes to components of the contractile systems Glycogen particles Binding of enzymes to the plasma membrane
Suggested Readings References Back to List of Chapters The cell is a dynamic system, in continuous change and in continuous motion. The underlying events must necessarily reflect molecular changes, namely chemical reactions. Most chemical reactions occurring in living systems are catalyzed by enzymes. As a result, the study of enzyme reactions of cells goes far in explaining the basis of their functional behavior. The genetic makeup of the cells and the processes involved in the expression of the genetic information determine the kinds of enzymes or structural proteins that are present. These enzymes and structural proteins are the ultimate functional units of the cell. This chapter addresses first chemical reactions, and enzyme-catalyzed reactions in particular, and then proceeds to a discussion of the properties of the enzymes, their regulation, and the organization in enzyme complexes.
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I. CHEMICAL REACTIONS In a population of molecules, the kinetic energy varies with each molecule. The distribution should be random and follow a pattern such as that represented in Fig. 1. In this plot, the fraction of the molecules, (dn/dv)nT-1, having velocities between v and v + dv, is represented as a function of the velocity v. A simple model describing the energy distribution in two dimensions is given by Eq. (1).
In Eq. (1), m corresponds to mass, ½mv2 is the kinetic energy, k is the Boltzmann constant (k = R/N, where R is the gas constant and N is Avogadro's number), and T is temperature. This relationship is represented graphically in Fig. 1. Representing the kinetic energy by E and dE = mv dv, Eq. (1) becomes
Fig. 1 Distribution of velocities of gas molecules or of molecules in an ideal solution. dn/nT is the fraction of molecules with velocity in the range of v to v + dv. The area under the curve has a value of unity.
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If we were to ask what fraction of the molecules has energy greater than a particular value of E, it becomes necessary to integrate under the curve [Eqs. (3) and (4)]. It would seem reasonable to consider that molecules need a critical minimal energy, termed the activation energy (E), in order to react. Equation (4) represents the proportion of molecules that surpass the activation energy at some specified temperature and hence the proportion of molecules that can react.
This idea is illustrated in Fig. 2a and b. Fig. 2a represents the energy of a molecule through the course of the reaction. The reaction is assumed to involve a single reactant. The molecules that can react are represented in Fig. 2b by the portion of the curve that is crosshatched. An increase in temperature increases the kinetic energy of the system. Accordingly, more molecules reach the critical energy level as represented in a diagram in which the bell-shaped distribution is shifted to the right (Fig. 2c). As implied by Eq.(4), the rate of the reaction should be proportional to n/nT. When the logarithm of the reaction rate constant (represented in this chapter as kr) is plotted as a function of 1/T, the slope of the line is -E/k. Generally, the relationship of Eq.(4) is not followed precisely. A number of other parameters are likely to come into play. For example, where more than one molecular species is involved, the two molecules must meet and, furthermore, they must meet in such a fashion that the reactive groups are specifically apposed. Therefore, we would expect the reaction rate to depend not just on temperature but on other factors as well. We would expect that the shape of the molecule or the nature or location of the reactive groups plays a significant role. Accordingly, the reaction rate constant is more closely predicted by a more complex equation. Here K is a constant, mostly empirical, and h is Planck's constant.
II. THE ROLE OF ENZYMES Since the metabolic activity of the cell is to a large extent the sum of its enzymatic activities, it is very much to the point to examine some of the properties of enzymes. Some insights into the control of metabolism can be gained by examining the biochemical properties of purified, soluble enzymes. However, this approach, while providing very useful information, is not likely to reveal a realistic pattern of control in the living cells because of the complexity of the intact system. The level of our ignorance has been illustrated in yeast by varying the concentrations of glycolytic enzymes through the introduction of the appropriate genes in multicopy vectors (Schaaff et al., 1989). The specific activity of individual enzymes was increased approximately 4 to 14 times over that of the wild-type. However, the rate of ethanol production by the yeast remained unchanged even with increases in enzymes catalyzing irreversible steps which have been presumed to play a regulatory role. Part of the reason for the lack of http://www.albany.edu/~abio304/text/13part1.html (3 of 17) [3/5/2003 8:20:26 PM]
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understanding results from (a) the complexity of the system, (b) the fact that many and perhaps all enzymes are present in multienzyme complexes and (c) they are present in specialized compartments or attached to specific structures which alter their properties and their environment (see Section VI). Almost all enzymes studied are proteins, generally ranging between 10 and 500 kDa in molecular weight. The reactants that interact with the enzymes are called substrates. Enzymes sometimes require small organic molecules (coenzymes) for activity. Carboxylase, for example, requires thiamine pyrophosphate and enzymes involved in acetylation require coenzyme A. Phosphorylase, which is discussed later in this chapter, requires pyridoxal 5-phosphate. In addition, many enzymes require certain metal ions for their activity. For example, Mg2+ is necessary for many enzyme-catalyzed reactions. The RNA portion of ribonuclease P (a ribonucleoprotein) has been found to have catalytic activity (Guerrier-Takada and Altman, 1984). In addition, a portion of an RNA intron of 395 nucleotides released by the self-splicing of a ribosomal RNA precursor (Zaug and Cech, 1986) acts as a ribonuclease and an RNA polymerase. Introns are the noncoding sequences of either a gene or the corresponding primary RNA transcript that are excised while the coding sequences are simultaneously linked during RNA processing to form mature mRNA (see Chapter 3). The large subunit of the ribosome catalyzes peptide bond formation. Although 30 proteins are present, the site at which the peptide bonds are formed is an RNA domain (see Ban et al., 2000). The RNA enzymes are sometimes referred to as ribozymes.
Fig. 2 Illustration of how an increase in temperature allows more molecules to reach the energy of activation, E.
The site primarily involved in catalysis, the active site, complexes with substrate and may interact with cofactors such as coenzymes and metal ions. It is but a very small portion of the enzyme molecule, as suggested for example, by the low molecular weight of many substrates. The specificity of the interaction between enzyme and substrate implies that a finite number of specific groups in the enzyme molecule are involved. The restricted nature of the active site has also been demonstrated directly. For example, low molecular weight analogs of the substrate can be covalently bonded to a single site in an enzyme molecule and thereby block its activity (Schoellmann and Shaw, 1963). The technique of X-ray crystallography has permitted the deduction of the structure of many http://www.albany.edu/~abio304/text/13part1.html (4 of 17) [3/5/2003 8:20:26 PM]
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macromolecules, including enzymes. In some cases, when the crystals of the enzyme are exposed to the substrate, electron density maps reconstructed from the diffraction data show an increased density at a discrete small site (e.g., see Ludwig et al. 1967). The location of this site generally implicates the same groups which were previously suggested by direct chemical studies. Why a large molecule is needed for biological catalysis is still a subject of debate. Some of the conformational rearrangements thought to occur during enzyme catalysis or during the regulation of enzyme activity may require the involvement of a macromolecule. Many proteins, such as enzymes, contain domains. Domains are portions of proteins that carry out specific functions (see Rashin, 1981). They are identified by computer algorithms that search in proteins data bases for segments (50 to 500 residues) with sequence similarity (see Khosla and Harbury, 2001). There are three kinds of modular enzymes: enzymes in which the substrate specificity and catalytic activity are separable (e.g., see Fig. 10), multisubstrate enzymes where the binding sites for each substrates are modular and multienzyme systems that catalyze metabolic pathways (see Section IIIF). The role of an enzyme consists of accelerating the rate of a given reaction. The tendency of a reaction to occur and its final equilibrium position are entirely expressed by the free energy, ∆G, of the reaction and remain unchanged in the presence of the enzyme. The thermodynamic considerations discussed in Chapter 12 apply whether a reaction is enzyme catalyzed or not. The enzyme may be regarded as increasing the rate of a reaction entirely by lowering the energy of activation. The principle can best be illustrated by comparing Fig. 2 and Fig. 3. Fig. 2, as we have seen, represents a hypothetical chemical reaction. Fig. 2a shows the energy as a function of the extent of the reaction, and Fig. 2b shows the proportion of molecules at any given energy. The proportion of molecules having sufficient energy to react is indicated by the crosshatched area under the curve. The energy of the molecules can be increased by heating (Fig. 2c). In contrast, introduction of an enzyme lowers the activation energy to the level represented in Fig. 3a. As a consequence, a larger proportion of the molecules now has sufficient energy to react, as indicated by the increase in the crosshatched area under the curve in Fig. 3b (compared to that in Fig. 2b) without a change in their energy. Catalysis of a chemical reaction is illustrated by the data in Table 1 (Koshland, 1956). In this table, Vo represents the rate or velocity of the reaction in the absence of enzyme and VE represents the velocity in the presence of enzyme. It is apparent that enzymes can speed up reactions as much as 1012 times the rate of the uncaltalyzed reaction.
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Fig. 3 Illustration of how an enzyme acts. The energy level of the molecules remains unchanged but the E is decreased.
Table 1 Rate of a Chemical Reaction in the Presence (VE) or Absence (V0) of the Appropriate Enzyme
Observerd (mol min-1 liter-1) Enzyme
Hexokinase
Substrate & Concentration
[ET] (Eq/liter)a
VO
VE
10-7
1 x 10-13
1.3 x 10-3
6 x 10-7
5 x 10-15
1.6 x 10-3
4 x 10-7
6 x 10-12
2.7 x 10-3
0.0003 M Glucose 0.002 M ATP
Phosphorylase
0.016 M Glucose 1phosphate 10-5 M Glycogen
Alcohol dehydrogenase
10-4 M NAD 0.04 M Ethanol
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Creatine kinase
0.024 M Creatine
3 x 10-9
3 x 10-9
4 x 10-5
0.004 M ATP
From D.E. Koshland, Journal of Comparative Physiology 47:217-234. Copyright ©1956 Alan R. Liss, Inc. a(Number
of moles/ liter) X (number of active sites per molecule)
It is possible to develop some insights into the mechanisms of enzyme catalysis by studying model compounds. These compounds are simple organic molecules that have been found to catalyze certain reactions, although with an efficiency much lower than that of enzyme catalysis. Studies with simpler molecules involve fewer complications and are generally easier to interpret. The possibility of synthesizing molecules that differ from each other in specific ways, permits the evaluation of various factors in the mechanisms of catalysis. In addition, from model reactions the contribution of various mechanisms that are suspected to play a role can be approximated. Cloning of DNA that codes specific proteins and site-directed mutagenesis (see Appendix) have made it possible to change systematically single amino acids in selected enzymes. An example of this approach as applied to the study of tyrosyl-tRNA synthetase (Wilkinson et al., 1984) is shown in Table 2. The substituted site, occupied by threonine in position 51 in the wild type, is shown in the first column, and the effects of the indicated substitutions on the kinetic constants kcat and Km are shown in the rest of the table. The constant kcat is the rate constant of the reaction and Km is the apparent Michaelis-Menten constant, discussed in Section III, A. The ratio of the two (kcat/Km) relates the reaction rate to the concentration of free enzyme and corresponds to an index of specificity in relation to alternative or competing substrates. The study of specifically altered enzymes is a very powerful method for elucidating enzyme mechanisms. However, model compounds still provide an important alternative. Alteration of a single amino acid in an enzyme may provide results that are difficult to interpret because it may produce alterations in the overall conformation of the enzyme, with dramatic effects on enzyme activity but only an indirect effect on the catalytic site. In a reaction involving two or more reactants, the lowering of the activation energy by the enzyme is in part the result of an increase in the number of potentially fruitful collisions between the substrate molecules. In this role, the enzyme orients the active groups of the substrates relative to each other in a manner most conducive for a reaction to occur (proximity effect). In contrast, the chances of the active groups free in solution coming into fruitful apposition is much lower. During enzyme catalysis, the active site, or some component held at the active site, may act as a nucleophilic (electron-donating) or electrophilic (electron-attracting) agent. The active site of the enzyme will make the reactive group more reactive by making it more electrophilic or nucleophilic. In many cases, this takes place by addition or removal of a proton.
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Residues at the active site act as acids (H+ donors) or bases (H+ acceptors) in general acid-base catalysis. The reactivity of the substrate may be altered by a nonpolar microenvironment at the active site. By virtue of its lower dielectric constant, the hydrophobic spot would alter the ionization of the substrate. The presence of oppositely charged residues (i.e., ion pairs) may also have an effect on the rate of the enzymecatalyzed reaction. The enzyme may contribute to the orientation of the substrates much more precisely than by the proximity effects already discussed. Perhaps the interaction of the active site substrate introduces a strain on the molecule by producing a distortion. The distortion could be produced by a straightforward interaction. Alternatively, a change in the conformation at the active site may distort the substrates and facilitate the reaction (the rack and strain theory). Generally, when an enzyme interacts with a substrate, the conformation of the enzyme changes (see Olson and Allgyer, 1973). In addition, changes in the shape of the enzyme molecule are frequently produced by substances involved in the regulation of enzyme activity, and these changes in conformation affect the ability of the enzyme to interact with the substrate (see Section III C). Table 2 Pyrophosphate Exchange Activity of Tyrosyl-tRNA Synthetasesa
Enzyme
kcat (s-1)
Km (ATP)
kcat/Km
(mM)
(s-1M-1)
TyrTS
7.6
0.9
8,400
TyrTS (Ala 51)
8.6
0.54
15,900
TyrTS (Pro 51)
12.0
0.058
208,000
From; Wilkinson et al (1984) Reproduced by permission from Nature 307:187-188. Copyright ©1984 Macmillan Magazines Ltd. a
Synthetase exchange activity was measured at 25oC, in 144 mM Tris-Cl, pH 7.8, 10 mM MgCl2, 0.1 mM phenylmethanesulfonyl chloride, 10 nM 2-mercaptoethanol, 2mM pyrophosphate, 50 µM tyrosine, and 100200 nM enzyme.
A number of facts that are known about enzymes can be summarized as follows. 1. The active site of an enzyme binds the substrate and interacts with the substrate to catalyze the reaction. 2. The active site is but a small portion of the total enzyme molecule.
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3. The enzyme itself is regenerated after the reaction. 4. The nature of the active group is such that the enzyme can interact only with specific substrates that have certain characteristic groupings. 5. The interaction brings about a lowering of the activation energy and thereby increases the proportion of the substrate molecules that can react at any one time. The result is an increase in the rate of the reaction. III. KINETICS OF ENZYME REACTIONS A. Michaelis-Menten Kinetics We have discussed how the rate of reaction depends on the number of molecules that have reached a critical energy level, permitting the reaction to take place. The number of molecules reacting will, therefore, be directly proportional to the concentration of molecules present. Representing the reactant as A and the product as P, the reaction can be represented as in Eq. (6a):
where k1 represents the rate constant of the forward reaction and k2 that of the reverse reaction. The ratio k1/k2 corresponds to the equilibrium constant. At the beginning of the reaction, with only A present, the backward reaction from P to A need not be considered, as shown in Eq. (6b). Equation (6c) represents the analogous relationship when the back reaction becomes significant.
Where more than one reactant is involved, the same reasoning applies. In addition, the different reactant molecules must meet before the reaction takes place. Again, the probability of their meeting will be proportional to their concentration. A reaction involving two reactants, A and B, can be represented by Eq. (7a). Accordingly, Eq. (7b) represents the rate of the reaction when the concentration of P is negligible.
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When an enzyme is involved in the reaction, the same considerations apply, except that the enzyme itself is regenerated as a product. In a reaction involving a single substrate, either the enzyme or the substrate could represent A or B. Generally, the enzyme is present only in very low concentrations. For this reason, its concentration becomes limiting in determining the rate of the reaction, and an increase in substrate concentration beyond a particular level will not increase the rate of the reaction. There is no free enzyme left to bind S. The equations describing the rate of an enzyme-catalyzed reaction can be modified to take this fact into consideration. An enzyme reaction involving a single substrate is represented by
Here, E and S represent enzyme and substrate, ES is the enzyme-substrate complex, and P is the product of the reaction. As we have seen, the rate of a reaction is proportional to the concentration of reactants and the formation of product (P) is proportional to k3(ES). Therefore, the rate of the reaction, conventionally called the velocity of the reaction (V), can be represented as
Under ordinary circumstances, the total amount of enzyme (ET) does not change (the enzyme is neither synthesized nor activated). Therefore, as in Eq. (9):
In addition, under steady-state conditions, which are established rapidly, the concentration of ES does not change significantly, i.e., d(ES)/dt = 0 or
On rearrangement, this becomes
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Substituting (k2+k3)/k1 = Km:
Since (E) = (ET) - (ES) [Eq. (9)], Eq. (l0c) can be rewritten in the form
The rate (V) of appearance of the product corresponds to k3(ES), as shown by Eq. (8b). Therefore, Eq. (10d) can be modified to give
k3(ET) represents the maximum possible rate [when (ES) = (ET)]; it is a constant that can be represented by Vm. Equation (11) now assumes the following form, known as the Michaelis-Menten equation:
A close examination of Eq. (12) is very revealing. When the amount of substrate is low, (S) becomes negligible in the denominator and the velocity approximates V = (Vm/Km)(S) (see Fig. 4a). In other words, the velocity varies linearly with substrate concentration. On the other hand, when (S) is very large compared to Km, Km can be dropped from the equation. Consequently, (S) is a factor in both numerator and denominator and cancels out, and V now equals the constant Vm: the velocity of the reaction is independent http://www.albany.edu/~abio304/text/13part1.html (11 of 17) [3/5/2003 8:20:26 PM]
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of the substrate concentration (horizontal line in Fig. 4a). The enzyme is entirely complexed to the substrate (it is saturated); (ES) approaches (ET). The velocity of the enzyme reaction as a function of increasing substrate concentration, expressed in Eq. (12), is represented in Fig. 4a and b. Enzyme reactions following this pattern are said to exhibit Michaelis-Menten kinetics. The constants Vm and Km are characteristic of the enzyme reaction and describe the kinetic properties of the system: Vm is directly proportional to the amount of enzyme present, and Km is characteristic of the enzyme in question. These two constants can be readily evaluated. The maximum number of substrate molecules transformed per unit time by a single enzyme molecule, Vm/(E), is the turnover number, a frequently used parameter of enzyme activity. The specific activity is the number of enzyme units (1 unit transforms 1 µmol of substrate per minute at 25oC) per milligram of protein.
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Fig. 4 Relationship between the various kinetic parameters in an enzyme-catalyzed reaction. (a) Rate of the reaction (V) as a function of substrate concentration (S). Initial slope and asymptote. (b) Rate of the reaction (V) as a function of substrate concentration (S). Determination of Km from (S) when V = Vm/2. (c) Lineweaver-Burk plot. (d) Rate of a reaction (V) as a function of substrate concentration (S). Curve 1, control; curve 2, competitive inhibition; curve 3, noncompetitive inhibition.
The constant Vm can be estimated by evaluating the asymptote from a graphical representation, such as Fig. 4b, and Km can be readily evaluated when V = ½Vm, where Km corresponds to the value of (S). It is common practice to evaluate the constant of the Michaelis-Menten relationship from a plot of the reciprocal of V against the reciprocal of (S) (known as a Lineweaver-Burk plot). The reciprocal of Eq. (12) takes the form
In a plot of 1/V as a function of 1/(S) the slope is Km/Vm. The 1/V intercept is 1/Vm and the 1/(S) intercept is 1/Km. None of these methods are entirely satisfactory. Software is now readily available to calculate the constants for standard treatments or for more complex equations. These programs generally include statistical error analysis (e.g., Software for Science, http://www.scitechint.com/scitech/). The presence of inhibitors could alter the kinetics represented in Fig. 4a. For example, the V may be lowered (e.g., Fig. 4d; compare curves 2 and 3 to curve 1) if the inhibitor combines with the enzyme to make less enzyme available for the reaction. This would result in a decrease in rate as well as Vm (noncompetitive inhibition, curve 3). On the other hand, the inhibitor could compete directly or indirectly for the active site. In this case, the substrate at a high enough concentration should be able to compete successfully, so the Vm would remain unaffected by the presence of inhibitor (curve 2). B. Sigmoidal Kinetics Equations (12) and (13) and Fig. 4a represent reasonably well the kinetics of many enzyme reactions. However, many other reactions do not follow these kinetics but, instead, a sigmoidal pattern, where the curve expressing rate as a function of substrate concentration is S shaped. The aspartate http://www.albany.edu/~abio304/text/13part1.html (13 of 17) [3/5/2003 8:20:26 PM]
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carbamoyltransferase reaction, which is one of these, takes place as shown in Eq. (14).
This reaction is the first in the pathway that converts carbamoyl phosphate and aspartate into cytidine triphosphate (CTP). The kinetics of the reaction, represented in Fig. 5 (Gerhart and Pardee, 1962), clearly do not follow the predictions of Eq. (12). In this figure, the rates of the reaction at various concentrations of aspartate are shown. The carbamoyl phosphate level is kept constant.
Fig. 5 Kinetics of aspartate carbamoyltransferase reaction. Velocity, units of activity (in this case micromoles of carbamoyl aspartate per hour) per milligram of protein x 10-3. Reaction mixture: 3.6 x 10-3 M carbamoyl phosphate and 9.0 x 10-3 µg of enzyme per ml. The mixture was in 0.04 M potassium phosphate buffer at pH 7.0. Reproduced with permission from J. C. Gerhart and A. B. Pardee, Journal of Biological Chemistry, 237:891-896. Copyright ©1962 The American Society of Biological Chemists, Inc.
At low concentrations of aspartate, the rate of the reaction does not vary linearly with substrate concentration. Rather, it increases more sharply, as if the effectiveness of the enzyme were increased at the higher concentration of substrate. A number of explanations could account for this phenomenon. The most http://www.albany.edu/~abio304/text/13part1.html (14 of 17) [3/5/2003 8:20:26 PM]
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likely one is that the reactivity of the enzyme does, in fact, change with increasing concentrations of substrate. It is difficult to visualize an effect of this kind unless we postulate that a combination of the substrate with the enzyme alters the structure of the enzyme so that it reacts more effectively with another molecule of substrate. This implies that each enzyme molecule has more than one functional unit and active site. A substrate at the active site can facilitate the reaction at another active site only if the two active sites are part of the same enzyme molecule. Many enzymes have been found to be made up of subunits that are held together by noncovalent bonds. In some enzymes these subunits are identical, whereas in others the subunits are dissimilar. Generally, but not always, the subunits alone have little or no activity. Reconstitution of the enzyme from its subunits restores the activity. The idea that these particular kinetics are the consequence of changes in reactivity of the carbamoyltransferase at different substrate concentrations, is supported by experiments in which the enzyme is manipulated chemically. The kinetic pattern is altered when the enzyme is treated with compounds that react with sulfhydryl groups or when it is heated to 60oC for short periods. This alteration is shown in Fig. 6 (Gerhart and Pardee, 1962) and consists of a conversion to a Michaelis-Menten curve from the original sigmoidal curve. The lower curve in Fig. 6 indicates results obtained with the untreated enzyme. The upper curve indicates results obtained after treatment with Hg(NO3)2. The rates in the upper curve are increased and the V is higher, evidence that the treatment has increased the activity of the enzyme. The lack of a sigmoidal shape for the kinetics of the treated enzyme can be explained by postulating that the altered enzyme can now react only maximally, and that the conformation of the enzyme has been changed by the treatment. There is considerable evidence that the conformation of some enzymes, changes when the enzymes interact with their substrates. In the case of aspartate carbamoyltransferase, the rate at which the enzyme sediments when centrifuged changes with the addition of succinate, a substrate analog (Gerhart and Schachman, 1968). The rate of sedimentation of macromolecules is a function of their size and shape (i.e., conformation). Presumably, the same changes would occur in the presence of aspartate. The presence of CTP has the opposite effect on the sedimentation of the enzyme. The metabolic significance of the regulation of aspartate carbamoyltransferase by CTP is examined in more detail in the next section (III, C). Changes produced by interactions with the substrate have also been detected by observing changes in the reactivity of some of the residues of the enzyme (e.g., -SH groups). These changes imply a change in the shielding of the residues and hence in the conformation of the enzyme. Similarly, conformational changes of some enzymes have been detected with X-ray diffraction and nuclear magnetic resonance (NMR) techniques. Recent crystallographic studies have demonstrated substrate induced changes in conformation in enzymes such as phosphoglycerate kinase (PGK). PGK plays a pivotal role in photosynthesis and glycolysis. This enzyme catalyzes a transfer of phosphate from 1,3 bisphosphoglycerate to ADP, producing ATP and 3phosphoglycerate (3PGA). The two substrates are bound to widely separated domains: one in the amino half of the molecule and the other in the carboxy-half of the molecule. However, the transfer of phosphate is direct since it takes place only when both substrates are present. The transfer could only take place with a substantial hinge-bending conformational change to bring the two substrates together in the catalytic process. Bernstein et al. (1997) have shown by crystallography that complexes of Mg, ADP, PGK and 3PGA are shifted as much as 27 Å compared to the unliganded enzyme with a domain rotation of 32o. Fig.7 shows the changes in conformation deduced from a variety of studies .
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Fig. 6 Dependence of the velocity of the aspartate carbamoyltransferase reaction on aspartate concentration after loss of feedback inhibition. Velocity units are activity per milligram of protein x l0-3 ( (untreated) enzyme; (
) Native
) 10-6 M Hg(NO3)2 present during assay (heavy metal-treated aspartate
carbamoyltransferase); ( ) enzyme heated for 4 min at 60C and cooled before assay; ( ) heated enzyme assayed in presence of 2 x 10-1 M CTP. The reaction mixture contained 3.6 x 10-3 M carbamoyl phosphate; aspartate varied as indicated; 2 x 10-1 M CTP when used; 0.04 M potassium phosphate, pH 7.0; and 9.0 x 10-1 µg of enzyme protein/ml. Reproduced with permission from, J. C. Gerhart and A. B. Pardee, Journal of Biological Chemistry, 237:891-896. Copyright ©1962 The American Society of Biological Chemists, Inc.
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Fig. 7 Substrate-induced conformational change in PGK catalysis. Enzymes from various sources are oriented by superimposing the amino-terminal domains. Reproduced with permission from Bernstein, B.E., Michels, P.A.M. and Hol, W.G.J. Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation, Nature 385:275-278. Copyright ©1997 MacMillan Magazines Ltd.
X-ray diffraction at very low temperatures (e.g., 100 o K will probably permit the study of various steps of enzyme catalyzed reactions (e.g., see Edman et al., 1999). Go to Part 2 REFERENCES Search the textbook
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Back to Part 1
C. Regulatory Mechanisms Allosteric interactions As previously mentioned, the fact that the presence of substrate facilitates the enzyme reaction argues for the presence of more than one functional unit or binding site per molecule of enzyme. In fact, treatment with mercurials (e.g., p-mercuribenzoate) separates the aspartate carbamoyltransferase enzyme into two types of subunits. One type is composed of two units containing the catalytic activity, whereas the other contains four units carrying the sites that react with the substances regulating the activity of the enzyme. A great number of enzymatic reactions can be inhibited or stimulated by compounds that are chemically quite distinct from the natural substrates. These substances interact at sites other than the active site. Such interactions are referred to as allosteric interactions. Allosteric inhibitions are frequently exerted by products of metabolic pathways and may be of great physiological significance by permitting precise control of the reaction by feedback inhibition (end product inhibition). In this manner, overproduction of end product leads to a slowing down of the pathway that produces it. The functional effect of such an inhibition is illustrated in Fig. 8a. In Fig. 8a and b, the hatched line and heavy arrow indicate stimulation and the black line and cross indicate inhibition. The pathway represented in Fig. 8a synthesizes the cell's uridine triphosphate (UTP) and CTP. Feedback inhibition by the end product (CTP) can adjust the rate of production, depending on the needs of the cell. Regulation can be much more complex, particularly where the pathways are complex. The diagram of Fig. 8b represents the biosynthetic pathway of the purine nucleotides: adenosine triphosphate (ATP) and guanosine triphosphate (GTP). The pathway is branched and its regulation includes feedback inhibition of the initial reaction before the branching point, as well as inhibition of the two separate branches independently. Furthermore, accumulation of the end product of one branch of the pathway stimulates the other branch. In this pathway, inosinic acid is converted into either ATP or GTP, depending on which branch of the reaction sequence is traversed. One of the early steps in the pathway, the first shown in the figure, involves transfer of an amino group to 5phosphoribosyl-1-pyrophosphate, forming 5-phosphoribosylamine (Fig. 8b). The aminotransferase catalyzing this reaction is inhibited by ATP, ADP, and AMP or, alternatively, GTP, GDP, and GMP, with the two groups of nucleotides acting at two separate regulative sites in the enzyme. The two divergent pathways are subject to separate feedback control at the point of divergence. The branch producing the G-containing nucleotides is controlled by GMP, whereas that producing the A-containing nucleotides is controlled by AMP. In addition, the end products of the two divergent pathways reciprocally facilitate each other: ATP facilitates the GTP pathway and GTP facilitates the ATP pathway. This mode of control ensures smooth integration of the two systems synthesizing the two purine nucleotides.
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The data available for the allosteric inhibition of aspartate carbamoyltransferase are shown in Fig. 9a (Gerhart and Pardee, 1962). In this experiment, the velocity of the reaction is measured at various concentrations of aspartate while the concentration of the other substrate, carbamoyl phosphate, is kept constant. The upper curve represents a control in the absence of CTP. The lower curve shows the same experiment carried out in the presence of CTP, the product of the metabolic pathway. Inhibitory compounds of this kind have been called negative effectors by some investigators. Other metabolites act as positive effectors; ATP acts as a positive effector in this reaction (Fig. 9b). Such a role may be of fundamental physiological importance. When the energy supply is plentiful (reflected in a high level of ATP), nucleic acid synthesis and, consequently growth, are favored by increasing the availability of the nucleic acid precursors. On the other hand, a decrease in ATP slows down the biosynthetic pathway.
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Fig. 8 (a) Feedback control of the aspartate carbamoyltransferase reaction by CAP and its stimulation by ATP. (b) Schematic diagram of the regulation of purine nucleotide metabolism.
Fig. 9 (a) Reversal of CTP inhibition of aspartate carbamoyltransferase by aspartate. (b) Effect of ATP on reaction velocity. Velocity, units of activity per milligram protein x 10-3. The reaction mixture contained http://www.albany.edu/~abio304/text/13part2.html (3 of 24) [3/5/2003 8:20:46 PM]
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3.6 x 10-3 M carbamoyl phosphate, 0.04 M potassium phosphate, pH 7.0, and 9.0 x 10-2 g of enzyme per ml. Reproduced with permission from J. C. Gerhart and A. B. Pardee, Journal of Biological Chemistry, 237:891-896. Copyright ©1962 The American Society of Biological Chemists, Inc.
The increase in enzymatic activity that occurs when the aspartate carbamoyltransferase is treated with mercurials (Fig. 9) is accompanied by loss of regulative ability. The inhibition by CTP is lost completely, as shown in Table 3, column 1. Columns 2 and 3 correspond to the apparent Vm and Km, respectively. Table 3 Selective Destruction of Feedback Inhibition
Treatment
1. None 2. 10-6 M Hg (NO3)2 3. 5 x 10-3 M
(1) Inhibition by 2 x 10-4M CTP (%)
(2) Maximal velocity (3) Aspartate for half(units/mg protein x saturation 10-3) (molarity x 10-2)
70 0
4.5 10.0
6 12
12
0
phydroxymercuribenzoate 4. 5 x 10-3 M mersalylb
0
5. 10-3 M AgNO3
0
6. Preheat 4 min at 60oC 7. 0.8 M urea
0
9
0
>4.5
Reproduced with permission from J.C. Gerhardt and A.B. Pardee, J. Biol. Chem. 237:891-896. Copyright ©1962 The American Society of Biological Chemists, Inc.
The fact that loss of regulative ability and loss of enzymatic activity do not go hand in hand speaks for two different reactive groups being responsible for the two effects. As mentioned, two different kinds of subunits are separated out from aspartate carbamoyltransferase upon treatment with p-mercuribenzoate. One kind of subunit involves enzymatic activity only, whereas the other, when complexed with the active portion, permits control by end product or end product analog inhibition (Fig. 10). Alternatively, the activity of the enzyme can be enhanced by ATP. The recoupling after removal or dilution of the mercurial compound is spontaneous.
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Fig. 10 (Gerhart and Schachman, 1965) represents a demonstration of the separation of the subunits of carbamoyltransferase by p-mercuribenzoate. It is possible to separate protein molecules on the basis of their size and density differences by means of centrifugation techniques. One such technique is centrifugation through a sucrose solution, forming a density gradient. The preparation, in this case the enzyme treated with p-mercuribenzoate, is layered on a tube containing the sucrose gradient. Centrifugation of the tube at very high speed causes the molecules to descend through the gradient, with the heaviest and largest molecules moving most rapidly. After centrifugation, the various layers of the gradient can be separated and sampled for enzymatic activity. The assay is carried out by mixing the layer with a substrate mixture of aspartate and carbamoyl phosphate. The appearance of the product of the reaction, carbamoyl aspartate, is measured by the appropriate chemical method. In Fig. 10, the distribution of the enzyme in the various layers is shown by the closed circles and dashed curve. The capacity of the fractions to inhibit the aspartate carbamoyltransferase (after dilution to decrease the concentration of p-mercuribenzoate) is shown by the filled triangles and line. The enzyme activities were assayed as follows: one of the fractions that is unable to respond to the negative effector (fraction 6) was mixed in the presence of CTP with the fraction to be sampled for inhibitory activity. Clearly, the enzymatic activity (filled circles) and the ability to respond to the inhibition (triangles) are in two separate molecules. The open circles represent the ultraviolet light absorption of each fraction (at a wavelength of 280 nm) and indicate protein concentration. In contrast to aspartate carbamoyltransferase, many of the enzymes studied so far do not have separate subunits responsible for regulation. One of these enzymes is glutamine synthetase from Escherichia coli. The enzyme synthesizes glutamine from glutamate, ATP, and ammonia. Glutamine is thought to serve as an important reserve of available nitrogen and is required by several metabolic pathways. Glutamine synthetase is made up of 12 identical subunits, each with a molecular weight of about 50 kDa (Shapiro and Stadtman, 1967). The activity of this enzyme is inhibited by at least eight separate feedback inhibitors which apparently attach to eight independent allosteric sites.
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Fig. 10 Subunits of aspartate carbamoyltransferase separated by sucrose gradient centrifugation. The layers contained 6-25% sucrose, 0.04 M potassium phosphate, pH 7.0, and 10 M p-mercuribenzoate. Samples were centrifuged for 20 h at 10oC at 38,000 rpm in a W-39 rotor of a Spinco model L centrifuge. Reproduced with permission from Gerhart and Schachman (1965) Biochemistry 4:1054-1062, copyright ©1965 by the American Chemical Society.
A model representing an enzyme made up of two identical subunits is shown in Fig. 11 (Koshland and Neet, 1968). Allosteric effectors, R2, interact with special regulator sites of the molecule (part B) as in the case of glutamine synthetase. In some cases, such as aspartate carbamoyltransferase, part B is a separate regulatory subunit. The shaded sites in part A of the molecule correspond to the active site bindings substrate, S, with two residues, X1 and X2. The effector molecule R1 can interact directly with the active site. The outside portion of the active site represents a flexible arm that is adjusted into position by the substrate itself and the effector R2 (the so-called induced fit). There is considerable evidence that induced fit plays a significant role in enzyme catalysis.
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Fig. 11 Hypothetical structure of an enzyme containing two identical subunits. The enzyme has two residues in the active site (X1 and X2). The substrate is denoted by S, and R1 and R2 are regulator molecules, functioning at the active site and at allosteric site, respectively. Modified from Koshland and Neet (1968). Reproduced, with permission, from the Annual Review of Biochemistry, Volume 37, 1968 by Annual Reviews Inc.
The model represented in this figure depicts neither a conformational change imposed by the allosteric effector nor a conformational change induced in one subunit by the interaction of the other with the substrate. The nature of the conformational changes in enzymes are still not entirely clear. As shown in Fig. 12 (Atkinson, 1965) the effects of positive and negative effectors are well suited for the regulation of enzyme activity. The presence of an effector can produce a major change in the reactivity of the enzyme. The small shifts in the curve, depicting the rate of the reaction as a function of substrate concentration, represent major changes in the rate of enzyme activity at certain fixed substrate concentrations (e.g., S') (compare 3, the control curve to 2 or 4 in Fig. 12). The bar diagrams (left side of the figure) show the rates at these critical substrate concentrations in the presence or in the absence of positive and negative effectors.
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Fig. 12 Generalized substrate response curve for an enzyme that is regulated by positive effectors (+) or negative effectors (-). The control (no effectors) is indicated by the letter c. Reproduced with permission from D. E. Atkinson, Science, 150:851-857. Copyright ©1965 by the AAAS.
Generally, allosteric effects can vary the activity of an enzyme over a 10-fold range. However, because in some cases the forward and backward reactions of a step are catalyzed by separate enzymes which are regulated independently, the actual regulation can result in a 100-fold change (see Chapter 14, Section IIC). Intrasteric interactions Regulation may involve internal sequences resembling the substrate and binding to the active site. This kind of regulation has been called intrasteric regulation (see Kobe and Kemp, 1999). Intrasteric regulation may result from allosteric interactions. The regulatory domain is referred to as a pseudosubstrate or intrasteric autoregulatory sequence (IARS). The pseudosubstrate domain may be in the protein itself or in another subunit of a complex and may even be in a separate protein molecule entirely (e.g., protein proteinase inhibitors, see Kobe and Kemp, 1999). Originally, intrasteric regulation had been observed only in protein kinases and phosphatases (Kemp and Pearson, 1991). However, intrasteric regulation occurs in a variety of enzymes and also in receptors and protein-targeting domains. Evidence for intrasteric regulation includes: (a) the inhibition of the enzyme by synthetic peptides corresponding to the pseudosubstrates, (b) absence of regulation with the removal of the pseudosubstrate domain, (c) or in the presence of antibodies against the regulatory domain, and (d) structural crystallographic studies that show interactions of a pseudosubtrate domain with the active site. IV. TRANSITIONS FROM INACTIVE TO ACTIVE FORMS
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Some of the regulative events of a cell's biochemical reactions seem to involve allosteric interactions, such as the end product inhibitions or simulations just discussed. However, enzyme activity may also be regulated by covalent modifications in which the enzyme is converted from an inactive form to an active form. Furthermore, at least in the case of phosphorylation, the modification may determine whether the enzyme can be controlled allosterically. A number of covalent modifications have been implicated, such as conversions from S-S to SH groups, acetylation or deacetylation, methylation or demethylation, adenylation or deadenylation. Activation of enzymes by proteolytic cleavage of the inactive form of the enzyme, the proenzyme, is involved in many physiological (Hall et al., 1979) and developmental processes (Hasilik and Tanner, 1978; Neurath and Walsh, 1976). At least in eukaryotic cells, many enzymes have been shown to undergo phosphorylative and dephosphorylative transitions. Table 4 (Krebs and Beavo, 1979) gives a relatively short list, and more are likely to be involved. Generally ATP, and in sometimes GTP, is implicated in phosphorylation. In some cases, the phosphorylated form is the more active form (as in the case of phosphorylase b), and in others it may be a less active form. Generally, the enzymes involved in a breakdown pathway are activated by phosphorylation, whereas enzymes involved in synthetic pathways are inactivated by phosphorylation. Some examples of these effects are shown in Table 5 (Cohen, 1980). Table 4 Initial Reports on Enzymes Undergoing Phosphorylation-Dephosphorylation
Enzyme
Year reported
Glycogen phosphorylase
1955
Phosphorylase kinase
1959
Glycogen synthase
1963
Hormone-sensitive lipase
1964,1970
Fructose-1,6-bisphosphatase
1966,1977
Pyruvate dehydrogenase
1969
Hydroxymethylglutaryl-CoA reductase
1973
Acetyl-CoA carboxylase
1973
DNA-dependent RNA polymerase
1973
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Pyruvate kinase (liver)
1974
Cholesterol ester hydrolase
1974
R subunit of type II cAMP-dependent protein kinase
1974 1975
Reverse transcriptase 1975 Phosphofructokinase (liver) 1975 Tyrosine hydroxylase 1975 Phosphorylase phosphatase inhibitor 1976 Phenylalanine hydroxylase 1977 elF2-kinase 1977 cGMP-dependent protein kinase 1977 Tryptophan hydroxylase 1978 NAD-dependent glutamate dehydrogenase (yeast)
1978
Glycerophosphate acyltransferase
From: Krebs and Beavo (1979) Reproduced with permission, from the Annual Review of Biochemistry, vol. 48 © 1979 by Annual Reviews Inc.
Phosphorylations are catalyzed by protein kinases; dephosphorylation by phosphatases (see Section V) These systems are under the control of hormonal or neural signals. Cyclic AMP, cyclic GMP, Ca2+ calmodulin complex, and other second messengers (see Chapter 6) have been implicated in some of these regulative events. Table 5 Enzymes Found in the Cytoplasm of Mammalian Cells That Are Regulated by Phosphorylation
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Types of protein kinase involved cAMP
Ca2+calmodulin
Other
Biodegradative pathway:
ACTIVATION BY PHOSPHORYLATION: Glycogen phosphorylase Phosphorylase kinase Myosin Triglyceride lipase
-
+
-
+
+
-
-
+
-
+
-
-
+
-
?
Glycogenolysis Glycogenolysis ATP hydrolysis Triglyceride breakdown
Cholesterol esterase
Cholesterol ester hydrolysis
INACTIVATION BY PHOSPHORYLATION:
Biosynthetic pathway:
Glycogen synthase
+
+
+
+
-
+ Fatty acid synthesis
Acetyl-CoA carboxylase Glycerol phosphate acyltransferase
+
-
Triglyceride synthesis
HMG-CoA reductase
Glycogen synthesis
-
+ Cholesterol synthesis
-
-
+ Protein synthesis
eIF
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From: P. Cohen, Molecular Aspects of Cell Regulation, vol 1, Copyright ©1980 Elsevier Science Publishers, Amsterdam.
A general role of the phosphorylation-dephosphorylation of proteins in cellular physiology has been proposed (Greengard, 1978); the process is likely to affect the functions of chromosomes, ribosomes, microtubules, and cell or intracellular membranes. A diverse enzyme regulatory role by ubiquitin conjugation, analogous to the phosphorylative mechanism, has also been proposed. Ubiquitin has been found in all cells examined (hence the name, which is derived from ubiquitous). Ubiquitin is an intracellular peptide which is ligated to proteins by a series of reactions involving several enzymes (Chapter 15, II, B). A regulatory role of ubiquitin conjugation is suggested by the presence of ubiquitin hydrolases, which remove ubiquitin from conjugates. The conjugation between ubiquitin and various proteins is involved in the pathway of protein degradation that takes place in all cells (Chapter 15, II, B). However, the role of ubiquitin is likely to touch a broader spectrum of the cell's activities (Jentsch et al., 1990). Metabolically stable ubiquitin-protein conjugates are found in eukaryotes. Furthermore, a large part of chromosomal histones are conjugated to ubiquitin, suggesting a role in gene expression. Several integral membrane proteins as well as actin, have also been found to be conjugated to ubiquitin. The conjugation to ribosomal enzymes enhances protein synthesis (e.g., see Spence et al., 2000). In addition, ubiquitin-conjugating enzymes appear to be involved in DNA repair and the control of the cell cycle (e.g., Goebl et al., 1988) (see Chapter 8). A general role of acetylation in regulation similar to the phosphorylation-dephosphorylation system has been envisioned (see Kouzarides, 2000). The role of acetylation of histones in gene expression is well understood (see Chapter 2, Section B and C). Many proteins have been modified by acetylation (such as transcription factors, nuclear import factors and α-tubulin). Acetylation has been found to regulate many functions including DNA recognition, protein-protein interaction and protein stability. Bromodomains recognize acetylated residues and may serve as signalling domains. In DNA-binding transcription factors, acetylation increases DNA binding. Acetylation inhibits protein-protein interactions and increases the half life of some proteins including α-tubulin and microtubules. Acetylated α-tubulin is present mostly stable microtubules and absent from dynamic cellular structures such as neuronal growth cones and the leading edges of fibroblasts. So far only one enzyme has been identified in the acetylationdeacetylation regulatory process, a tubulin deacetylase ( Hubbert et al., 2002). The enzyme is cytoplasmic and associates with the microtubule motor complex containing p150glued ( see Chapter 24, Section IV B and Section IV C). V. REGULATION OF PHOSPHORYLATION AND DEPHOSPHORYLATION We saw that phosphorylation and dephosphorylation have a key role in the regulation of enzymatic activity. Furthermore, the transcriptional and translational machinery of the cell is regulated by phosphorylation-dephosphorylation. Phosphorylation appears to function in both repression, positive http://www.albany.edu/~abio304/text/13part2.html (12 of 24) [3/5/2003 8:20:46 PM]
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regulation, stability, localization and the ability to interact with other proteins or DNA (see Holmberg et al., 2002). The phosphorylation of proteins at multisite sites permits the precise tuning of activity. For example, p53 which mediates cell-growth block and apoptosis has 16 phosphorylation sites (see Holmberg et al., 2002) leading to different effects (e.g., see Vousden, 2002). The concerted activity of protein kinases (PKs) and phosphatases (PPs) has a central role in this mechanism of regulation. About 200 PKs and 100 PPs have been identified. Approximately 30 of the PPs are protein tyrosine phosphatases (Mauro and Dixon, 1994). PKs and PPs are relatively nonspecific, at least in vitro, and can even catalyze reactions with artificial substrates. Their specificity may rest in a specific localization. Present information suggests that these enzymes, in addition to a catalytic domain, contain a targeting domain which directs them to specific targets in the cell (see also Chapter 10, Section VI). An additional role of this domain may be regulatory, via an allosteric interaction. The sections that follow address these question in more detail. The regulation of protein kinases and phosphatases is also discussed in some detail in relation to intracellular signals in Chapter 7. A. Protein Kinases PKs mediate the interaction between extracellular ligands and biological activity (Chapter 6 and 7). At least two classes of protein kinases have been shown to be regulated by targeting subunits: cAMPdependent protein kinases (PKAs), which are activated by the presence of the second messenger cAMP (see Chapter 7) and cyclin-dependent kinases. Cyclins are protein molecules involved in the regulation of cell division (Chapter 8). Protein kinases have been found to be profoundly affected by domains that allow them to interact docking sites on their substrate. Interactions with substrate docking sites Protein kinases have been found to complex tightly to their substrates even when inactive. The enzyme and the complex may interact directly. The small domain in the substrate molecule that binds to the enzyme is known as a docking site (see Table in Holland and Cooper, 1999). Alternatively, the interaction may be through a third protein. This binding increases the efficiency of the kinase (see e.g., Jacobs et al., 1999; Gavin and Nebreda, 1999; Smith et al., 1999). Several docking sites can be present and are modular: when experimentally attached to different substrates they change their specificity for the kinase. The presence of substrate docking sites has several consequences. It may determine the location of the kinase within the cell. For example, the MSAP kinase p38 is localized in the nucleus when not active because of its binding to its substrate, the MAPKAP kinase 3 (Ben-Levy et al., 1998). Docking sites increase the specificity of the kinases which normally would be capable of reacting with a variety of substrates. Furthermore, the preassembly of kinase and substrate allows for very rapid enzymatic action http://www.albany.edu/~abio304/text/13part2.html (13 of 24) [3/5/2003 8:20:46 PM]
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after activation (see Holland and Cooper, 1999). The tightness of the interaction between substrate and enzyme would slow down the reaction if it were not for the fact that the complex of the two is destabilized by the phosphorylation of the substrate (Zhao et al., 1996; Waskiewicz et al., 1997) so that there is an increase in Vmax accompanying the increase in affinity (see Jacobs et al., 1999) cAMP-dependent protein kinases PKAs are present in the cytoplasm as an inactive tetramer of two catalytic and two regulatory subunits. In this form, the enzymes are excluded from the nucleus. Two cAMPs bind to each regulatory subunit; this binding induces the dissociation of the complex into a regulatory dimer and two catalytic monomers. The free catalytic monomers can enter the nucleus (Fantozzi et al., 1992). In the nucleus, they regulate transcription factors such as the cAMP response element binding protein (CREB)(see Chapter 7, Fig. 18). The regulatory subunit can also serve as a targeting device. Regulatory subunits of type II PKAs keep the enzyme at specific locations. For example, the regulatory subunit of PKA type II sequesters most of the cAMP of the brain and binds to the microtubular associated protein 2 (MAP-2). Cyclin-dependent protein kinases The cyclin dependent PKs are activated at different stages of the cell cycle (see Chapter 8). These enzymes are heterodimers. The catalytic subunit attaches to a cyclin, its regulatory subunit, which activates the enzyme and targets it to particular sites. B. Protein Phosphatases The PPs have been studied less than the PKs. Obviously, they must be as significant in regulation as the better understood PKs. A general discussion of PPs will be found in Chapter 7. Several of the PPs involved in the regulation of metabolism have been studied in detail. PP1 associated with glycogen particles is a protein serine/threonine phosphatase. In vitro it is capable of dephosphorylating proteins that have been phosphorylated by different PKs. However, in vivo its specificity is assured by its association with glycogen. The next section will discuss this kind of PP1 (PP1 G). PP1G is present as a heterodimer containing a catalytic subunit of 31 kDa and a glycogen targeting subunit of 124 kDa (Strålfors et al., 1985, Tang et al., 1991). The regulatory subunit also contains a sarcoplasmic reticulum targeting domain and several phosphorylated sites (see Hubbard and Cohen, 1993). PP1G binds glycogen with high affinity and is present exclusively in its bound form. Phosphorylase kinase and glycogen synthase are also bound to glycogen. VI. MULTIENZYME COMPLEXES http://www.albany.edu/~abio304/text/13part2.html (14 of 24) [3/5/2003 8:20:46 PM]
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The organization of functionally related enzymes in complexes concentrates catalytic activity and allows for a high steady-state concentration of intermediates so that substrates are transferred from one active site to another with a minimum of diffusional effects or dilution. Furthermore, the presence of enzymes in a complex makes possible coordinated allosteric control of several activities. α-ketoacid dehydrogenase and fatty acid synthetase are soluble multienzyme complexes that have been studied in some detail. In the next section, the pyruvate dehydrogenase complex of E. coli will serve as an example of a complex of several distinct enzymes. The combination of enzymes into complexes involving membranes is discussed primarily in relation to oxidative phosphorylation (Chapter 16), photosynthesis (Chapter 17), and the reactions involved in the translocation of solutes across the cell membrane (Chapters 19, 20, and 21). Many other enzymes are present in multimolecular assemblies. As our knowledge gains in detail, the number of known complexes increases. The splicing of mRNA is carried out by spliceosome, and transcription takes place in transcriptional complexes. Protein synthesis involves a variety of enzymes, mRNA and ribosomes, forming translational complexes. The degradation of proteins involve proteosomes. In addition, there is evidence for multienzyme systems in other pathways (referred to as metabolons), such as glycolysis, nucleotide synthesis (e.g., Jones, 1980), urea biosynthesis, tricarboxylic acid cycle, fatty acid oxidation and amino acid metabolism. The sections that follow will first discuss pyruvate dehydrogenase. Other cases that have been studied less will be discussed in later sections. A. The Pyruvate Dehydrogenase Complex In E. coli, the pyruvate dehydrogenase reaction is carried out by enzyme aggregates made up of three types of enzymes with a total molecular weight of about 4,000 kDa. Similar complexes are thought to cata1yze these reactions in other organisms, including mammals. The reactions catalyzed by the pyruvate dehydrogenase complex are represented in Fig. 13, in which part (a) represents the overall reaction and part (b) some of the details. As shown in this figure, pyruvate in the presence of coenzyme A and NAD+ is oxidized and decarboxylated to form acetyl coenzyme A, reduced NAD, and CO2. This pyruvate dehydrogenation involves several separate reactions and the three enzymes represented in Fig. 13b. These enzymes are held together in the pyruvate dehydrogenase complex. In reaction 1, pyruvate reacts with pyruvate dehydrogenase (E1). The reaction involves the coenzyme thiamine pyrophosphate (TPP). Decarboxylation of the pyruvate forms the α-hydroxyethylTPP-E1 complex. Reaction 2 involves lipoyl reductase-transacetylase (E2). The lipoyl moiety of E2 oxidizes the hydroxyethyl residue that is transferred from E1-TPP as the acetyl moiety. In reaction 3, the acetyl-reduced lipoyl-E2 transfers the acetyl group to coenzyme A, and, in reaction 4, the lipoyl-E2 molecule reduced in reaction 2 is oxidized by the flavoprotein dihydrolipoyl dehydrogenase (E3-FAD). The reducing equivalents from this reaction are transferred to NAD+ (reaction 5) and eventually oxidized by the cytochrome chain. In these reactions, the lipoyl moiety of E2 (abbreviated Lip) interacts with both E1-TPP (reaction 1) and E3 (reactions 3 and 4). It serves to transfer reducing equivalents as well as acetyl groups (see Fig 13). http://www.albany.edu/~abio304/text/13part2.html (15 of 24) [3/5/2003 8:20:46 PM]
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Fig. 13 Diagrammatic representation of the pyruvate dehydrogenase reaction. E1, Pyruvate dehydrogenase; E2, dihydrolipoyl transacetylase; E3, dihydrolipoyl dehydrogenase.
The three enzymes can be separated out from the pyruvate dehydrogenase complex by relatively gentle means since they are held together by noncovalent bonds. The separated molecules reassemble spontaneously when mixed together at neutral pH. Alone, each kind of enzyme molecule forms an active complex made up of several subunits; these complexes have a specific morphology. Complexes are also formed between the molecules of pyruvate dehydrogenase (E1) or dihydrolipoyl dehydrogenase (E3) and the transacetylase (E2). The characteristic appearance of these complexes and that of the entire dehydrogenase complex have permitted their reconstruction. A reconstruction of the entire pyruvate dehydrogenase complex is shown in Fig. 14. Figure 14a shows an electron micrograph of the complex of the three enzymes after negative staining with phosphotungstate. In this procedure, the particles, suspended in a solution of phosphotungstate, are placed on a grid in a thin layer by a suitable procedure and dried. When viewed with the electron microscope, the molecules appear white against the dense background of the phosphotungstate (Fig. 14a). A model based on various observations is shown in Fig. 14b.
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Fig. 14 (a) Electron micrographs of the E. coli pyruvate dehydrogenase complex and its componenent enzymes (bar corresponds to 75 nm), A, Pyruvate dehydrogenase complex; B, dihydrolipoyl transacetylase (E2); C, pyruvate dehydrogenase (E1); D, dihydrolipoyl dehydrogenase (E3). (b) Interpretative model of E. coli pyruvate dehydrogenase complex: A, Model viewed along the fourfold axis of the dihydrolipoyl transacetylase (E2) core, illustrating the proposed architectural organization of the complex; B, stereoscopic drawings of a portion of the model showing the spatial relationships of the components. The eight trimers of E2 binding domains are represented by large spheres, and the single E2 lipoyl domain shown is represented by an ellipsoid. Pyruvate dehydrogenase (E1) chains are represented by tetrahedra consisting of four lobes (small spheres) bound along the edges of the cubelike "inner" core of E2 binding domains. In this projection of the model, the 12 E1 chains shown are superimposed over 12 other similarly positioned chains of E1 (not shown). The dimers of dihydrolipoyl dehydrogenase (E3) are located on the faces of the cubelike E2 "inner" core and are represented by pairs of cylinders. From R. M. Oliver and L. S. Reed, Electron Microscopy, 2:1-48, 1982, with permission of Academic Press.
The molecules of each of the three enzymes occupy fixed positions in the complex. Because the active groups correspond to a very small portion of the enzyme, it is difficult to see how an interaction can take place between the dihydrolipoyl transacetylase active site and the other two enzymes unless parts or all of the molecule move in relation to the others, first to react with one enzyme (pyruvate dehydrogenase)
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and then with the other (dihydrolipoyl dehydrogenase). The dihydrolipoyl transacetylase portion is composed of 24 polypeptide chains each bearing two lipoyl moieties, which function in both acetylation and redox reactions (see Fig. 15). The interaction could result from the swinging of the lipoyl-lysyl 1.4 nm arm of the dihydrolipoyl transacetylase as shown in Fig. 15a. Alternatively, the polypeptides to which the lipoyl moiety is attached could be mobile (Collins and Lester, 1977; Perham and Duckworth, 1981). These two models (A and B) are shown in Fig. 15b.
Fig. 15 (a) Schematic representation of the possible rotation of a lipoyl-lysyl moiety between αhydroxyethylthiamine pyrophosphate (TPP-Ald) bound to pyruvate dehydrogenase (D) (the site for acetyl transfer to CoASH) and the reactive disulfide of the flavo-protein (F). From Oliver and Reed (1982), with permission. (b) Model illustrating movement of lipoyl domains (and not simply rotation of lipoyl moieties) to span the physical gaps between catalytic sites on the complex. A dihydrolipoyl transacetylase (E2) subunit is represented by a sphere (subunit binding domain) and its attached ellipsoid (lipoyl domain). The catalytic site for transacetylation resides on the subunit binding domain, whereas the two lipoyl moieties are on the lipoyl domain. It is visualized that movement of lipoyl domains permits their covalently attached lipoyl moieties (not shown) to service the catalytic sites on the complex. Each E1, E2, http://www.albany.edu/~abio304/text/13part2.html (19 of 24) [3/5/2003 8:20:47 PM]
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and E3 subunit is serviced by at least two lipoyl moieties, which apparently reside on two separate lipoyl domains. The E1, E2, and E3 subunits shown need not be adjacent to each other in the complex. From Stepp et al. (1981). Reproduced with permission from Biochemistry 20:4555-4560. Copyright ©1981 American Chemical Society.
In the previous section, we saw that enzyme complexes made up of identical subunits undergo conformational changes during enzyme-catalyzed reactions. In this section, we saw how the larger multienzyme complexes discussed must also involve some movement, either of the molecules of the complex in relation to each other, or of a portion of one of the molecules. These movements, or conformational rearrangements, appear to be a fundamental property of many enzyme-catalyzed systems and may be involved in a wide range of biological mechanisms. The molecular mechanisms of muscular contraction or other events linked with cellular motility, the changes in shape of cells, and the mechanisms of solute transport across biological membranes, may well involve specialized modifications of these conformational rearrangements. B. Other Multienzyme Complexes Some of the evidence for the presence of metabolic enzymes associated in complexes comes from indirect information. For example, the centrifugation of intact cells has been shown to sediment most cytosolic proteins (Zalokar, 1960; Kempner and Miller, 1968); this observation suggests that a large portion of the proteins of the cell must be present as aggregates. It has also been argued that the high concentration of proteins in the mitochondrial matrix (calculated to be 56% by weight) (Hackenbrock, 1968) corresponds to the densest possible packing for proteins, i.e., that of the maximum packing of identical spheres. This suggests that these proteins are present in a semi-crystalline state and therefore must be in the form of interacting complexes. Similar arguments can be made for the glycolytic enzymes in muscle, where most of the space not occupied by the contractile apparatus is likely to be occupied by glycolytic enzymes (Pette and Brandau, 1962; Sigel and Pette, 1969). Pette and Brandau estimate that in the muscle of Locusta migratoria, the concentration of these enzymes by themselves corresponds to 0.2 g per ml. Other evidence is more direct and is discussed below. A strong argument for the idea that glycolytic enzymes are organized in assemblies comes from experiments with permeabilized fibroblasts, where the efficiency of the conversion of [14C]-glucose to [14C] lactate (Clegg and Jackson, 1990) suggests that the endogenously produced intermediates are channeled as if they were in complexes. Mitochondrial enzymes The complexes involved in electron transport and phosphorylation in mitochondria (Chapters 16 and 18) and chloroplasts (Chapters 17 and 18) will be discussed separately. Evidence exists for the organization of mitochondrial matrix enzymes in complexes and that in some cases the localization of certain enzymes play an important role in metabolism.
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Each mitochondrial enzyme of the citric acid cycle is able to bind the next enzyme in the sequence. Furthermore, they do not bind to isoenzymes catalyzing the same reaction in other locations (e.g., fumarase binds only the mitochondrial malate dehydrogenase and not the cytoplasmic enzyme, Beekmans and Kanarek, 1981). The interactions are summarized in Table 6. The enzymes are listed in sequential order, beginning with citrate synthase. All enzymes are present either in the first or second column, with the exception of succinate dehydrogenase. Together with the fact that the enzymes are present in very high concentrations, as discussed above, the results suggest very strongly that the enzymes are present in a complex. In addition to the ability to form complexes, the tricarboxylic acid cycle enzymes also bind to the inner mitochondrial membrane (D'Souza and Srere, 1983). These experiments took advantage of the inner mitochondrial vesicle preparations that are inside-out. Isoenzymes that catalyze the same reactions but correspond to other organelles (e.g.,+ in yeast citrate synthase from peroxisomes) (Kispal and Srere, 1991) do not bind to these vesicles. Although there was no binding of the enzymes to liposomes free of protein components, mitochondrial malic dehydrogenase did bind to liposomes in which the mitochondrial Complex I was incorporated (Sumegi and Srere, 1984). Complex I is capable of accepting electrons from dehydrogenases (see Chapter 16). Mitochondria disrupted by ultrasound (as demonstrated by the accessibility of citrate synthase to its antibody or of malic dehydrogenase to high molecular weight blue dextran) contain tricarboxylic acid cycle enzymes (Robinson et al., 1985). This association supports the view that they are bound to the inner mitochondrial membrane. In these complexes, the rate of metabolism is much higher than in the solubilized state (Robinson et al., 1987; Sumegi et al., 1991), suggesting that the enzyme properties are quite distinct when bound. The localization of mitochondrial components is also important. Apparently, the respiratory rate of cardiac mitochondria is under the control of creatine kinase, which catalyzes the phosphate exchange between creatine and ATP. This enzyme is present between the mitochondrial inner and outer membranes (Jacobus et al., 1983). Furthermore, the mitochondrial outer membrane has binding sites for hexokinase (Felgner et al., 1979) probably at sites at which the inner and outer membrane make contact (the so-called contact sites), so the ATP synthesized can be used without delay to phosphorylate hexoses. Table 6 Interactions of TCA Enzymes
Enzyme citrate synthase
Binds to malate dehydrogenase, aconitase, pyruvate dehydrogenase citrate transporter
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Reference Halper and Srere, 1977; Tyiska et al., 1986; Sumegi and Alkonyi, 1983; Persson and Srere, 1992
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aconitase
NADH-isocitrate dehydrogenase
Tyiska et al., 1986
succinyl-CoA synthase
α-ketoglutarate dehydrogenase complex
Porpaczy et al., 1983
fumarase
malate dehydrogenase
Beekmans and Kanarek, 1981
Binding of glycolytic enzymes to components of the contractile systems The location of enzymes at specific sites is shown in muscle, where most glycolytic enzymes (but not hexokinase, which is localized primarily in mitochondria) are located within the I-band, a site shared with F-actin (Dolken et al., 1975; Sigel and Pette, 1969). A number of glycolytic enzymes have been shown to bind to F-actin (Arnold and Pette, 1968, 1970; Arnold et al., 1971). The binding is most pronounced in the presence of the regulatory proteins tropomyosin and troponin (Clarke and Morton, 1976; Clarke and Masters, 1975, 1976; Stewart et al., 1980). Binding to actin was observed for fructose1,6-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. In Drosophila flight muscle, glycerophosphate dehydrogenase (GPDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase can be localized at Z discs and M lines using immunofluorescence (Wojtas et al., 1997). The Z lines were identified using anti-α-actinin, a Z-disc protein. The position of the M-lines can be deduced from the fact that they are equidistant between Z discs. GPDH is not a glycolytic enzyme. Its substrate, glycerophosphate, is generated from the breakdown of lipids. However, it is oxidized to dihydroacetone phosphate and the latter, after an isomerization reaction, produces glyceraldehyde 3-phosphate, an intermediate in glycolysis. Transgenic flies able to synthesize only one of the GPDH isoforms (GPDH-3) lacked the characteristic distribution of GPDH, GADPH and aldolase, and were unable to fly although they contained the normal complement of glycolytic enzymes (Wojtas, 1997). GPDH-3 differs from the normal GPDH-1 only in lacking a tripeptide at the carboxy terminal. These observations show that the organization of enzymes in relation to cytoplasmic structures is of fundamental importance in function and that the distribution of related enzymes is codependent. The glycolytic reaction sequence catalyzed by aldolase, triosephosphate isomerase, glyceraldehydephosphate dehydrogenase and phosphoglycerate kinase have been found in isolated muscle triads (see Chapter 24). In the triad, these enzymes synthesize ATP in the presence of glyceraldehyde 3-phosphate http://www.albany.edu/~abio304/text/13part2.html (22 of 24) [3/5/2003 8:20:47 PM]
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or fructose 1,6-bisphosphate (Han et al., 1992). The synthesized ATP appears associated with the triad and is not in equilibrium with the bulk ATP. Phosphofructokinase (Vertessy et al., 1997; Volker and Knull, 1997) and aldolase (Vertessy et al., 1997) have been found associated with microtubules. Glycogen particles Glycogen particles isolated from skeletal muscle contain the enzymes of glycogen metabolism. These include: phosphorylase b and its kinase and phosphatase; glycogen synthase and its respective kinase and phosphatase (Meyer et al, 1970); as well as the debranching enzyme, glycogen synthase kinase 2, protein phosphatase 2, and inhibitors 1 and 2 (Nimmo et al., 1976). The glycogen particles themselves are closely associated to the sarcoplasmic reticulum near the thin filaments (Wanson and Drochmans, 1968). The kinetic behavior of the enzymes in the particles is distinct from those in solution (Fischer, et al., 1971). Binding of enzymes to the plasma membrane Certain enzymes are located in the cell's periphery. At least some of this binding is to the cytoplasmic domain of integral membrane proteins. In the red blood cell, Glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase bind to Band 3 protein, the anion transporter (see Yu and Steck, 1975; Tsai et al., 1982). In the case of glyceraldehyde-3-phosphate dehydrogenase, the binding exhibits 1:1 stoichiometry. The bound enzymes have different kinetic properties from the free enzymes. For example, phosphofructokinase, upon binding, loses its allosteric inhibition by ATP (Karadsheh and Uyeda, 1977) and glyceraldehyde-3-phosphate dehydrogenase is inhibited by binding (Tsai et al., 1982). The presence of organized multienzyme complexes or clusters offers significant opportunities for integrated activity and regulation that are just beginning to be appreciated (Welch, 1977; Paul et al., 1989). There are indications, for example, that at least portions of the enzymes glyceraldehyde-3phosphate dehydrogenase and phosphoglycerate kinase function at the inner surface of the red cell membrane to provide ATP to Na+,K+ -ATPase but not to the rest of the cell (Mercer and Dunham, 1981). SUGGESTED READING Introductory Creighton, T.E. (1993) Proteins, Structures and Molecular Properties, W.H. Freeman, New York, Chapter 9. http://www.albany.edu/~abio304/text/13part2.html (23 of 24) [3/5/2003 8:20:47 PM]
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Kraut, J. (1988) How do enzymes work? Science 242:533-540.(Medline) Saier, M. H., Jr. (1987) Enzymes in metabolic pathways. Harper Row, New York, Chapters 3 and 4. Stryer, L. (1994) In Biochemistry. 4th ed. Freeman, New York Chapters 8 and 9. More Advanced Fersht, A. (1985) Enzyme Structure and Mechanism 2nd Ed., Freeman, New York Chapters 1-3, 8, 10 and 15. Schachman, H. K. (1988) Can a simple model account for the allosteric transitions of aspartate transcarbamoylase? J. Biol. Chem. 263:18583-18586.(Medline) Special aspects James, H.A. and Turner, P.C. (1995) Ribozymes, Essays in Biochemistry, 29:175-192.(Medline) REFERENCES Search the textbook
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Chapter 13: References
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REFERENCES Arnold, H. and Pette, D. (1968) Binding of glycolytic enzymes to structure proteins of the muscle, Eur. J. Biochem. 6:163-171.(Medline) Arnold, H. and Pette, D. (1970) Binding of aldolase and triosephosphate dehydrogenase to F-actin and modification of catalytic properties of aldolase, Eur. J. Biochem. 15:360-366.(Medline) Arnold, H., Henning, R. and Pette, D. (1971) Quantitative comparisons of the binding of glycolytic enzymes to F-actin and the interaction of aldolase with G-actin, Eur. J. Biochem. 22:121-126.(Medline) Atkinson, D. E. (1965) Biological feedback control at the molecular level, Science 150:851857.(Medline) Ban, N., Nissen, P., Hansen, J., Moore, P.B. and Steitz, T.A.. (2000) The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution, Science 2890:905-920. (MedLine) Beekmans, S. and Kanarek, L. (1981) Demonstration of physical interactions between consecutive enzymes of the citric acid cyclic and of the aspartate-malate shuttle, Eur. J. Biochem. 117:527535.(Medline) Ben-Levy, R., Hooper, S., Wilson, R., Paterson, H.F. and Marshall, C.J. (1998) Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2, Curr. Biol. 8:10491057.(Medline) Bernstein, B.E., Michels, P.A.M. and Hol, W.G.J. (1997) Synergistic effects of substrate-induced conformational changes in phosphoglycerate kinase activation, Nature 385:275-278.(Medline) Clarke, F.M. and Masters, C.J. (1975) On the association of glycolytic enzymes with structural proteins in skeletal muscle, Biochim. Biophys. Acta 381: 37-46.(Medline) Clarke, F.M. and Masters, C.J. (1976) Interactions between muscle proteins and glycolytic enzymes, Inter. J. Biochem. 7:359-365.
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Chapter 13: References
Clarke, F.M. and Morton, C.J. (1976) Aldolase binding to actin-containing filaments, Biochem. J. 159: 797-798.(Medline) Clegg, J.S. and Jackson, S.A. (1990) Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells, Arch. Biochem. Biophys. 278:452-460.(Medline) Cohen, P. (1980) Protein phosphorylation and the coordinate control of intermediate metabolism, Mol. Aspects Cell. Regul.1:255-268. Collins, J.H., and Lester, L.J. (1977) Acyl group and electron pair relay system: a network of interacting lipoyl moieties in pyruvate and α-ketog1utarate dehydrogenase complexes from Echerichia coli, Proc. Natl. Acad. Sci. USA. 74:4223-4227.(Medline) Dölken, G., Leisner, E. and Pette, D. (1975) Immunofluorescent localization of glycogenolytic and glycolytic enzyme proteins and of malate isozymes in cross-striated skeletal muscle and heart of rabbit, Histochemistry 43:113-121.(Medline) D'Souza, S.F. and Srere, P.A. (1983) Binding of citrate synthase to mitochondrial inner membranes, J. Biol. Chem. 258:4706-4709.(Medline) Edman, K., Nollert, P., Royant, A., Belrhali, H., Pebay-Peyroula, E., Hajdu, J., Neutze, R. and Landau, E.M. (1999) High-resolution X-ray structure of an early intermediate in the bacteriorhodopsin photocycle, Nature 401:822-826.(Medline) Fantozzi, D.A., Taylor, S.S., Howard, P.W., Maurer, R.A., Feramisco, J.R. and Meinkoth, J.L. (1992) Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase, J. Biol. Chem. 267:16824-16828. (MedLine) Felgner, P. L., Messer, J. and Wilson, J. E. (1979) Purification of a hexokinase-binding protein from the outer mitochondrial membrane, J. Biol. Chem. 254:4946-4949.(Medline) Fischer, E.H., Heileyer, L.M.G. Jr. and Hasche, R.H. (1971) Phosphorylase and the control of glycogen degradation, Curr. Top. Cell. Reg. 4:211-251. Gavin, A.C. and Nebreda, A.R. (1999) A MAP kinase docking site is required for phosphorylation and activation of p90rsk/MAPKAP kinase-1, Curr. Biol. 9:281-284.(Medline) Gerhart, J.C. and Pardee, A.B. (1962) The enzymology of control by feedback inhibition, J. Biol. Chem.237: 891-896.
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Gerhart, J.C. and Schachman, H.K. (1965) Distinct subunits for the regulation and catalytic activity of aspartate transcarbamoylase, Biochemistry 4:1054-1062.(Medline) Gerhart, J.C. and Schachman, H.K. (1968) Allosteric interactions in aspartate transcarbamoylase. II. Evidence for different conformational states of the protein in the presence and absence of specific ligands, Biochemistry 7:538-552.(Medline) Goebl, M.G., Yochem, J., Jentsch, 5., McGrath, J. P., Vashavsky, A. and Byers, B. (1988) The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme, Science 241:1331-1335.(Medline) Greengard, P. (1978) Phosphorylated proteins as physiological effectors, Science 199:146-152.(Medline) Guerrier-Takada, C. and Altman, S. (1984) Catalytic activity of an RNA molecule prepared by transcription in vitro, Science 223:285-286.(Medline) Hackenbrock, C.R. (1968) Chemical and physical fixation of isolated mitochondria in low and high energy states, Proc. Natl. Acad. Sci. USA 61:598-605.(Medline) Hall. E.R., McCully, V., and Cottam, G.L. (1979) Evidence for proteolytic modification of pyruvate kinase in fasted rats, Arch. Biochem. Biophys. 195:315-324.(Medline) Halper, L.A. and Srere, P.A. (1977) Interaction between citrate synthase and malate dehydrogenase in the presence of polyethylene glycol, Arch. Biochem. Biophys. 184:529-534.(Medline) Han, J.W., Thieleczek, R., Varsanyi, M. and Heilmeyer, L.M. Jr. (1992) Compartmentalized ATP synthesis in skeletal muscle triads, Biochemistry 31:377-384.(Medline) Hasilik, A. and Tanner, W. (1978) Biosynthesis of the vacuolar yeast glycoprotein carboxypeptidase Y. Conversion of precursor into enzyme, Eur. J. Biochem. 85:599-608.(Medline) Holland, P.M. and Cooper, J.A. (1999) Docking sites for kinases, Curr Biol. 9:R329-331. (MedLine) Holmberg, C.I., Tran, S.E., Eriksson, J.E. and Sistonen, L.(2002) Multisite phosphorylation provides sophisticated regulation of transcription factors, Trends Biochem. Sci. 27:619-627. (MedLine) Hubbard, M.J. and Cohen, P. (1993) On target with a new mechanism for the regulation of protein phosphorylation, Trends Biochem. Sci. 18:172-177.(Medline) Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito. A., Nixon, A., Yoshida, M., Wang, X.F. and Yao, T.P. (2002) HDAC6 is a microtubule-associated deacetylase. Nature417:455-458. (MedLine) http://www.albany.edu/~abio304/ref/ref13.html (3 of 8) [3/5/2003 8:20:52 PM]
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Jacobs, D., Glossip, D., Xing, H., Muslin, A.J. and Kornfeld, K. (1999) Multiple docking sites on substrate proteins form a modular system that mediates recognition by ERK MAP kinase, Genes Dev. 13:163-175.(Medline) Jacobus, W.E., Moreadith, R.W., and Vandegaer, K.M. (1983) Control of heart oxidative phosphorylaton by creatine kinase in mitochondrial membranes, Ann. N. Y. Acad. Sci. 414:73-89.(Medline) Jentsch, S., Seufert, W., Soomer, T. and Reins, H.-A. (1990) Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells, Trends Biochem. Sci. 15:195-198.(Medline) Jones, M.E. (1980) Pyridine nucleotide biosynthesis in animals: genes, enzymes and regulation of UMP biosynthesis, Ann. Rev. Biochem. 49:253-279.(Medline) Karadsheh, N.S. and Uyeda, K. (1977) Changes in the allosteric properties of phosphofructokinase bound to erythrocyte membranes, J. Biol. Chem. 252:7418-7420.(Medline) Kemp, B.E. and Pearson, R.B. (1991) Intrasteric regulation of protein kinases and phosphatases, Biochim. Biophys. Acta. 1094:67-76.(Medline) Kempner, E.S. and Miller, J.H. (1968) The molecular biology of Euglena gracilis. IV. Cellular stratification by centrifuging, Exp. Cell Res. 51:141-149.(Medline) Khosla, C. and Harbury, P.B. (2001) Modular enzymes, Nature 409:247-252. Kispal, G., and Srere, P.A. (1991) Studies of yeast peroxisomal citrate synthase, Arch. Biochem. Biophys. 286:132-137.(Medline) Kobe, B. and Kemp, B.E. (1999) Active site-directed protein regulation, Nature 402:373-376.(Medline) Koshland, D.E., Jr. (1956) Molecular geometry in enzyme action, J. Cell. Comp. Physiol. 47 (Suppl. 1):217-234. Koshland, D.E., Jr. and Neet, K.E. (1968) The catalytic and regulatory properties of enzymes, Annu. Rev. Biochem. 37:349-410. Kouzarides T. (2000) Acetylation: a regulatory modification to rival phosphorylation? EMBO J. 19:11761179. (MedLine) Krebs, E.G. and Beavo, J.A. (1979) Phosphorylation-dephosphorylation of enzymes, Annu. Rev. http://www.albany.edu/~abio304/ref/ref13.html (4 of 8) [3/5/2003 8:20:52 PM]
Chapter 13: References
Biochem. 48:923-959.(Medline) Ludwig, M.L., Hartsuck, J.A., Steitz, T.A., Muirhead, H., Coppola, J.C., Reeke, G.N. and Lipscombe, W.N. (1967) The structure of carboxypeptidase A. IV. Preliminary results at 2.8 Å resolution and a substrate complex at 6 Å resolution, Proc. Natl. Acad. Sci. U.S.A. 57:511-514. Mauro, L.J. and Dixon, J.E. (1994) 'Zip codes' direct intracellular protein tyrosine phosphatases to correct cellular 'address', Trends in Biochem. Sci. 19:151-155.(Medline) Mercer, R.W. and Dunham, P.B. (1981) Membrane bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesiclcs from human red cell membranes, J. Gen. Physiol. 78:547-568. (MedLine) Meyer, F., Heilmeyer, L.M.G., Jr., Haschke, R.H. and Fischer, E.H. (1970) Control of phosphorylase activity in a muscle glycogen particle. I. Isolation and characterization, J. Biol. Chem. 245:66426648.(Medline) Neurath, H. and Walsh, K.A. (1976) Role of proteolytic enzymes in biological regulation, Proc. Natl. Acad. Sci. U.S.A. 73:3825-3832.(Medline) Nimmo, H.G., Proud, C.G. and Cohen, P. (1976) The phosphorylation of rabbit skeletal muscle glycogen synthase by glucose synthase kinase 2 and adenosine-3,5-monophosphate-dependent protein kinase, Eur. J. Biochem. 68:31-44.(Medline) Oliver, R.M. and Reed. L.J. (1982) Multienzyme complexes. In Electron Microscopy of Proteins, Vol. 2 (Harris, J.R., ed.) Academic Press, New York, pp.1-48. Olson, M.S. and Allgyer, T.T. (1973) The regulation of nicotinamide adenine dinucleotide-linked substrate oxidation in mitochondria, J. Biol. Chem. 248:1582-1597.(Medline) Paul, R.J., Hardin, C.D., Raeymaekers, L., Wuytack, F. and Casteels, R. (1989) Preferential support of Ca2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade, FASEB J. 3:2298-2301.(Medline) Perham, R.N. and Duckworth, H.W. (1981) Mobility of polypeptide chain in the pyruvate dehydrogenase complex revealed by proton NMR, Nature 292:474-477.(Medline) Persson, L.-O. and Srere, P.A. (1991) Purification of mitochondrial citrate transporter in yeast, Biochem. Biophys. Res. Comm. 183:70-76. (MedLine)
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Pette, D. and Brandau, H. (1962) Intracellular localization of glycolytic enzymes in cross-straited muscle of Locusta migratoria, Biochem. Biophys. Res. Comm. 9:367-370. Porpáczy, Z., Sümegi, B. and Alkonyi, I. (1983) Association between the α-ketoglutarate dehydrogenase complex and succinate thiokinase, Biochim. Biophys. Acta 749: 172-179.(Medline) Rashin, A.A. (1981) Location of domains in globular proteins, Nature 291:85-87. (MedLine) Reed, L. J., and Cox, D. J. (1966) Macromolecular organization of enzyme systems, Annu. Rev. Biochem. 35:57. Robinson, J.B. Jr. and Robinson, J.B., Jr. and Srere, P.A., (1985) Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria, J.Biol. Chem. 260:10800-10805.(Medline) Robinson, J.B. Jr., Inman, L., Sumegi, B. and Srere, P.A. (1987) Further characterization of the Krebs tricarboxylic acid cycle-metabolon, J. Biol. Chem. 262:1786-1790.(Medline) Smith, J.A., Poteet-Smith, C.E., Malarkey, K. and Sturgill, T.W. (1999) Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo, J. Biol. Chem. 274:2893-2898.(Medline) Srere, P.A. (1985) Organization of Krebs tricarboxylic acid cycle enzymes in mitochondria, J. Biol.Chem.260:10800-10805.(Medline) Schaaff, I., Heinisch, J. and Zimmermann, F.K. (1989) Overproduction of glycolytic enzymes in yeast, Yeast 5:285-290.(Medline) Schoellmann, G. and Shaw, E. (1963) Direct evidence for the presence of histidine in the active center of chymotrypsin, Biochemistry 2:252-255. Shapiro, B.M., and Stadtman, E.R. (1967) Regulation of glutamine synthetase. IX. Reactivity of the sulfhydryl groups of the enzyme from Escherichia coli, J. Biol. Chem. 242:5069-5079.(Medline) Sigel, P. and Pette, D. (1969) Intracellular localization of the glycogenolytic and glycolytic enzymes in white and red rabbit skeletal muscle. A gel film method for coupled enzyme reactions in histochemistry, J. Histochem. Cytochem.17:225-237.(Medline) Spence, J., Gali, R.R., Dittmar, G., Sherman, F., Karin, M. and Finley, D. (2000) Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain, Cell 102:67-76. (MedLine)
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Srere, P.A. (1993) Wanderings (wonderings) in metabolism, Hoppe-Seyler 374:833-842.(Medline) Stepp, L.R., Bleile, D.M., McRorie, D.K., Pettit, F.H. and Reed, L.J. (1981) Use of trypsin and lipoamidase to study the role of lipoic acid moieties in the pyruvate and α-ketoglutarate dehydrogenase complexes, Biochemistry 20:4555-4560.(Medline) Stewart, M., Morton, D.J. and Clarke, F.M. (1980) Interaction of aldolase with actin-containing filaments. Structural studies, Biochem. J. 186:99-104.(Medline) Strålfors, P., Hiraga, A. and Cohen, P. (1985) The protein phosphatases involved in cellular regulation. Purification and characterization of glycogen-bound form of protein phosphatase-1, Eur. J. Biochem. 149:295-303.(Medline) Sumegi, B. and Alkonyi, I. (1983) A study of the physical interaction between the pyruvate dehydrogenase complex and citrate synthase, Biochem. Biophys. Acta 749:163-171.(Medline) Sumegi, B. and Srere, P.A. (1984) Complex I binds several mitochondrial NAD-coupled dehydrogenases, J. Biol. Chem. 259:15040-15045.(Medline) Sumegi, B., Propaczy, Z. and Alkonyi, I. (1991) Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and complexes of the respiratory chain, Biochim. Biophys. Acta 1081: 122128. Tang, P.M., Bondor, J.A., Swiderek, K.M. and DePaoli-Roach, A.A.(1991) Molecular cloning and expression of the regulatory (RG1) subunit of the glycogen-associated protein phosphatase, J. Biol. Chem. 266: 15782-15789.(Medline) Tsai, I.-H., Murthy, S.N.P. and Steck, T.L. (1982) Effect of red cell membrane binding on the catalytic activity of glyceraldehyde-3-phosphate dehydrogenase, J. Biol. Chem. 257: 1438-1442.(Medline) Tyiska, R.L., Williams, J.S., Brent, L.G., Hudson, A.P., Clark, B.J., Robinson, J.B. Jr. and Srere, P.A. (1986) in Organization of Cell Metabolism (Welch, G.R. and Clegg, J.S., eds), NATO Series A: Life Sciences 127:177-189. Vertessy, B.G., Orosz, F., Kovacs, J. and Ovádi, J. (1997) Alternative binding of two sequential glycolytic enzymes to microtubules. Molecular studies in the phosphofructokinase/aldolase/microtubule system, J. Biol. Chem. 272:25542-25546.(Medline) Volker, K.W. and Knull, Hr. (1997) A glycolytic enzyme binding domain on tubulin, Arch. Biochem. Biophys. 338:237-243.(Medline)
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Vousden, K.H. (2002) Activation of the p53 tumor suppressor protein, Biochim. Biophys. Acta 1602:4759. (MedLine) Wanson, J.-C. and Drochmans, P. (1968) Rabbit skeletal muscle glycogen, A morphological and biochemical study of glycogen -particles isolated by the precipitation-centrifugation method, J. Cell Biol. 38:130-150.(Medline) Waskiewicz, A.J., Flynn, A., Proud, C.G. and Cooper, J.A. (1997) Mitogen-activated protein kinases activate the serine/threonine kinases Mnk1 and Mnk2, EMBO J.16:1909-1920.(Medline) Welch, G.R. (1977) On the role of organized multienzyme systems in cellular metabolism: a general synthesis, Prog. Biophys. Mol. Biol. 32:103-191. Wilkinson, A.J., Fersht, A.R., Blow, D.M.. Carter, P. and Winter, G. (1984) A large increase in enzyme substrate affinity by protein engineering, Nature 307:187-188.(Medline) Wojtas, K., Slepecky, N., von Kalm, L. and Sullivan, D. (1997) Flight muscle function in Drosophila requires colocalization of glycolytic enzymes, Mol. Biol. Cell 8:1665-1675.(Medline) Yu, J. and Steck, T.L. (1975) Association of band 3, the predominant polypetide of the human erythrocyte membrane, J. Biol. Chem. 250: 9176-9184. Zaug, A.J. and Cech, T.R. (1986) The intervening sequence RNA of Tetrahymena is an enzyme, Science 231:470-475.(Medline) Zhao, Y., Bjorbaek, C. and Moller, D.E. (1996) Regulation and interaction of pp90(rsk) isoforms with mitogen-activated protein kinases, J. Biol. Chem. 271:29773-29779.(Medline) Zalokar, M. (1960) Cytochemistry of centrifuged hyphae from Neurospora, Exp. Cell Res, 19:114-132.
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14. Regulation of Metabolism I. Problems in Applying In Vitro Observations to Intact Cells A. Organization of Enzymes B. Mitochondrial Matrix Enzymes C. Binding of Glycolytic Enzymes to Actin D. Glycogen Particles E. Binding of Enzymes to the Plasma Membrane II. Regulatory Elements A. Cascades Role of AMP-activated kinase Hormonal control: the case of epinephrine B. Role of Exergonic Steps C. Separate Enzymes for Forward and Backward Reactions D. Effectors Sensitive to Small Changes E. AMP as a Metabolic Effector F. F2,6P G. G1,6P III. Control of Glycolysis A. ∆G and Regulation B. Substrate Cycling IV. Control of the Tricarboxylic Acid Cycle A. Substrate Availability B. Inhibition by Products and Intermediates V. Control of Oxidative Phosphorylation and Glycolysis in Tandem A. Regeneration of NAD + B. Energy Balance C. Control of Oxidative Phosphorylation in Mitochondria Near equilibrium and translocase hypotheses Regulation of mitochondrial permeability The uncoupling proteins VI. Control of Carbohydrate Metabolism in Plants VII. Regulation via the Redox State of the Cell A. Control of metabolism in plants http://www.albany.edu/~abio304/text/chapter_14.html (1 of 34) [5/5/2002 6:51:21 PM]
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B. Role in animal cells Suggested Reading Web Resources References Back to List of Chapters Chapter 13 discussed how enzyme activity is regulated allosterically by positive and negative effectors. An additional mechanism is provided by the cycling of the enzyme molecules between active and inactive states, frequently involving phosphorylation and dephosphorylation of the enzyme. The present chapter will concentrate on the control of energy yielding reactions of cell metabolism on moment-to-moment adaptive changes. I. PROBLEMS IN APPLYING IN VITRO OBSERVATIONS TO INTACT CELL The metabolism of cells and its regulation are so complex that it is difficult to arrive at a quantitative evaluation of the effect of control mechanisms on the overall economy of the cell. Much is known about the properties of the purified soluble enzymes involved in the cell's metabolic pathways. Although some inferences can be drawn from this information, the control is complex and involves factors that have not been studied in detail. The enzymes of the glycolytic pathway have been studied in detail and their regulation might be thought to involve few surprises. However, some experiments suggest that our understanding is very limited. One experiment illustrates this point effectively. In yeast, the concentration of glycolytic enzymes can be increased by introducing the appropriate genes in multicopy vectors (Schaaff et al., 1989). This process increases the activity of individual enzymes approximately 4 to 14-fold compared to the wild-type enzymes. However, the rate of ethanol production by the yeast remains unchanged, even with increases in enzymes catalyzing irreversible steps that are thought to have a key regulative role (see below). This observation may be explained by the presence of enzymes in "assemblies" where the enzymes are held in precise stoichiometry. The free excess enzyme is simply left out of the pathway. Many biochemical pathways interact in ways which are unpredictable with our present knowledge. Most are branched at least to some extent. The kinetics of even the simplest hypothetical pathway have unsuspected complexities. For example, a linear pathway with no branching has kinetics that depend on the constants of each step, except for the reactions subsequent to an irreversible step. Similarly, when the concentration of one substrate in a sequence is very large (for example, provided by a side feeder reaction) the rate of the pathway depends not only on the rate of that one step but also on the rates of all the proceeding steps (Hearon, 1948, 1949). Furthermore, the information obtained in vitro is not likely to be very realistic. The organization of the cytoplasm, both structurally and functionally, is very complex. Isolated enzymes in solution are not likely to duplicate the in situ conditions. http://www.albany.edu/~abio304/text/chapter_14.html (2 of 34) [5/5/2002 6:51:21 PM]
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A. Organization of Enzymes Enzymes are not free in solution; they are frequently attached to structures or are present in multienzyme complexes. In intact cells, the properties of enzymes are much different from those of isolated, purified forms. When attached to structures they acquire very different properties. When bound to actin, the maximal enzymatic rate (Vm) of aldolase doubles and the Km for fructose 1,6-bisphosphate (Arnold and Pette, 1970) increases. Upon binding to structures, phosphofructokinase loses its allosteric inhibition by ATP (Karadsheh and Uyeda, 1977) and glyceraldehyde-3-phosphate dehydrogenase is inhibited (Tsai et al., 1982). Organization in structures and specific sites have other unexpected results. The effective local concentration of substrates or regulatory molecules may attain unsuspected levels. The Ca2+ concentration at axon terminals may reach concentrations as high as 0.1 mM, whereas it is well below the µM level in most of the cell (see Chapter 22). At the synapse, Ca2+ triggers neurotransmitter release. In most cells, Ca2+ initiates many other functions as a second messenger (see Chapter 7 and 24). The organization of enzymes in assemblies may well be the rule rather than the exception. Their behavior is distinct from that of their individual components and not readily predictable from conventional models. As our knowledge gains in detail, the number of known enzyme assemblies increases. Spliceosomes carry out the splicing of mRNA; transcriptional complexes carry out transcription; and translational complexes which contain a variety of enzymes, mRNA and ribosomes, are involved in protein synthesis. Similarly, proteasomes containing several enzymes, are responsible for the degradation of proteins. Evidence abounds for multienzyme complexes (referred to as metabolons) playing a role in metabolic pathways. These pathways include glycolysis, nucleotide synthesis (e.g., Jones, 1980), urea biosynthesis, tricarboxylic acid (TCA) cycle, fatty acid oxidation (e.g., Gillevet and Dakshinamurti, 1982) and amino acid metabolism. Some of the evidence for the presence of metabolic enzymes associated in complexes comes from indirect information. For example, the centrifugation of intact cells has been shown to sediment most cytosolic proteins (Zolokar, 1960; Kempner and Miller, 1968). This observation suggests that a large portion of the proteins of the cell must be present as aggregates. The very high concentration of proteins suggests that they are not in solution and may well be present in a semicrystalline state. In the mitochondrial matrix, the protein concentration has been calculated to be 56% by weight (Hackenbrock, 1968), a proportion which corresponds to the densest possible packing of proteins, assuming them to be identical spheres. Similar arguments can be made for the glycolytic enzymes in muscle, where most of the space not occupied by the contractile apparatus is likely to be occupied by glycolytic enzymes (Pette and Brandau, 1962; Sigel and Pette, 1969). Pette and Brandau estimate that in the muscle of Locusta migratoria the concentration of these enzymes by themselves corresponds to 0.2 g per ml. Other evidence is more direct. The discussion
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below will address the question for metabolic enzymes. B. Mitochondrial Matrix Enzymes Each enzyme of the citric acid cycle is able to bind the next enzyme in the sequence, indicating that they are likely to be present in a complex. Citrate synthase associates specifically with mitochondrial malate dehydrogenase, but not cytoplasmic malate dehydrogenase (Halper and Srere, 1977). Similarly, citrate synthase binds to mitochondrial, but not cytoplasmic aconitase (Tyiska et al., 1986), the pyruvate dehydrogenase complex (Sumegi and Alkonyi, 1983), and the citrate transporter (Persson and Srere, 1991). Similar results are obtained with pure enzymes. Enzymes which are sequential in the cycle bind to each other: mitochondrial aconitase binds to NADH-isocitrate dehydrogenase (Tyiska et al., 1986); fumarase binds to mitochondrial malate dehydrogenase (but not the cytoplasmic enzyme) (Beekmans and Kanarek, 1981); and the αketoglutarate dehydrogenase complex binds succinyl-CoA synthase (succinate thiokinase) (Porpaczy et al., 1983). An additional level of organization is revealed by the observation that the tricarboxylic acid cycle enzymes bind to inside-out inner mitochondrial membrane vesicles (D'Souza and Srere, 1983). The binding is specific; isoenzymes that catalyze the same reactions in other organelles (such as citrate synthase from peroxisomes) do not bind (Kispal and Srere, 1991). In addition, mitochondrial malic dehydrogenase binds to liposomes containing the mitochondrial Complex I (Sumegi and Srere, 1984), with which it must interact for the oxidation of the NADH generated by this enzyme. Binding is also demonstrated by the observation that mitochondria disrupted by ultrasound do not lose the citric acid cycle enzymes, even though citrate synthase has become accessible to an anti-synthase antibody and malic dehydrogenase to high molecular weight blue dextran (Robinson and Srere, 1985). The properties of these bound enzymes suggest that the organization in the mitochondrial matrix has an important role in determining the metabolic rate. The bound enzymes have a much higher rate than those in soluble form (Robinson et al., 1987; Sumegi et al., 1991). C. Binding of Glycolytic Enzymes to Actin In muscle, F-actin and most glycolytic enzymes are located within the I-band (Dolken et al., 1975; Sigel and Pette, 1969). The exception is hexokinase, which is localized primarily in mitochondria. A number of glycolytic enzymes have been shown to bind to F-actin (Arnold and Pette, 1968, 1970; Arnold et al., 1971). The binding is most pronounced in the presence of the regulatory proteins tropomyosin and troponin (Clarke and Morton, 1976; Clarke and Masters, 1975; Stewart et al., 1980). Binding to actin has been observed for fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase. The implications of these findings are not easyly assessed.
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D. Glycogen Particles Glycogen particles isolated from skeletal muscle contain the enzymes of glycogen metabolism. These include: phosphorylase b and its kinase and phosphatase, glycogen synthase and its respective kinase and phosphatase (Meyer et al., 1970), as well as its debranching enzyme, glycogen synthase kinase 2, protein phosphatase 2, and inhibitors 1 and 2 (Nimmo and Cohen, 1976). The glycogen particles themselves are closely associated with the sarcoplasmic reticulum near the thin filaments (Wanson and Drochmans, 1968). The kinetics of the enzymes in the particles are distinct from that in solution (Fischer, et al. 1971). E. Binding of Enzymes to the Plasma Membrane Certain enzymes are located in the cell's periphery, and some of them have been shown to bind to the cytoplasmic domain of integral membrane proteins. In the red blood cell, glyceraldehyde-3phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase bind to the Band 3 protein, the anion transporter (see Yu and Steck, 1975; Tsai et al., 1982). In the case of glyceraldehyde-3phosphate dehydrogenase, the binding exhibits 1 to 1 stoichiometry. The functional significance of these associations is not clear. II. REGULATORY ELEMENTS The regulation of enzyme specific activity (in contrast to regulating the number of enzyme molecules present) can take place by different mechanisms. Regulation may occur by the phosphorylation-dephosphorylation of enzymes by specific kinases (Chapter 13). The kinases respond to the level of a metabolite or, when responding to hormonal signals, to second messengers. This kind of regulation generally affects key entry reactions. In this chapter we will discuss the regulation of glycogen phosphorylase, which is activated by phosphorylation, and that of pyruvate dehydrogenase, which is inhibited by phosphorylation. These two enzymes control the entry into the glycolytic pathway (when glycogen is the substrate) and the TCA cycle, respectively. The control of enzyme activity can also be through allosteric regulation. Sometimes the allosteric regulator is a component of the pathway. At other times the regulator is synthesized in a side reaction of the pathway. The function of this side reaction is probably solely to provide regulation. The presence of regulatory cycles (originally called futile cycles) is thought to offer a very effective regulatory mechanism, for example in glycolysis. Substrate availability and feedback inhibition are also significant. Some of these regulatory mechanisms require more thorough examination in light of the information considered in Section I. http://www.albany.edu/~abio304/text/chapter_14.html (5 of 34) [5/5/2002 6:51:21 PM]
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The general features of metabolic regulation will be discussed first (A to D) followed by a more specific discussion of the regulation of metabolic reactions (Sections III-VI). A.Cascades The activity of enzymes or enzymatic pathways are controlled by complex mechanisms. A signal can be amplified by a series of biochemical reactions (a cascade mechanism). This kind of effect was examined in Chapter 7 in relation to intracellular signals. The initial signal may be a hormone or a regulative metabolite. The activity of an enzyme may be through phosphorylationdephosphorylation reactions as also discussed in Chapter 7. We will examine two cascades that reflect these two distinct triggers: the AMP-activated protein kinase and the mobilization of glucose by the hormone epinephrine. AMP-activated protein kinase. AMP-activated protein kinase (AMPK) occupies an important position in the regulation of metabolism because AMP is a sensitive sensor of metabolic states as discussed in Section IIE. The precise mechanisms are not always clear at this time but the basic role of AMPK is well recognized. AMPK inhibits anabolic pathways to limit the use of ATP (Hardie and Carling, 1997) because it inhibits enzymes involved in glycogen, fatty acid and cholesterol synthesis. In contrast, AMPK accelerates reactions generating ATP, such as those of fatty acid oxidation in striated muscle and heart. In the heart, 60-70% of metabolism originates from fatty acid oxidation. AMPK, a heterotrimer, is activated by high AMP and low ATP in a cascade which involves allosteric regulation, facilitation of phosphorylation by an upstream protein kinase (AMPK kinase), and inhibition of dephosphorylation. The activity of the AMPK kinase and two phosphatases depends on the concentration of AMP (Davies et al., 1995). This protein-kinase cascade represents a sensitive system, which responds to the depletion of ATP. AMPK is equivalent to the yeast SNF1 protein-kinase complex. SNF1 is activated by glucose starvation which in yeast leads to ATP depletion. Apparently, it is involved in derepression of glucose-repressed genes. AMPK phosphorylates and activates hydroxymethylglutaryl-CoA reductase and acetyl-CoA carboxylase, key regulatory enzymes of sterol synthesis and fatty acid synthesis, respectively. It probably phosphorylates many other target enzymes as well. Inhibition of acetyl-CoA carboxylase by AMPK reduces malonyl-CoA. In this way, it relieves the allosteric inhibition of mitochondrial carnitine palmitoyl transferase (CTP1) (e.g., see Kudo et al., 1995). CTP1 is the rate-limiting enzyme involved in mitochondrial uptake of fatty acids transport into mitochondria (McGarry et al., 1989). In mammals, AMPK also induces an increase in glucose transport following exercise by a mechanism independent of the action of insulin (Hayashi et al., 1998). In muscle, phosphocreatine has a role in maintaining the ATP concentration constant. AMPK phosphorylates and inhibits creatine kinase (Ponticos et al., 1998) thereby blocking the exchange of high energy phosphate between creatine and ATP, possibly shutting off a high energy phosphate sink. AMPK http://www.albany.edu/~abio304/text/chapter_14.html (6 of 34) [5/5/2002 6:51:21 PM]
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is itself regulated by a mechanism involving phosphocreatine, creatine and pH. The creatine kinasecreatine phosphate system is clearly of great significance in the energy balance of the cell (see Wallimann et al., 1992). However, the significance of the AMPK regulation is not obvious particularly since the phosphocreatine system is compartmentalized and the physiological effect will depend on the location of the enzyme. The hormone leptin, secreted by adipocytes, has been found to increase the oxidation of fatty acids, the uptake of glucose and to prevent the accumulation of lipids in nonadipose tissues. The effect of leptin on fatty acid oxidation rests on its activation of AMPK in muscle which inhibits the synthesis of malonyl-CoA , thereby favoring fatty acid oxidation (Minokoshi et al., 2002). Leptin is a hormone secreted by adipocytes. It has a role in food intake and in controlling energy expenditure and neuroendocrine function (see Friedman and Halaas, 1998). Hormonal control: the case of epinephrine. Glycogen phosphorylase of skeletal muscle is controlled by the cascade shown in Fig. 1 (Fisher et al., 1970). The less active form of phosphorylase, phosphorylase b, is converted to the active phosphorylated species, phosphorylase a (Fig. 1). In turn, phosphorylase a can be inactivated by the appropriate phosphatase. Phosphorylase a releases glucose-1-phosphate from glycogen. Glucose-1-phosphate can then be metabolized by the muscle cell's glycolytic machinery. The phosphorylase cascade is very significant in the mobilization of the carbohydrate stores of muscle. The conversion of phosphorylase b to phosphorylase a is indirectly regulated by the hormone epinephrine (also called adrenaline), secreted by the adrenal medulla and other components of the sympathetic nervous system. In muscle, epinephrine activates adenyl cyclase (see Fig. 1), which catalyzes the conversion of ATP to cyclic AMP (cAMP). cAMP, in turn, activates the kinase that phosphorylates phosphorylase kinase (i.e., a kinase kinase). In the presence of Ca2+, the phosphorylated active form of phosphorylase kinase, along with ATP, phosphorylate the phosphorylase b, thereby producing phosphorylase a. The binding of a small amount of epinephrine has triggered the production of many molecules of glucose-1-phosphate and thereby stimulated the major metabolic pathway of muscle through a cascade of reactions. The activity of phosphorylase is also under the control of several effectors, such as ATP, glucose-6phosphate, glucose, and uridine diphosphoglucose.
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Fig. 1 Control of glycogen phosphorylase (Fisher et al., 1970). Reproduced by permission from Essays in Biochemistry 6:23-68, copyright ©1970 The Biochemical Society, London.
The mechanism involving muscle phosphorylase increases the glucose available for glycolysis in muscle. In liver, phosphorylase is also involved in the regulation of blood glucose and hence in the metabolism of the organism as a whole. The control of the release of glucose by epinephrine in muscle is but one of the reactions involved in mobilizing glucose in mammals (Exton et al., 1971). cAMP inhibits phosphorylase phosphatase, thereby prolonging the presence of phosphorylase in its active form. cAMP also mediates the transformation of glycogen synthetase to a less active form, thereby blocking the storage of glucose favoring its release. Epinephrine is not the only hormone involved in the release of cAMP. Glucagon also tends to increase the concentration of cAMP, while insulin decreases the concentration. The interactions between the effects of the three hormones are responsible for the final effect on glucose metabolism. Both glucagon and insulin are pancreatic endocrine hormones. http://www.albany.edu/~abio304/text/chapter_14.html (8 of 34) [5/5/2002 6:51:21 PM]
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A number of key enzymes controlled by a variety of signals are also regulated through a phosphorylation-dephosphorylation mechanism; an example is pyruvate dehydrogenase. However, in contrast to the phosphorylase, the dephosphorylated pyruvate dehydrogenase is the active form. B. Role of Exergonic Steps Most of the component reactions in a pathway proceed rapidly and are close to equilibrium (i.e., ∆G = 0 ). Therefore, they are not subject to regulation. The metabolic pathways themselves are highly exergonic. They are virtually irreversible because at least one step highly exergonic. This step has been called the first committed step and is frequently at the beginning of a metabolic pathway. Since it is far from equilibrium, it can be regulated. The regulation, through the control of a rate-limiting exergonic step, is analogous to the way in which a dam controls the flow of a whole river by adjusting the flow of the water passing through the dam itself. Besides the requirement that the step be exergonic, other conditions must also be met. The enzyme must be capable of responding to the appropriate regulator. Furthermore, the substrate concentration should be in a range allowing regulation; the rate of enzyme activity cannot be increased when the enzyme is saturated. In the regulated steps, generally forward and backward reactions are catalyzed by separate enzymes, as discussed in the next section. C. Separate Enzymes for Forward and Backward Reactions In key metabolic steps, frequently one enzyme catalyzes the forward reaction and another the backward reaction. This feature allows irreversible steps in either direction and the independent regulation of the forward and backward pathways. In addition, the continuous formation and degradation of substrate (substrate cycling) has an important regulatory role. Its purpose was not originally understood and the process is undoubtedly wasteful in terms of energy. For these reasons, the cycling was originally called futile cycling. Whereas allosteric regulation allows variation of the rate of a step over a ten-fold range, substrate cycling permits regulation over a hundred-fold range. Let us examine this process in more detail (Newsholme et al., 1984). The flow of metabolites through a pathway, that is, the flux (Jnet) in moles per unit time through a rate-limiting step, can be represented as the difference between the forward (Jf) and the backward flux (Jb). The two fluxes are catalyzed different enzymes. This can be represented as: Jnet = Jf - Jb (1) Selecting reasonable values in arbitrary units [based on the regulation of the phosphorylation of
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fructose-6-phosphate (F6P) to form fructose-1,6-bisphosphate (F1,6P)], Jf may be 10 and Jb, 9. Jnet will be 1. If Jf is increased tenfold and Jb decreased tenfold, Jnet will be 99, i.e., approximately a one hundredfold increase. In the case of this metabolic step, the forward reaction is catalyzed by phosphofructokinase (PFK), whereas the reverse reaction is catalyzed by fructose-1,6bisphosphate phosphatase (FBPase). The two enzymes are regulated by AMP, which stimulates the kinase and inhibits the phosphatase. The stimulation of PFK by AMP actually results from blocking an inhibition by ATP (Mansour and Ahlfors, 1968). This metabolic step is also sensitive to a variety of other metabolites, illustrating how complex a regulative pattern can be. One of the allosteric activators of PFK is cAMP, a second messenger under hormonal control. The fact that, in the case discussed, the regulating metabolite is primarily AMP leads us to another basic principle of regulation. The effector must be a chemical which changes in concentration significantly with physiological conditions. This question will be discussed in the next section. D. Effectors Sensitive to Small Changes Effectors are produced in two distinct ways. The pathway itself may provide a metabolite that can serve as a sensitive indicator of metabolic conditions. Basically this mechanism corresponds to feedback inhibition, discussed in Chapter 13. Alternatively, the enzymatic machinery of the cell can produce an effector from side reactions, sometimes under the control of a hormone. The first reaction of the glycolysis illustrates feedback control. The phosphorylation of glucose to form glucose-6-phosphate (G-6-P), is catalyzed by hexokinase (Hk). Hk is inhibited allosterically by G-6-P (McDonald et al., 1979). In some cells, this effect is very important, and the reaction, the gateway to glycolysis, regulates the whole pathway. A side reaction may also provide a sensitive indicator of metabolic state. The concentration of AMP, which we discussed above, serves as an indicator of the energy balance. Fructose-2,6bisphosphate (F2,6P), one of the most powerful allosteric regulators now known, is also generated in a step that is not in the main metabolic pathway. The regulative role of F2,6P differs with the function of the tissue. Glucose-1,6-phosphate (G1,6P), which has an important role as a cofactor, is also a significant effector in glycolysis. The regulation by AMP, F2,6P, and G1,6P are discussed in more detail below. E. AMP as a Metabolic Effector In muscle, as we saw for PFK, it is AMP, not ATP, that is the major controlling effector. Why not ATP, which is directly involved in supplying the power to cells? Many enzymes (including PFK) are, in fact, regulated by ATP. However, in this case, and particularly in heart muscle, the effect of AMP is preeminent. The answer lies in the fact that the ATP and ADP concentrations remain http://www.albany.edu/~abio304/text/chapter_14.html (10 of 34) [5/5/2002 6:51:21 PM]
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relatively constant (Helmreich and Cori, 1965). In vertebrate muscle and nerve, ATP is rapidly regenerated by two reactions: (a) the creatine kinase reaction where ATP is formed from ADP and phosphocreatine as shown in Eq. (2) and (b) the adenylate kinase reaction that generates ATP and AMP from 2 ADPs, as shown in Eq. (3). creatine-P + ADP→ATP + creatine (2) 2 ADP→ATP + AMP (3) The adenylate kinase reaction rate is rapid and can be considered at equilibrium. In turn, AMP concentration plays a very important physiological role. The equilibrium constant Keq = (ATP) (AMP) / (ADP)2 ~1. The concentrations of ADP and ATP are rather large compared to AMP. A small increment in (ATP) will produce a very significant decrease in AMP. Actual calculations assuming a decrease in ATP of 10% will produce a large decrease in AMP (approximately 4-fold), so that AMP can serve as a sensitive indicator of the energy stores of the cell. F. F2,6P Fructose 2,6-bisphosphate (F2,6P) is an important regulator of glycolysis in several mammalian tissues (Hers et al., 1982). In liver, it acts to block glycolysis, thereby favoring the release of glucose produced by the concurrent degradation of glycogen. The liver is the key organ responsible for regulating the concentration of blood glucose. The regulation of the glucose level via F2,6P is part of this mission. In heart muscle, which degrades glycogen for its own glycolysis, F2,6P instead, stimulates glycolysis. The level of F2,6P depends on the balance between the enzyme activity responsible for the phosphorylation of F6P (phosphofructokinase 2, PFK-2) and the enzyme activity responsible for its hydrolysis (fructose-2,6-bisphosphatase, FBPase-2). The same protein is responsible for the two activities. In liver, the cAMP-dependent phosphorylation of the enzyme by a protein kinase inhibits PFK-2 and activates the phosphatase. In heart, the opposite is true: phosphorylation activates PFK-2 and blocks FBPase-2. G. G1,6P Glucose 1,6-bisphosphate (G1,6P) is a cofactor of the phosphoglucomutase reaction which converts glucose-1-phosphate (G1P) to glucose-6-phosphate (G6P). It is generated by the phosphorylation of G1P and it is degraded by a specific phosphatase. G1,6P is an activator of PFK, pyruvate kinase (PK), and is an inhibitor of HK and FBPase. G1,6P also releases phosphoglucomutase from ATP and citrate inhibition. Because its level is subject to a variety of physiological controls, including the presence of several second messengers (summarized in Table http://www.albany.edu/~abio304/text/chapter_14.html (11 of 34) [5/5/2002 6:51:21 PM]
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1), it must act as a significant physiological signal (Beitner, 1984). III. CONTROL OF GLYCOLYSIS The regulation of glycolysis is fairly complex, involving most of the mechanisms discussed. Many of these regulatory effects are summarized in Fig. 2, part A. A. ∆G and Regulation Insights into the regulation of a metabolic pathway require information that is not always available. As discussed above, regulated steps must be exergonic, i.e., far from equilibrium. Therefore, ∆G of the various reactions can predict in what steps the regulation may occur inside cells. However, ∆G alone merely indicates feasibility of regulation. As already mentioned, the enzyme catalyzing the step must be capable of responding to the appropriate effectors. As we saw in Chapter 12, calculation of ∆G requires knowing not only the ∆Go of the reactions (and this can be calculated, see Chapter 12), but also the actual substrate concentrations. Fortunately, data is available for some tissues, for example, heart muscle cells (Newsholme and Start, 1973). The calculated ∆Gs show that regulation is likely to occur only at three glycolytic steps: the HK (∆G = 6.5 kcal), PFK (∆G = - 6.2 kcal), and PK (∆G = -3.3 kcal) reactions. The major negative effectors of these enzymes are G6P for HK and ATP for PK. ATP and citrate are inhibitors of PFK. Only PFK has positive effectors, among which is AMP. As we saw in heart muscle, AMP is very sensitive to the energy balance of the cell. Furthermore, the regulatory effect is enhanced by the mechanism of substrate cycling, permitting as much as a one hundred-fold activation. For these reasons, in most mammalian tissues, PFK is thought to be the most important glycolytic control site (e.g., see Bosca and Corredor, 1984). These regulatory steps are indicated in Fig. 2, part A.
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Fig. 2 Representation of the control of metabolism. The oxidation-phosphorylation steps of mitochondria were left out to simplify the diagram.
B. Substrate Cycling Substrate cycling occurs not only in the F6P to F1,6P step (e.g., Clark, D.G. et al., 1973; Clark, M.G. et al., 1973; Rognstad and Katz, 1980; Challis et al., 1984), but also in glucose to G6P (Surholt and Newshome, 1983) and the PK step (Freidmann et al., 1971), suggesting that in at least some of the systems studied, these steps can be regulated through this mechanism. The substrate cycling steps are indicated in Fig. 2 on the left of part A of the diagram. Table 1 Factors and Conditions That Control Cellular G1,6P Levels.
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Effectors
Tissues
Changes in G1,6P levels
Cyclic AMP
Diaphragm Cultured muscle
↑↑
Cyclic GMP
Diaphragm Cultured muscle
↓↓
Ca2+
Diaphragm Cultured muscle
↓↓
Epinephrine
Diaphragm Cultured muscle
↑↑
Epinephrine + propranolol
Cultured muscle
↓
Hormones:
Vasopressin
Cultured muscle
↓
Serotonin
Skeletel muscle Skin
↓↓
Bradykinin
Skeletal muscle Skin
↓↓
Phospholipase A2
Diaphragm
↓
Lysolecithin
Diaprhagm
↓
Muscular dystrophy
Skeletal muscle
↓
Fasting
Skeletal muscle Normal Dystrophic
↓↓
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Refeeding
Skeletal muscle Normal Dystrophic
↑↑
Anoxia
Diaphragm
↓
Ischemia
Brain
↓
Aerobiosis
Diaphragm
↑
Differentiation (fusion)
Cultured muscle
↑
Growth
Skeletal muscle Heart Brain Skin
↑↔↓↓
Old age
Skeletal muscle
↓
Glucose
Pancreatic islets
↑
Pharmacological agents Local anesthetics Lithium
Trifluoroperazine
Diaphragm Skeletal muscle Normal; Dystrophic Diaphragm Brain Liver Skeletal muscle
↓ ↓↓↓↓↔↑
↑ = increase ; ↓=decrease; ↔ = no change From Beitner (1984). Reproduced with permission from International Journal of Biochemistry 16. Beitner, R., Control levels of glucose 1,6-bisphosphate, copyright ©1984, Pergamon Journals, Ltd.
IV. CONTROL OF THE TRICARBOXYLIC ACID CYCLE The tricarboxylic acid (TCA) cycle acts in concert with NADH oxidation and ATP synthesis and http://www.albany.edu/~abio304/text/chapter_14.html (16 of 34) [5/5/2002 6:51:21 PM]
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is, therefore, inexorably linked to these processes. Glycolysis and other sources of acetyl-CoA provide one of the needed substrates. Because it provides NADH to be oxidized by the electron transport chain, the cycle depends on the rate of oxidation by the mitochondrial complexes. Therefore, for many reasons it would be more appropriate to examine the regulation of the tricarboxylic acid cycle in relation to the whole metabolic system, as done in the next section (V). However, some of the details presented in this section facilitate our understanding of the whole. Regulation of the TCA cycle primarily involves the regulation of the availability of the entry substrate and feedback inhibition (Hansford, 1980) (see Fig. 2, part B). Because the entry substrates, acetyl-CoA and oxaloacetate, are present at less than saturation level, their influx into the system can control the flow through the cycle. A major product of the cycle is NADH, and as might be expected, acts as a feedback inhibitor. A. Substrate Availability Pyruvate dehydrogenase (PDH), a multienzyme complex, is responsible for the production of one molecule of acetyl-CoA and one molecule of NADH from the oxidation of each pyruvate. This reaction controls the entry into the TCA cycle of substrate from the glycolytic pathway (see Randle et al., 1978). PDH is inactivated by phosphorylation catalyzed by a specific kinase. The kinase, in turn, is activated by NADH and acetyl-CoA (see Reed et al., 1985). NADH and acetylCoA, the products of the PDH reaction, also act directly on PDH as feedback inhibitors. Both mechanisms then inhibit the production of acetyl-CoA in response to excess products. PDH is also regulated by nucleotides (GTP inhibiting and AMP activating), and its inhibition by phosphorylation is increased by high ATP/ADP ratios. Acetyl-CoA and oxaloacetate are the substrates of citrate synthase, the enzyme which catalyzes the first reaction of the TCA cycle. Oxaloacetate is produced by malate dehydrogenase. The reaction is in equilibrium so that: Keq = (oxaloacetate) (NADH) / (NAD+) (malate) (4) As shown in Eq. (4), the oxaloacetate concentration varies inversely with that of NADH. Conversely, the increase in NAD+ (and consequent decrease in NADH) will increase the level of oxaloacetate. This reaction serves as a delicate sensor of the redox state of NAD+ and responds by adjusting the amount of oxaloacetate fed into the TCA cycle. The two substrates of the entry reaction to the TCA cycle are therefore under tight regulation by at least two separate mechanisms. The citrate synthase reaction itself is also inhbited by a high NADH/NAD+ ratio (see below). B. Inhibition by Products and Intermediates http://www.albany.edu/~abio304/text/chapter_14.html (17 of 34) [5/5/2002 6:51:21 PM]
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As already discussed, regulation is likely to occur at exergonic steps in the TCA cycle, such as the citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase steps. In fact, these appear to be control steps (see Fig. 2, part B). NADH produced by the cycle inhibits all three. In addition, citrate inhibits citrate synthase, while succinyl-CoA inhibits α-ketoglutarate dehydrogenase and citrate synthase. As might be expected, ADP and ATP have opposite roles. ADP stimulates isocitrate dehydrogenase, whereas ATP inhibits it. An interesting role is played by Ca2+, a second messenger (see Chapter 7), which stimulates pyruvate, isocitrate and α-ketoglutarate dehydrogenase, thereby tending to increase the metabolic rate. The mechanism controlled by Ca2+ can override all other factors. The regulation of metabolism by Ca2+ is also discussed in Chapter 7 (section IA). V. CONTROL OF OXIDATIVE PHOSPHORYLATION AND GLYCOLYSIS IN TANDEM The interactions of the various components of the metabolic reactions cannot be appreciated without examining them together. As we saw in the discussion of glycolysis and the TCA cycle, the availability of substrates and regulation by indicators of energy metabolism are important components. This section will discuss these factors in some detail. A. Regeneration of NAD+ One of the products of glycolysis is NADH, which is formed from NAD+ by the glyceraldehyde-3phosphate dehydrogenase reaction as shown schematically in Fig. 3. The total amount of NAD, reduced or oxidized, is finite, and the NAD+ has to be regenerated if glycolysis is to proceed. During oxidative metabolism, NADH can be oxidized by the electron transport chain (see Chapter 16 and discussion below). However, when the oxidative metabolism cannot keep up with the recycling of NADH, NAD+ is regenerated by an anaerobic reaction. In mammalian tissues, lactate is produced from pyruvate by lactate dehydrogenase, as shown in Fig. 3 and Eq. (5). In yeast, the decarboxylation of pyruvate by the pyruvate decarboxylase (Eq. 6) is followed by the reduction of acetaldehyde to produce ethanol (Eq. 7). CH3COCOO- + NADH + H+→ CH3CHOH + NAD+ (5) CH3COCOO-→ CH3CHO + CO2 (6) CH3CHO + NADH + H+→ CH3CH2OH (7)
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Fig. 3 Involvement of NADH in anaerobic glycolysis.
Fig. 4 Representation of shuttle carrying reducing equivalents from cytoplasmic NADH to intramitochondrial NAD+.
Mitochondrial NADH can be oxidized readily by the electron transport system (Chapter 16). However, glycolysis occurs in the cytoplasm. How does the NADH reach the NADH-coenzyme Q reductase (complex I) of mitochondria? This multienzyme complex reacts with the NADH only on the matrix side of the mitochondrial inner membrane. Apparently, the oxidation of the cytoplasmic NADH is mediated by shuttles. Two possible shuttles are likely to take place. Dihydroxyacetone phosphate (DHAP), one of the intermediates of glycolysis, can be reduced to form http://www.albany.edu/~abio304/text/chapter_14.html (19 of 34) [5/5/2002 6:51:21 PM]
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glycerophosphate (GP), a reaction catalyzed by glycerol phosphate dehydrogenase (Eq. 8). CH2COCH2OPO3H2 + NADH + H+ → CH2OHCHOHCH2OPO3H2 + NAD+ (8) The mitochondria from muscle can oxidize GP to regenerate DHAP, which can then be transferred to the cytoplasm (Fig. 4). A similar shuttle may occur in liver, where β-hydroxybutyrate can be formed from acetoacetate in the cytoplasm, which can then be oxidized in the mitochondria, regenerating the β-hydroxybutyrate. Shuttles such as these may play an important role in the regulation of metabolism. For example, inhibition of the GP shuttle would favor the formation of lactic acid. Lactic acid is favored in some cells, for example, tumor cells. This effect has been referred to as the Crabtree effect. The actual mechanism for the Crabtree effect is not known, and a number of other possibilities have been proposed. In other systems, under aerobic conditions, no lactic acid is accumulated, a phenomenon referred to as the Pasteur effect. Again, the mechanism is not well understood, and it has been suggested that a competition between the glycolytic and the electron transport systems for NADH, ADP, and Pi may be responsible. Pi may be involved in the expression of both the Crabtree and the Pasteur effect in some cells (Krebs, 1972). In some tissues, the oxidation of NADH by peroxisomes may have an effect on the regulation of metabolism. B. Energy Balance In Chapter 13 we saw the importance of the adenosine phosphates acting as allosteric regulators in directing metabolites in the purine nucleotide biosynthetic pathway. In this role, ATP is a positive effector of reactions favoring the synthesis of macromolecules. The role of ATP, ADP, and AMP in metabolic regulation was discussed above from the limited perspective of the individual pathway, for example, glycolysis and the TCA cycle. These compounds, by indicating the energy balance of the cell, may serve as ideal general regulators of metabolism. Therefore, a more global perspective is very useful. The role of these compounds in regulating metabolism has long been recognized and referred to as the energy charge (Atkinson, 1977). A high energy charge (high ATP concentration) inhibits ATP-generating reactions and facilitates energy-yielding reactions. Generally, ATP acts in the opposite direction of AMP or ADP, although different reactions may be the target of the individual adenine nucleotide. The examination of the regulation of the individual steps clearly indicates that ATP, ADP and AMP play a role. For example, in the PK reaction, ATP present in high concentrations favors the formation of the inactive form of the enzyme, whereas ADP has the opposite effect. This, in effect, can regulate the amount of pyruvate that can enter the TCA cycle. AMP or ADP (depending on the organism) favor the hydrolysis of polysaccharide by the polysaccharide phosphorylase reaction, thereby permitting the mobilization of glucose. They speed up the machinery of glycolysis through the activation of PFK and decrease the formation of http://www.albany.edu/~abio304/text/chapter_14.html (20 of 34) [5/5/2002 6:51:21 PM]
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polysaccharide stores by inhibiting FBPase. Through the activation of isocitrate dehydrogenase, AMP or ADP stimulate the turnover of the TCA cycle. In the same way, Pi stimulates succinate dehydrogenase and fumarase. ATP, in contrast, has the opposite effect by activating FBPase, which favors the synthesis of polysaccharide. Likewise, ATP inhibits isocitrate dehydrogenase and tends to slow down the TCA cycle. We have already seen that the ATP concentration does not vary significantly in muscle, because it can be regenerated from creatine phosphate, with AMP being a much more sensitive indicator of the energy level. However, phosphocreatine (PC), which decreases during muscular activity, can also act as a regulative signal. An increased concentration of PC activates FDPase (Fu and Kemp, 1973) and inhibits PFK (Uyeda and Racker, 1965) and PK (Moyed, 1961). Molecules that are interconvertible with ATP may also have an effect; for example, GTP inhibits α-ketoglutarate dehydrogenase (Olson and Algyer, 1973). There are other controls superimposed on this basic pattern. G6P is a positive effector for the polysaccharide synthase reaction: favoring the laying down of carbohydrate stores while discouraging glycolysis through its inhibitory effect on hexokinase. Citrate, which inhibits citrate synthase, stimulates fatty acid synthesis from acetyl-CoA, thereby favoring the formation of fat deposits. Succinate dehydrogenase is inhibited by oxaloacetate (Pardee and Potter, 1948) and activated by succinate, ATP, reduced CoQ, and succinylCoA (Pardee and Potter, 1948; Kearney et al., 1972). Some of these reactions, other reactions previously discussed, and their regulation are shown in Fig. 2. The heavy arrows indicate positive effects, whereas lighter lines with crosses indicate negative effects. For simplicity, the figure leaves out the regulation by F2,6P and G1,P. C. Control of Oxidative Phosphorylation in Mitochondria In eukaryotic cells, mitochondrial oxidative phosphorylation provides 95% of the total ATP required. Furthermore, any event affecting oxidative phosphorylation will have repercussions in the pathways that precedes it by oxidizing their metabolic products. Therefore, understanding how the mitochondrial system is regulated is of paramount importance to understand the regulation of metabolism. The mitochondrial electron transport chain is responsible for accepting reducing equivalents from NADH and reduced flavoprotein (FPH2) and oxidizing them to ultimately produce water and ATP. The chain is a multienzyme system consisting of four complexes embedded in the mitochondrial inner membrane (see Chapter 16). The appropriate dehydrogenases and the ATP synthase complexes are located on the inner face of the mitochondrial inner membrane. The transfer of ATP to the cytoplasm, where it is mostly used, requires the adenine nucleotide translocator, an integral protein of the inner membrane which mediates the exchange between the mitochondrial ATP4- and the cytoplasmic ADP3-.
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Near equilibrium and translocase hypotheses Two major hypotheses have been advanced to explain the regulation of mitochondrial oxidative phosphorylation. One hypothesis proposes that the first two sites of oxidative phosphorylation (in complexes I and III) are near equilibrium. The regulation of the system results from the virtually irreversible reactions in which reduced cytochrome c (2 cyt c2+) is oxidized by O2 in complex IV. This proposal has been referred to as the near equilibrium hypothesis (see Erecinska and Wilson, 1982). An alternative hypothesis the translocase hypothesis (Klingenberg, 1980a) proposes a key role of the adenine nucleotide translocase. The transport mediated by the adenine nucleotide translocase (the ADP/ATP transporter, seeChapter 5) is rate limiting and is responsible for setting the metabolic rate. The concept of the near equilibrium hypothesis can be illustrated most simply by representing the events of the first two phosphorylative sites as follows: NADH + 2 cyt c3+ + 2 ADP + Pi→ NAD+ + 2 cyt c2+ + 2 ATP (9) In this representation, cyt c3+ and cyt c2+ represent the oxidized and the reduced species of cytochrome c. If we now assume that the reactions are at equilibrium: Keq = [(NAD+)/(NADH)]½ (ATP)/(ADP)(Pi) (cyt c2+)/(cyt3+) (10) then at equilibrium, (cyt c2+)/(cyt c3+) = (NADH)½/(NAD+)½ (ADP) (Pi)/(ATP) (1/Keq) (11) As shown in Eq. (11), any increase in the NADH/NAD+ or (ADP) (Pi)/ATP ratios will be reflected in an increase in cyt c2+. Therefore, this increase in cyt c2+ will lead to an increase in oxidation, because the cyt c-oxidase is not saturated and the reaction is irreversible. The key and contrasting points of the two models have to do with the equilibrium position. The near equilibrium hypothesis assumes equilibrium for the first two phosphorylating steps, which include adenylate translocase. The translocase hypothesis assumes that the adenylate translocase is not at equilibrium. Can we support one model over the other at this time? Examination of the available data shown in Table 2 indicates that, at steady state, in many biological systems these reactions appear to be close to equilibrium (Erecinska and Wilson, 1978). The table calculates the redox potential for the http://www.albany.edu/~abio304/text/chapter_14.html (22 of 34) [5/5/2002 6:51:21 PM]
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reactions between NADH and cyt c by subtraction. The values were calculated from the experimentally determined concentrations for the system listed in the first column. The corresponding ∆G values are listed in the fourth column. When the ∆G for the synthesis of ATP, also calculated from the experimentally determined concentration (column 5), is subtracted from this value (displayed in column 6 and represented as ∆∆G), the results show little deviations from equilibrium (i.e., ∆G = 0). ∆G (expressed as ∆∆G) for the whole system is close to zero. Unfortunately, arguments for or against either model depend crucially on the accuracy of the rather complex measurements needed to calculate concentrations. Furthermore, some of the components may be bound and not free in solution. Therefore, at this time it is difficult to reach a firm conclusion (see Erecinska and Wilson, 1982 for a discussion). Regulation of mitochondrial permeability Mitochondrial metabolism may also be regulated by mechanisms affecting the accessibility of the mitochondrial internal compartment to substrates. NADH and NADPH reduce the permeability of the outer mitochondrial membrane to ADP (Lee et al., 1994) apparently by closing the outer mitochondrial membrane channel VDAC (voltage dependent anion channel). The permeability to other substrates may also be affected. Uncoupling proteins Uncoupling proteins stimulate heat production in mitochondria by uncoupling respiration from ATP synthesis (e.g., see Klingenberg, 1990). Presumably, the proteins act as channels that discharge the proton-electrochemical gradient in mitochondria. The uncoupling protein-1 (UCP1), the first to be discovered, is a mitochondrial protein of brown adipose tissue (BAT) and is essential for nonshivering thermogenesis (see Himms-Hagen, 1990). Shivering is the immediate mechanism for thermogenesis on exposure to cold, whereas nonshivering thermogenesis increases metabolism for long periods by affecting the metabolic machinery. UCP-1 plays an important role in arousal from hibernation, in cold adaptation in rodents and in neonatal animals. A role in humans is still in question. Two other uncoupling proteins similar to UCP1 have been discovered. UCP-2 has a 59% amino acid correspondence to UCP1. In contrast to UCP1, UCP2 is widely distributed in animal and human tissues. It is present in white fat, skeletal muscle, heart and tissues rich in macrophages. The expression of UCP2 mRNA is strongly influenced by dietary factors, for example, it is sharply increased by fasting (Boss et al., 1997) or a fat diet (Fleury et al., 1997). It is presently thought to have a role in body weight regulation with implications for obesity and hyperinsulinemia. The brain distribution of UCP2-mRNA in mouse brain (Richard et al., 1998) suggests that UCP2 is present in neurons, perhaps having a role in neuroendocrine functions (e.g., it is abundant in the hypothalamus), autonomic responses and arousal.
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UCP3 is highly expressed in skeletal muscle of rodents and humans (Boss et al., 1998a). In a mouse myoblast cell line, overexpression of UCP3 by transfection dissipated part of the mitochondrial proton motive force. A similar uncoupling was shown when incorporated into yeast (Gong et al., 1997). In contrast to UCP2, which showed little hormonal regulation, UCP3-RNA levels are regulated hormonally. Muscle UCP3 levels were decreased 3-fold in hypothyroid rats and increased 6-fold in hyperthyroid rats, suggesting that UCP3 may be responsible for the the effect of thyroid hormone on thermogenesis. In the skeletal muscle of rodents, UCP3-mRNA expression did not change in response to cold exposure. However, it decreased by 81% with food restriction and increased 5.6-fold with fasting. Skeletal muscle is an important site of catecholamine and diet-induced thermogenesis in rats and in humans and, therefore, might play an important role in whole body thermogenesis. In rats, both UCP3 and UCP2 mRNAs in muscle are downregulated by excercise, presumably to direct the energy expenditure to the motor activity (Boss et al., 1998b). A role of UCP-3 in lipid metabolism has been suspected for some time (see Muzzin et al., 1999; Samec et al., 1998). A role in regulation of metabolism is supported by experiments in which transgenic mice (see Chapter 1) overproduced UCP-3 in skeletal muscle (Clapham et al., 2000). The mice ate excessively but maintained a low body weight and a reduced adipose tissue mass. It has been proposed that the UCPs could function in reducing the concentration of reactive oxygen species which produce oxidative damage (see Chapter 2). In agreement with this thesis, superoxide has been found to increase mitochondrial proton conductance through effects on UCP1, UCP2 and UCP3 (Echtay et al., 2002). This activation required the presence of fatty acids and was inhibited by purine nucleotides. Table 2 Free-Energy Relationships Between the Oxidation-Reduction Reactions of the Respiratory Chain and ATP Synthesis. From Erecinska and Wilson, 1978.
Material
Eh(V) cyt c
Eh(V) NAD
∆Eh
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∆Gox-red (kcal/2e)
∆GATP (kcal/2ATP)
∆∆G (kcal)
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Liver cells (no substrate)
0.272
-0.242
0.514
23.7
23.8
-0.1
0.269
-0.260
0.529
24.4
24.2
0.2
0.253
-0.263
0.516
23.8
22.6
1.2
0.260
-0.270
0.530
24.4
23.6
0.8
0.271
-0.252
0.523
24.1
24.2
-0.1
0.251
-0.236
0.487
22.5
22.4
0.1
0.276
-0.244
0.520
24.0
24.1
-0.1
0.253
-0.313
0.566
26.1
26.3
-0.2
0.270
-0.343
0.613
28.4
29.8
-1.4
Liver cells (lactate + ethanol) Perfused liver (no substrate) Ascites tumor cells Cultured kindey cells Tetrahymena pyriformis Paracoccus dentrificans Perfused heart (80 cm H2O) Pigeon heart mitochondria (succinate)
E = Eh NAD-Eh cyt c see Chapter 12. Reproduced from Trends in Biochemical Sciences vol. 3, M. Erecinska and D.F. Wilson, pp.219-223, copyright ©1978 with permission from Elsevier Science Publishers, Amsterdam.
VI. CONTROL OF CARBOHYDRATE METABOLISM IN PLANTS In plants, the energy captured from light is trapped and converted into ATP (synthesized from ADP and Pi) and NADPH (reduced from NADP+) (see Chapter 17). Eventually, after a series of reactions that fix CO2, the energy is stored in the synthesis of starch or sucrose. Starch is formed in the chloroplasts, whereas sucrose is formed in the cytoplasm. The pathways generate dihydroxyacetone phosphate (DHAP) or triose phosphates derived from DHAP. In the production of starch, DHAP is converted first to hexose phosphate. In the production of sucrose, triose phosphate is first transported across the chloroplast envelope to the cytoplasm. In the absence of light, the plant cells can draw on energy reserves by breaking down starch to form triose phosphates, which can be metabolized further. These two alternatives are summarized in Fig. 5 http://www.albany.edu/~abio304/text/chapter_14.html (25 of 34) [5/5/2002 6:51:21 PM]
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(Buchanan, 1984). As shown, the enzymes that synthesize starch and those that catalyze its breakdown, coexist in the chloroplast. For this reason, the system is tightly regulated. The enzymes involved in synthesis are activated by light (when energy is available from light) and the degradative enzymes are deactivated by light, so that in plants, carbon assimilation predominates during the day and carbohydrate degradation during the night. Light regulates the enzymes indirectly, functioning of the photosynthetic machinery of the chloroplast. Ferredoxin, a component of the electron transport chain of the thylakoid vesicles, is reduced during photosynthesis (see Chapter 17). Light controls the enzymes through the redox changes of ferredoxin as discussed in the next section.
Fig. 5 Role of chloroplasts in producing and storing energy-rich compounds in the plant. From B.B. Buchanan, BioScience, 34:378-383 Copyright ©1984 by the American Institute of Biological Sciences.
VII. REGULATION VIA THE REDOX STATE OF CELLS The redox state of cells is emerging as an important regulatory factor. The redox state of any cellular compartment depends on the redox state of certain molecular species such as protein thiols, glutathione, pyridine nucleotides (e.g., Gilbert 1990; Chance et al., 1962). This state is regulated by enzymes that sense and respond to the redox state of cells. In animal cells, redox signaling has been shown to have effects on metabolism as well as in the regulation of transcription, translation and apoptosis (see e.g., Xanthoudakis et al., 1994; Hampton et al., 1998; Sen, 1998). http://www.albany.edu/~abio304/text/chapter_14.html (26 of 34) [5/5/2002 6:51:21 PM]
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A. Control of metabolism in plants. In plants, regulation by the redox state of the system is responsible for the activation of the chloroplast's Calvin cycle enzymes and inhibition degradative reactions by light (e.g., see Buchanan, 1994; Jacquot et al., 1997). The enzyme ferredoxin-thioredoxin reductase (FTR), an iron sulfur protein, is key to this system. During photosynthesis, PSI reduces ferredoxins which produces NADPH catalyzed by NADP+-ferredoxin reductase. In addition, the reduced ferredoxin is used by FTR to reduce thioredoxins. Thioredoxins (Trxs) are responsible for the activation of the enzymes of the Calvin cycle and other enzymes (see Jacquot et al., 1997). Trxs correspond to a group of small proteins about 12 kDa in size, probably present in all organisms. The thiol groups of Trxs undergo redox changes so that 2SH are oxidized to S-S. The FTR of plants is distinct from either mammalian or bacterial FTR. The plant FTR is an ironsulfur enzyme heterodimer of a catalytic subunit (β) of 13 kDa and a redox active disulfide and a 4FeS-4S center. The α subunit is of similar size but differs from organims to organism. FTRs are disulfide reductases which use as an electron donor, [Fe2S2]2+/+ ferredoxin and an FeS cluster as a prosthetic group (e.g., see Staples, 1996, 1998). The FTR molecule is thin and flat, with the iron sulfur cluster in close contact with the iron-sulfur center (Dai et al., 2000). The functioning of the ferredoxin-thioredoxin system in plants is summarized in Fig. 6. Reduced thioredoxin activates the enzymes of carbohydrate biosynthesis and those of the reductive pentose phosphate cycle, and deactivates glucose-6-phosphate dehydrogenase, which is needed for degradation through the pentose phosphate cycle. The reductive pentose phosphate and the regulation by light are shown in Fig. 7 (Buchanan, 1984). Table 3 summarizes the enzymes which are activated by light or dark (Anderson et al., 1982). Thioredoxin acts by reducing or oxidizing the enzymes that can also undergo thiol-disulfide transitions. The action of two different varieties of Trx is summarized in Fig. 8. As shown, the ferredoxin-thioredoxin system also activates NADPmalate dehydrogenase (NADP-MDH) and ATP synthase (CF1ATPase). In non-photosynthesizing tissues, Trx can be reduced by NADPH.
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Fig. 6 (a) Ferredoxin-thioredoxin system of enzyme photoregulation (light activation/ dark deactivation). From B. B. Buchanan, BioScience, 34:378-383. Copyright ©1984 by the American Institute of Biological Sciences. (b) Outline of the mechanism of activation and inactivation of NADP-MDH. E, Enzyme; Td, thioredoxin; Fd, ferredoxin.
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Fig. 7 Steps of the reductive pentose phosphate cycle regulated by light through the Trx system. The light dependent reactions are indicated by h and the heavy arrows. From B. B. Buchanan, BioScience, 34:378-383. Copyright ©1984 by the American Institute of Biological Sciences.
Fig. 8 Enzymes regulated by the ferredoxin-thioredoxin system. Reproduced from Biochimica et Biophysica Acta, vol.853, Cseke and Buchanan, pp.43-63. Copyright ©1986 with permission
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from Elsevier Science Publishers, Amsterdam.
NADP-MDH, which catalyzes the reduction of oxalate by NADPH, can serve as an example for the scheme discussed and summarized in Fig. 6. The reaction proceeds as shown in Eq. 12. Oxaloacetate + NADPH + H+ → malate + NADP+ (12) The enzyme is rapidly inactivated when leaves are kept in the dark and is reactivated by light (Johnson, 1971; Johnson and Hatch, 1970; Scheibe and Anderson, 1981). Added thiols keep the enzyme active in the presence of oxygen and reactivate the enzyme when it has been inactivated. When the activation is produced by light, it is blocked by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) (Hatch, 1977) that blocks the electron transport chain of the chloroplast. Activation of the inactive enzyme requires the presence of thioredoxin (Kagawa and Hatch, 1977), that also undergoes dithiol-disulfide transitions. Table 3 Light Modulation in Different Plants Reproduced with permission from Anderson et al., 1982.
Prokaryote
cyanobacterium
Eukaryotes
Algae
Anacystis nidulans A.Green B.Brown Light-activated enzymes
C3 plants
C4 plants
CAM
A.Pisum B.Spinacia
A.Zea B.Tidestromia
Kalanchoe
14 x
50 3.3
1.7
2.4 5
2 3.1
2
4.4
Light stimulatioin xfold
NADP-linked malic dehydrogenase NAPD-linked glyceraldehyde-3-P dehydrogenase
Nil
Ribulose-5-P kinase
2
7.7 3.2
1.6 4
Nil
1.7 2.2
Nil
Fructose-1,6-P2 phosphatase
~2 Nil
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Nil
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Sedoheptulose-1,7-P2 phosphatase
Nil
1.8 1.7
Pyruvate, orthophosphate dikinase
~12
NAD-linked malic dehydrogenase
1.7
Dark-activated enzymes
Glucose-6-P dehydrogenase
1.7
Dark stimulation xfold 4.8
2 3
3
1.4 Phosphofructokinase
2 11
Phosphorylase
1.6
Phosphoglucomutase
3.7
Phosphoglucoisomerase
3.6
Apart from the ferredoxin-thioredoxin regulation in plants, there is evidence that the redox state of cells plays an important role in a variety of systems (Ziegler, 1985). Sulfides and sulfhydryl groups are pervasive in enzymes. Similarly, there are a variety of enzymes which catalyze transitions between the two. In addition, many cells are rich in gluthione. Trx is also thought to play a regulatory role in mammalian cells, including the control of growth (Holmgren, 1989). B. Role in animal cells. The precise role of redox state in animal cells, frequently through thiol-reactive proteins, such as Trx, is far reaching. However, the physiological implications of this regulation are still far from clear. The role of Trxs in animal cells is undeniable. In mammals, mutant redox-inactive forms of Trx are unable to stimulate cell growth or inhibit apoptosis (Oblong et al., 1994; Freemerman et al. 1999). Increases in gene expression for thioredoxin reductases (TrxRs) and Trx, are thought to play a protective role against oxidative damage (see Mustacich and Powis, 2000). In addition to protecting the cell from oxidative stress by maintaining Trx in its reduced state, TrxR is likely to http://www.albany.edu/~abio304/text/chapter_14.html (31 of 34) [5/5/2002 6:51:21 PM]
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be responsible for the recycling of ascorbate. In humans, this role is very important since humans lack the ability to synthesize ascorbic acid. The mammalian TrxRs are a family of flavoproteins containing selenium that are pyridine nucleotide-disulfide oxidoreductases with mechanistic and sequence identity (see Mustacich and Powis, 2000). They include lipoamide dehydrogenase, glutathione reductase and mercuric ion reductase. The redox activity of this catalytic site is necessary for their biological activity. In mammals, TrxRs are the only enzymes known to reduce oxidized Trx. TrxRs are homodimers where each monomer includes an FAD prosthetic group, an NADPH binding site and an active site containing a redox-active disulphide. Electrons are transferred from NADPH via FAD to the activesite disulphide of TrxR, which then reduces the substrate (see Williams, 1995). Substrates other than Trx are reduced by TrxRs, including lipoic acid, lipid hydroperoxides, vitamin K3, dehydroascorbic acid and the tumor-suppressor p53. However, the physiological role of these reactions is not known. Trx proteins supply reducing equivalents to enzymes such as ribonucleotide reductase (Laurent et al., 1964) and thioredoxin peroxidase (Chae et al., 1994). They also reduce cysteine residues in certain transcription factors, increasing their binding to DNA, thereby altering gene transcription. In mammalian cells low oxygen increases the transcription of genes that favor the delivery of oxygen (e.g., angiogenesis, erythropoiesis and vasomotor function) or that permit metabolic adjustments to low oxygen (e.g., regulation of glycolytic enzymes and glucose transport). The responses to low oxygen are through the transcription factor know as the hypoxia inducible factor (HIF) (see Semenza, 1999). HIF-1 is also needed for normal development. HIF-1 has two subunits, one of 91-94 kDa and the other of 120-130 kDa.. HIF-1 is stable at low oxygen but is degraded at higher oxygen concentration via the E3 ubiquitin-ligase system (see Chapter 15),which contains the von Hippel-Lindau (pVHL) tumor suppressor protein. pVHL binds to a domain of HIF-1 when the proline of this domain is hydroxylated ( Ivan, et al., 2001; Jaakkola et al., 2001). The hydroxylation requires Fe2+ and molecular oxygen. The ubiquitination system, responsible for various physiological functions including protein degradation (see Chapter 15, Section IIB) is regulated by the cellular ratio of oxidized to reduced glutathione (GSSG:GSH ratio) (Jahngen-Hodge et al., 1997; Obin et al., 1998). In eukaryotes several transcription factors such a AP-1 and NF-κ B respond to oxidative stress (see Dalton et al. 2000). In addition, kinases and phosphatases (see Section V, Chapter 13; Section IV, Chapter 7) are thought to have a significant role in signal regulation by the redox state. The activity of some serine/threonine and tyrosine phosphatases (e.g., calcineurin, PP1 and PP2A) depend on the redox state of their active sites (see Rusnak and Reiter, 2000) such as that of the Fe ion (e.g., Yu et al., 1997; Merkx and Averill, 1998) for the former and cysteine residues for the latter (e.g., Denu and Tanner, 1998). These phosphatases could therefore play a central role in the http://www.albany.edu/~abio304/text/chapter_14.html (32 of 34) [5/5/2002 6:51:21 PM]
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regulation of signal transduction and responses to oxidative stress caused by the presence of ROSs (Huie et al., 1999). Several systems involved in transcription of certain genes have been found to be regulated by nicotinamide adenine nucleotides. The carboxyl-terminal binding protein (CtBP) is a corepressor with an important role in development in Drosophila and vertebrates. It has been shown to have a role in growth and differentiation in Drosophila and vertebrates. The binding of CtBP transcriptional repressors is facilitated by NAD+ and NADH, the reduced form being two to three orders of magnitude more effective (Zhang et al., 2002). The clock mechanism of the mammalian forebrain has been found to be regulated by the redox state of nicotinamide adenine dinucleotide cofactors, supposedly in response to metabolic conditions. The DNA-binding activity of the Clock:BMAL1 and NPAS2:BMAL1 is increased by the presence of NADH and NADPH and inhibited by the oxidized forms. (see see Chapter 15). Sir2 proteins in mice and yeast have been found to be nicotinamide adenine dinucleotide-dependent histone deacetylases (Imai et al., 2000). The acetylation state of the histones have been shown to have a role in gene expression (see Chapter 2). This finding suggests that Sir2 proteins might be sensors of the redox-state of the cell. The deacetylase activity of Sir2 proteins has a role in the mechanism by which Sir2 silences transcription of several genes, suppresses recombination of rDNA and promotes longevity in yeast. SUGGESTED READING Beitner, R. (1984) Control of levels of glucose 1,6-bisphosphate, Int. J. Biochem. 16:579585.(Medline) Boss, O., Muzzin, P., Giacobino, J.P. (1998) The uncoupling proteins, Eur. J. Endocrinol. 139:19.(Medline) Cséke, C. and Buchanan, B. B. (1986) Regulation of the formation and utilization of photosynrthate in leaves, Biochim. Biophys. Acta 853:43-63. Erecinska, M., and Wilson, D. F. (1982) Regulation of cellular energy metabolism, J. Membr. Biol. 70: 1-14.(Medline) Goodwin, T. W. and Mercer, E. I. (1983) Introduction to Plant Biochemistry, 2nd ed. Pergamon. New York. See pp. 139-159. Jacquot, J.-P., Lancelin, J.-M., Meyer, Y. (1997) Thioredoxins: structure and function in plant cells, New Phytol. 136:543-570. Klingenberg, M. (1979) ATP shuttle of the mitochondrion, Trends in Biochem. Sci. 4:249-252.
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Mustacich, D. and Powis, G. (2000) Thioredoxin reductase, Biochem. J. 346:1-8. (Medline) Newsholme, E. A., Challis, R. A., and Crabtree, B. (1984) Substrate cycles: their role in improving sensitivity in metabolic control, Trends in Biochem. Sci. 9:227-280. Ricquier, D. (1998) Neonatal brown adipose tissue, UCP1 and the novel uncoupling proteins, Biochem. Soc. Trans. 26:120-123.(Medline) Saier, M. H., Jr. (1987) Enzymes in Metabolic Pathways., Chapters 5 and 6. Harper and Row, New York. WEB RESOURCES Diwan, J.J., Molecular Biochemistry REFERENCES Search the textbook
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Chapter 14: References
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Chapter 14: References
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15. Regulation by Synthesis and Degradation of Macromolecules
15. Regulation by Synthesis and Degradation of Macromolecules I. Production and Degradation of Specific Enzymes II. The Degradative Pathways A. Degradation as a General Regulatory Mechanism B. Biochemical Pathways of Proteolysis Multiple proteolytic pathways The ubiquitin pathway Proteasomes Degradation of membrane proteins Caspases C. Degradation of Receptors III. Folding and Quality Control IV. Regulation of Enzyme Synthesis A. Transcriptional Control B. Posttranscriptional Control C. Regulation in Skeletal Muscle V. Circadian Rhythms A. Clock mechanisms B. Entrainment Suggested Reading References Back to List of Chapters As discussed in Chapters 13 and 14, metabolic activity can be regulated by adjusting the rates of the cell's biochemical reactions either by activating previously inactive enzymes or, more subtly, by adjusting the activity of the appropriate enzymes. Alternatively, cells may alter the concentration of enzymes and hence the rate of certain metabolic reactions. In eukaryotes, the concentration of any one enzyme is controlled by the balance between the ability of cells to synthesize the enzyme and their ability to degrade it. In bacteria, enzymes are generally stable. However, because of their much shorter generation time, in exponentially growing bacterial cells, unneeded enzymes are rapidly diluted by cell division.
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The synthesis of specific enzymes can be regulated at several biochemical levels in the various processes occurring between transcription of the gene and the actual synthesis of the enzyme. The present chapter will concentrate on exploring the regulation of these events in eukaryotes. In bacteria, the adjustment of the enzymatic composition to face an environmental challenge, such as the presence of a new nutrient in the medium, is a major function of the regulatory mechanisms. Regulation is primarily at the transcriptional and translational levels. Lower eukaryotes, such as yeast, may also be subject to the same nutritional challenges. To some extent, the problem is different for multicellular organisms. Since their tissues are specialized, only some (for example, the liver) have to adjust to nutritional changes. The environment and nutrient composition of other tissues are maintained relatively constant. However, protein and enzyme synthesis is regulated by hormones or growth factors. Furthermore, the protein composition of a cell depends on its developmental stage. Each stage reflects differential gene expression that eventually results in differentiation. In the differentiated state, only some of the proteins encoded by the genome are fully expressed, as discussed in Chapter 2. In multicellular eukaryotes, the control of protein or enzyme levels is very complex. As in prokaryotes, protein and enzyme synthesis is controlled by transcriptional and posttranscriptional mechanisms. In addition, however, specific enzymes and their corresponding mRNAs are differentially degraded, and the transfer of mRNA from the nucleus may be regulated. This is the topic of this chapter. Some of the molecular events of transcriptional and posttrascriptional regulation are discussed in Chapter 3. One of the heat shock proteins, Hsp90, has been found to have a role in the folding of certain key proteins so that it regulates the formation and localization of nuclear hormonal receptors, transcription factors, protein kinases involved in signaling, and translational events (see Chapter 7). Hsp is also needed for the maturation and maintenance of the chloride channel CFTR (Loo et al., 1998). The proteins requiring Hsp90 are often labile, have very complex folding patterns, or have to be maintained in a particular conformation to insert a cofactor or other ligand (see Mayer and Bukau, 1999). Hsp90 and co-chaperones remain associated with the multiprotein complexes after maturation and may be involved in facilitating ligand induced conformational changes (see Mayer and Bukau, 1999). I. PRODUCTION AND DEGRADATION OF SPECIFIC ENZYMES The balance between production and breakdown of enzymes plays an important role in the regulation of the cell's metabolic machinery in eukaryotes. The concentration of an enzyme can be increased in response to the presence of its substrate, to the secretion of a hormone, or to general nutritional conditions. In rat liver, the level of the enzyme tryptophan pyrrolase (also called tryptophan 2,3-dioxygenase) is increased sharply by administering hydrocortisone or a large concentration of tryptophan. The enzyme catalyzes the reaction shown in Eq. (1). (1)
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This reaction is the first in one of the two major biochemical pathways responsible for the breakdown of tryptophan. The formation of the product of the reaction, N-formylkynurenine with time, can be followed spectrophotometrically. The adrenal cortex secretes a number of steroid hormones, some of which increase the breakdown of proteins to produce glucose (gluconeogenesis) in the intact animal. Steroid induction of enzymes such as tryptophan pyrrolase, tyrosine transaminase, serine dehydratase, serine deaminase, and phosphoenolpyruvate carboxykinase, is likely to be part of this catabolic effect. The hormonal control of tyrosine transaminase and serine dehydratase is discussed in more detail later in this chapter. In the experiment shown in Table 1 (Schimke et al., 1965), adrenalectomized rats (i.e., rats whose adrenal gland was surgically removed) were used to avoid the complication of endogenous release of the steroid hormone by the adrenal cortex. The tryptophan pyrrolase activity in a liver extract increased with the addition of tryptophan and hydrocortisone (column 3 of Table 1). Other enzyme activities also increased (columns 4 and 5), although more moderately. Compared to an untreated control (in which a NaCl solution was injected), both hydrocortisone- and tryptophan-treated animals had large increases in the activity of tryptophan pyrrolase. The two treatments administered together had an even greater effect. Since a latent form of tryptophan pyrrolase is known, the increase in enzyme activity could result from activation of the enzyme rather than de novo synthesis. An immunological assay can resolve which of the two possible mechanisms is responsible for the effect. Immunological techniques depend on the interaction between antibodies and their corresponding antigens. Serum antibodies specific for regions (i.e., epitopes) of an enzyme molecule can be produced by injecting the pure protein into animals, generally rabbits. The amount of antibody-antigen complex formed after the addition of excess antibody to an extract, quantitatively measures the amount of enzyme present. In the commonly used radioimmunoassay technique, radioactive iodine is introduced into the antibody molecule and the presence of antibody-antigen complex is assayed in the precipitate by simply measuring its radioactivity. Similarly, if the proteins of an animal are made radioactive by injection of radiolabeled amino acids, an enzyme can be recognized with an antibody in the same way. Experiments using this latter approach will be discussed later. Table 1 Effect of Repeated Doses of Tryptophan and Hydrocortisone on Liver Weight and http://www.albany.edu/~abio304/text/15part1.html (3 of 19) [3/5/2003 8:21:17 PM]
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Tryptophan Pyrrolase, Formylkynurenine Formylase, and Arginase of Rat Liver
Enzyme activity (µmol product/h per g liver weight)
a
(1)
(2)
(3)
(4)
(5)
Treatment
Total liver weight (g)a
Tryptophan pyrrollase
Formylase
Arginase
NaCl
25.4
3.0
1800
13,800
Hydrocortisone (HC)
27.4
38
1810
19,740
Tryptophan (Try)
28.7
28
2060
15,200
HC + Try
25.0
116
2180
20,160
Combined weight of 4 animals
Reproduced with permission from R.T. Schimke et al., J. Biol. Chem. 240:322-331. Copyright © 1965 American Society of Biological Chemists, Inc.
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Fig. 1 Immunological analysis of tryptophan pyrrolase activity of rat liver extracts. Adrenalectomized rats weighing 150-170 g were given repeated administrations of 0.85% NaCl (control) or hydrocortisone 2lphosphate, L-tryptophan, or both. At the end of 16 h, the livers were removed. Two rats that had received both hydrocortisone and tryptophan were killed 3 h later, at a time when the enzyme level was falling. The dashed line indicates that no enzyme activity was detectable in the supernatant fluid. Reproduced with permission from R. T. Schimke, et al., Journal of Biological Chemistry 240:322-331, Copyright © 1965 American Society of Biological Chemists, Inc.
In the experiment of Fig. 1 (Schimke et al., 1965), aliquots of liver homogenate were added sequentially to a fixed amount of antibody. The supernatant was sampled for the enzyme activity that remains after precipitation of the antibody-enzyme complex. This manipulation is analogous to an analytical procedure in which aliquots of a reagent are added to a sample until an end point is reached-in this case, the appearance of the enzymatic activity in the supernatant. The enzyme activity of the aliquots, before addition to the antibody, is shown on the abscissa and the enzyme activity remaining in solution after precipitation is shown on the ordinate. As soon as the added enzyme is in excess of the antibody present, the activity appears in the supernatant. Presumably, the antibody will interact with the enzyme whether the latter is active or inactive. Inactive enzyme should remove antibody but will not be detected in the assay of the activity of the homogenate. Any masked enzyme would therefore show up in a graph, such as that of Fig. 1, as an earlier appearance of activity (at a lower homogenate activity) because there will be less antibody to bind the active enzyme. In this experiment, regardless of whether the animal receives only weak salt solution (the control) or is treated with hydrocortisone, tryptophan, or both, the results are the same. The appearance of enzymatic activity in the supernatant occurs at precisely the same level of http://www.albany.edu/~abio304/text/15part1.html (5 of 19) [3/5/2003 8:21:17 PM]
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added enzyme activity in all experimental preparations, indicating that the active enzyme and the antigen bound by the antibody are precisely equivalent. Therefore, there is no inactive enzyme before induction. This indicates that the increase in activity corresponds to an increase in the enzyme present. Other criteria (see below) are in agreement with the conclusions of this study. Since the amount of enzyme has increased, either more enzyme is being made or less is being broken down. After [14C]-labeled amino acid (in these experiments [14C]-leucine) is injected into rats, immunological precipitation of the enzyme from homogenates serves as a measure of its synthesis, assuming that [14C] leucine inside the cells remains the same under the different experimental conditions. The results of an experiment depicted in Fig. 2 (Schimke et al., 1965) show that this assumption is correct. Curve 2 in the figure represents the amount of radioactive trichloroacetic acid-soluble material (TCA extract) that corresponds primarily to free amino acid. The radioactivity in the protein corresponds to the material precipitated with TCA (curve 1). Each point corresponds to a different group of rats treated in the same way. In Fig. 2, the squares and the triangles represent the radioactivity present in liver extracts of hydrocortisone- and tryptophan-treated animals, respectively; the circles represent controls. The radioactivity is equivalent in all three cases. Therefore, the concentration of [14C] leucine incorporated represents the same degree of protein synthesis in all three cases.
Fig. 2 Effect of hydrocortisone 21-phosphate and tryptophan administration on free pool of administered L-[14C]leucine and its incorporation into liver protein. Reproduced with permission from R. T. Schimke, et al., Journal of Biological Chemistry, 240:322-331. Copyright © 1965 American Society of Biological Chemists, Inc.
The incorporation of a label into immunologically precipitated tryptophan pyrrolase is shown in Table 2 (Schimke et al., 1965). The label incorporated in the enzymes of the saline-treated control animals http://www.albany.edu/~abio304/text/15part1.html (6 of 19) [3/5/2003 8:21:17 PM]
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corresponds to about 1400 counts/min (column 4). The incorporation in the hydrocortisone-treated animals (column 4) is considerably higher, about 9500 counts/minute. Since this corresponds approximately to a sevenfold increase in incorporation, the system behaves as if there were an increase in the synthesis of the enzyme in response to the presence of the hormone. Table 2 Immunological Precipitation of Tryptophan Pyrrolase from Liver Extracts of Rats that Received [14C]L-lysinea
Total enzyme activity in (units/hour) (1)
(2)
(3)
(4)
Homogenate
Supernatant
DEAEcellulose eluate
Corrected incorporation (total counts/min)
NaCl
41.1
26.0
23.1
1406
Hydrocortisone
189
118
107
9466
Tryptophan
80.1
56.7
51.3
1954
Treatment
a
Adrenalectomized rats weighing 150 to 160 g each were given single intraperitoneal injections of component in 0.85% NaCl. After 3 h and 20 min each animal was given intraperitoneally injection of 20 µCi of L-[14C]lysine (specific activity 80 mCi/mmol) in 1 ml od 0.85 NaCl. After 40 min the animals were killed, and extracts prepared by immunological precipitation. Reproduced with permission from R.T. Schimke et al., J. Biol. Chem. 240: 322-331 Copyright © 1965 American Society of Biological Chemists.
In contrast, the incorporation in the tryptophan-treated animals (column 4) is very small, about 40% more than the control value, despite the fact that the activity of the enzyme doubled (compare tryptophan and control values in columns 1, 2, and 3). Does the increase in available enzyme result from a decrease in its breakdown? This question can be examined experimentally by observing the temporal disappearance of the label from the previously labeled tryptophan pyrrolase molecule. The results of these experiments are shown in Fig. 3 (Schimke et al., 1965). After a single injection of [14C]leucine, some of the animals were sacrificed at the times shown on the abscissa. The radioactivity of the total unfractionated protein remains the same in the controls (Fig. 3a, squares) and the tryptophanhttp://www.albany.edu/~abio304/text/15part1.html (7 of 19) [3/5/2003 8:21:17 PM]
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treated animals (Fig. 3b). However, the decrease in radioactivity of the enzyme with time (Fig. 3a, triangles) is rapid in the controls, while the enzymatic activity remains constant (Fig. 3a, circles). The rate of synthesis must correspond precisely to the rate of breakdown. In contrast, in the tryptophantreated animals, the radioactivity does not decrease with time (Fig. 3b, triangles), while enzyme activity increases (Fig. 3b, circles). The increase in activity must result from inhibition of the degradation of the pyrrolase. Rat liver serine dehydratase exhibits a similar response to a dietary intake and the presence of the hydrocortisone (Jost et al., 1968). This enzyme catalyzes the dehydration and deamination of serine to produce pyruvate. The pyruvate is subsequently oxidized or used in the production of glucose. Administration of a mixture of amino acids to rats, results in introduction of the enzyme unaccompanied by changes in the total protein content of the liver. Glucagon, a polypeptide hormone that is secreted by the pancreas and favors gluconeogenesis, has a similar effect. Administration of glucagon or the amino acids produces an increase in the incorporation of [14C]-valine into serine dehydratase molecule, as shown by an immunological precipitation similar to that in the experiment of Table 1. In contrast, glucose inhibits production of the enzyme and increases its rate of breakdown (shown by experiments analogous to those of Fig. 3).
Fig. 3 Effect of L-tryptophan administration on loss of tryptophan pyrrolase prelabeled with L[14C]leucine. The values represent total enzyme activity present in the combined extracts from two animals
. Counts per minute represent total radioactivity present in protein precipitated by the
or in total cellular protein from two livers. Reproduced with tryptophan pyrrolase antiserum permission from R. T. Schimke, et al., Journal of Biological Chemistry, 240:322-331 Copyright © 1965 American Society of Biological Chemists, Inc.
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II. THE DEGRADATIVE PATHWAYS The previous section showed how synthesis and degradation play an important role in the regulation of the concentration of the enzyme. This section will concentrate on the degradative processes. The degradation of proteins contributes to the turnover of the cellular components. Under basal conditions, i.e., at rest and with a sufficient food supply, protein turnover has been estimated to account for 15% of the energy expenditure of humans. In addition to its role in the regulation of enzyme concentration, proteolysis is also thought to function in removing abnormal proteins produced by mistakes in translation (Goldberg, 1972). Such proteins would fulfill no function, and their breakdown allows their use as a metabolic fuel. We have seen how degradation plays a role in the regulation of enzyme activity. In yeast, changes in the supply of nutrients, such as addition of glucose or removal of carbon sources (carbon starvation), irreversibly inactivate certain enzymes, and at least glutamine synthetase (Ferguson and Sims, 1974) is degraded. The enzyme activity returns after a return to the original conditions, but only if protein synthesis is not blocked by inhibitors such as cycloheximide. A. Degradation as a General Regulatory Mechanism In eukaryotes, the rate of proteolysis is as significant as the rate of synthesis. This conclusion is drawn from the demonstration that most enzymes have a characteristic lifetime. At steady state, the balance between synthesis and degradation is generally expressed as a half-time (t1/2). The t1/2, can be estimated from data such as those shown in Fig. 3a representing the decay of the radioactivity in the enzyme molecule (E). This rate, dE/dt, can be expressed as a function of the rate constant for synthesis ks (the rate of synthesis will not change if short synthetic periods are considered) and a degradation rate constant kd as shown in Eq. (2). dE/dt = ks - ksE (2) If the original radioactivity has been rapidly diluted by adding unlabeled amino acid (i.e., by a chase) ks = 0. Equation (9.2) can be integrated to the form loge E/E0 = -kdt (3) where Eo represents the total amount of radioactivity present at time zero. When half of the enzyme has decayed, the half-time can be expressed as shown by Eq. (4); kd can be readily calculated from a logarithmic plot of E/E0 as a function of time, where it would correspond to the slope.
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t1/2 = loge 2 / kd (4) The regulation of an enzyme through its turnover rate must be reflected in a characteristic t1/2 for the enzyme. Table 3 (Ballard, 1977) shows that enzymes indeed have characteristic half-times, in agreement with this idea. Therefore, proteolytic degradation seems to choose specific targets. What mechanism or mechanisms provide such precise specificity? It is possible to clarify these processes by examining the properties of known cellular proteinases (Barrett, 1980; Matern and Holzer, 1979). These enzymes differ significantly from each other, but many have a common characteristic: they must be activated or they need special conditions for their action. However, aside from the fact that they are frequently located at specific sites (e.g., microsomal or mitochondrial membranes, the Z band of the sarcomere), nothing suggests that they can act on certain proteins and not on others. An initial inactivation has frequently been found to precede the actual hydrolysis. There are indications that some enzymes are marked for proteolysis by phosphorylation. In other cases, other forms of modification, such as the inactivation by oxidation, may mark an enzyme for proteolysis (e.g., Fucci et al., 1983; Rivett, 1985). Most of the degradation of proteins follows complex and specialized pathways, which are discussed in some detail in the next section. Table 3 Characteristic Half-Times of Enzymes
t1/2 in vivo (days) Lactate dehydrogenase
6.0
Fructose bisphosphate aldolase
4.9
Glucose-6-phosphate dehydrogenase
1
Glucokinase
1
Phosphoenolpyruvate carboxykinase
0.3
Thymidine kinase
0.1
Ornithine decarboxylase
0.008
RNA polymerase I
0.05
Tyrosine aminotransferase
0.06
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Tryptophan oxygenase
0.08
Phosphoenolpyruvate carboxykinase
0.25
Acetyl-CoA carboxylase
2
Glyceraldehyde phosphate dehydrogenase
3.4
Arginase
4.5
From Ballard (1977 and 1978), Reproduced with permission from Essays in Biochemistry 13:1-37 copyright © 1977 The Biochemical Society, London.
B. Biochemical Pathways of Proteolysis A good deal is known about the biochemical pathways of protein degradation. Present evidence indicates that there are two distinct processes, one relatively unspecific, the other exquisitely specific. Multiple proteolytic pathways Perfusion of rat liver or incubation of cell cultures with a medium devoid of amino acids, induces an increase in the breakdown of proteins. This enhancement of proteolysis can be blocked by supplementing the medium with amino acids (Woodside and Mortimore, 1972) or by introducing insulin (Mortimore et al., 1973) or some other growth factor. The breakdown of a number of components after endocytosis has been shown to occur in lysosomes (Chapter 9). Therefore, a general role of lysosomes in proteolysis would not be surprising. In fact, proteolysis, which accompanies amino acid deprivation, correlates well with the enlargement of lysosomes and the presence of autophagic vacuoles (Mortimore and Schworer, 1977), i.e., vacuoles that enclose cellular structures and function in their digestion. Conversely, these events are prevented by the presence of amino acid supplements. How can this problem be studied in more detail? As many cell organelles continue to function after isolation, it would seem possible to examine lysosomal activity after isolation of the vesicle. Under a variety of conditions, including amino acid deprivation and amino acid supplementation (Mortimore and Ward, 1981), lysosomes isolated from amino acid-deprived animals release amino acids or acid-soluble material at a rate compatible with the overall protein breakdown (Mortimore et al., 1973). Despite this observation, it is difficult to imagine that lysosomes are responsible for the very specific breakdown of the rapidly turning over enzymes we discussed. In contrast, it would be easy to imagine the involvement of lysosomes in the nonspecific hydrolysis of protein at a lower rate, in response to a generalized signal http://www.albany.edu/~abio304/text/15part1.html (11 of 19) [3/5/2003 8:21:17 PM]
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(however, see exceptions discussed below). The fate of these two kinds of proteins could be followed after a short pulse of a radioactive amino acid. Such a pulse should predominantly label the rapidly turning over proteins. A more prolonged pulse would label both kinds of protein. The view that there are two different processes is supported by the finding that inhibitors of lysosomal proteases, which have little or no effect on the proteolysis of shortlived or abnormal proteins (Neff et al., 1979), do block the proteolysis of the longer-lived proteins. Furthermore, a temperature-sensitive mutant has been found for one of the enzymes of the ubiquitin conjugation system (Finley et al., 1984). Reactions involving ubiquitin correspond to a second proteolytic pathway and, as we shall see later in this section, are responsible for the rapid turnover of proteins. In the mutant, at a permissive temperature, 70% of the rapidly turning over proteins are degraded in 4 h (Ciechanover et al., 1984). However, only 15% of this fraction is degraded at the nonpermissive temperature. The effect of protease inhibitors, which inhibit lysosomal proteolysis, is shown in Table 4 (Neff et al., 1979). The experiment was carried out with cultured hepatocytes. Part A of the table shows the appearance of [3H]-labeled material in the medium of cells that have either been labeled for long periods (column 1), pulse-labeled with [14C]-leucine (column 2), or pulse-labeled with an analog that will be incorporated to form an abnormal protein (column 3). The inhibitors have no effect on the pulse-labeled proteins or abnormal proteins, but inhibit the proteins that have been labeled for a long period. Although the effect is small, it is significant. Table 4B shows a much more marked effect for some of the inhibitors - as much as 54% inhibition. In addition, the effect is not additive, suggesting that the inhibitors are acting on the same mechanism. The amount of inhibition is not complete, possibly because of permeability barriers to the inhibitors. More significantly, the inhibitors failed to inhibit the breakdown of defective protein, even when its concentration was sharply increased by prolonged incubation. The results are therefore consistent with the presence of two independent pathways: one for the slowly turning over proteins broken down by lysosomes and the other for the breakdown of rapidly turning over proteins including abnormal proteins (however, see Gronostajski et al., 1985). Three pathways of lysosomal proteolysis have been recognized. Microautophagy takes place in wellnourished cells and is non-selective. The lysosomal membrane invaginates at multiple locations (Marzella and Glaumann, 1987) and internalizes proteins which are then digested by lysosomal proteases. Macroautophagy (Seglen et al., 1990) which is induced in cell cultures, for example, by removal of growth factors, is similarly nonspecific. Autophagic vacuoles are formed in the cytoplasm in which newly formed membranes sequester cytoplasmic components. These are then exposed to the lysosomal hydrolytic enzymes. An additional pathway, induced by serum deprivation in confluent cultured cells in which growth is arrested, is selective. In this mechanism, proteins with a specific sequence of five amino acids are imported into the lysosomes (Dice et al., 1990) and subsequently digested. Table 4A Effects of Protease Inhibitors on Degradation of Different Classes of Cellular Proteins
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Normal
Inhibitor
Anaolg containing
(1)
(2)
(3)
Chronically labeled
Pulse labeled
Pulse labeled
Protein degraded/ (% of total) None
1.65±0.13
21.2±0.7
26.7±2.6
Percent inhibition Leupeptin
21
0
0
Chymostatin
27
0
0
Antipain
18
0
0
Pepstatin
0
-----
-----
Reproduced by permission from Neff et al., J. Cell. Physiol. 101: 439-458. Copyright © 1979 Alan Rl Liss, Inc.
Table 4B Lack of Additivity of Inhibitors on Protein Degradation
Inhibitor None Leupeptin Chymostatin Antipain Leupeptin + chymostatin Leupeptin + chymostatin + antipain
Protein degraded/h (%of total)
Inhibition (%)
3.32±0.11 2.62±0.17 1.72±0.09 2.97±0.16 1.54±0.08 1.72±0.05
----21 48 11 54 48
Reproduced by permission from N. Neff et al., J. Cell. Physiol. 101: 439-458. Copyright © 1979 Alan R. Liss, Inc.
The ubiquitin pathway The nonlysosomal pathway has been partially elucidated by use of a special strategy (Gronostajski et al., 1985). All protein breakdown in the cell requires energy. Therefore, proteolysis can be arrested in extracts by the simple expedient of leaving ATP out of the incubation medium. This would allow the fractionation and subsequent reconstitution of the system, which can then be activated at will by the http://www.albany.edu/~abio304/text/15part1.html (13 of 19) [3/5/2003 8:21:17 PM]
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addition of ATP. Reticulocyte extracts have proved particularly useful because they synthesize hemoglobin almost exclusively and also possess a very active proteolytic system to dispose of abnormal globin. This approach can best be illustrated by showing the results of the original experiments which defined the ubiquitin proteolytic pathway and were carried out with the reticulocyte extract (Table 5) (Ciechanover et al., 1978). The degradation of [3H]-globin, previously labeled by incubation of the intact cells with [3H]-labeled amino acids, provides a measure of proteolytic activity. As shown in line 1, ATP is required for proteolysis. The two fractions derived from the extract, fractions I and II, have little activity when alone, as shown by lines 2 and 3. However, the two together reconstitute the proteolytic system as long as ATP is present, as shown in line 4. Further extraction of fraction I demonstrates the presence of a heat-stable factor with a molecular weight of 10 kDa (Ciechanover et al., l980a) and containing 76 amino acids. In the complete system, this low molecular weight component is found to bind to protein when the system was incubated in the presence of ATP and fraction II (Ciechanover et al., 1980b). The binding is covalent, since the association is resistant to severe treatment such as heat denaturation, exposure to acid and alkali, or exposure to a chemical reducing agent. The heat-resistant protein corresponds to a form of ubiquitin containing two extra glycine residues. Several molecules of this factor bind a higher molecular weight protein. The ubiquitin attaches through its carboxyl terminal to the amino groups of the lysines in the higher molecular weight proteins. Table 5 Resolution of the ATP-Dependent Cell-Free Proteolytic System into Complementing Activities Degradation of [3H] globin (%/h) Enzyme fraction
-ATP
+ATP
1.5
10.0
2. Fraction I
0
0
3.Fraction II
1.5
2.7
4. Fractions I and II
1.6
10.6
1. Extract
Reproduced by permission from Ciechanover et al., 1978.
When ATP is removed from the extract, covalent binding of diglycine ubiquitin to proteins ceases. However, the extract continues to break down the protein portion of the protein-ubiquitin complex (Hershko et al., 1980). In this fashion, ubiquitin is regenerated and, in the presence of ATP, can be used again (Ciechanover et al., 1984). A version of this cycle is illustrated in Fig. 4 (Hershko, 1988), where E1, E2, and E3 correspond to a family of enzymes required for ubiquitin ligation and protein breakdown. In order to be efficiently degraded, the proteins targeted for destruction must be polyubiquinated. An http://www.albany.edu/~abio304/text/15part1.html (14 of 19) [3/5/2003 8:21:17 PM]
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additional factor, E4 (UFD2 in yeast) (Koegl et al., 1999) is required for polyubiquination. Conjugation of proteins to ubiquitin has been observed in many mammalian tissues (e.g., Ciechanover et al., 1984), so the ubiquitin-proteolytic system seems to be a general mechanism not limited to reticulocytes. Furthermore, mutations in two of the three yeast genes coding for E2-proteins greatly reduce protein degradation (Seufert and Jentsch, 1990). Deletion of all three is lethal (Seufert et al., 1990). It follows that the ubiquitin-linked proteolysis system plays a vital role.
Fig. 4 Proposed sequence of events in ubiquitin-mediated protein breakdown. Ub, Ubiquitin; CF1, CF2, and CF3, conjugate-degrading factors 1-3, respectively; E1, E2, and E3 are enzymes. Reproduced with permission from A. Hershko, Journal of Biological Chemistry, 263:15237-15240. Copyright © 1988 American Society of Biological Chemists, Inc.
Despite the important advances in our understanding of the proteolytic degradation pathways, the precise role of the ubiquitin system and the mechanism that imparts specificity is still not entirely clear. The multiplicity of E2s and E3s may have a role in providing specificity to the protein degradation system. In Saccharomyces cerevisiae there are thirteen different E2s. In some cases, the E2s can transfer the ubiquitin directly to the substrate. More generally the transfer is carried out by E3s, the ubiquitin-protein ligases. There are several kinds of E3s that differ significantly in sequence and mechanism, and may be the ones capable of most substrate recognition. Some E3s facilitate transfer form E2s to substrates. Others provide thioester intermediates. At least some of the E3 are in large multicomponent complexes (see Hochstrasser, 1996). Specificity of the E3 ligases is provided by the presence of many substrate specificadapters that recruit the proteins to the core ubiquitination complexes. There are several reports of E3 ubiquitin ligase complexes containing adapters (e.g., see Peters, 1998): the SCF, APC and VCB families of complexes (Kamura et al., 1999; Skowyra et al., 1999 and Stebbins et al., 1999).
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The SCF family complexes contain adapter subunits called F-boxes. They recognize different substrates through specific protein-protein binding domains (Bai et al., 1996). The presence of the F-box containing proteins in protein data bases, suggests that many proteins may be targeted for degradation by the SCF pathway. In fact, cyclins, CDK inhibitors and transcriptional regulators have been found to be substrates of SCF complexes (see Patton et al., 1998; Maniatis, 1999). APC is a second kind of E3 ligase complex that uses different adapters for different substrates. Their substrates include cyclins and other proteins involved in the cell cycle (see Peters, 1998). The VCB complex contains the von Hippel-Lindau tumor suppressor protein (VHL) and associated proteins. At this time, a role of this complex in E3 ligase is suspected but not yet established. The VCB complex recognizes proteins that contain a SOCS-box (see Starr and Hilton, 1999). A new aspect of the functioning of the ubiquitination system is provided by recent developments involving the Nedd4 protein family. Nedd4 is a gene expressed in various tissues (e.g., Kumar et al., 1997). At this time, members of the Nedd4 family have been found in yeast, mouse, rat and human. The Nedd4 proteins regulate a variety of functions. A group of proteins of the Nedd4 family (see Harvey and Kumar, 1999) are ubiquitin ligases with a Ca2+-lipid-binding domain (C2 domain) and three repeats of approximately 40 amino acids, the WW domains known to be protein-protein binding modules (see Sudol, 1996; Staub and Rotin, 1996). In addition, the carboxy-terminal region of Nedd4 was found to be similar to the oncoprotein E6-associated protein (E6-AP) which is an E3 ubiquitin ligase involved in the ubiquitination of the protein p53 (Scheffner et al., 1993; Scheffner et al., 1995). The epithelial Na+ channel is down regulated by Nedd4 probably by ubiquination (Goulet et al., 1998). In humans, malfunction in this regulation leads to hypertension (Liddle's syndrome). The equivalent protein in Saccharomyces cerevisiae (Rsp5p/Npi1p) has varied functions. It is involved in the ubiquitin mediated regulated turnover of various permeases (see Harvey and Kumar, 1999). Rsp5p/Npi1p ubiquitinates RNA polymerase II (Huibregtse et al., 1997). Human nEDD4 and Rsp5p are needed for the hormonal activation of transcription by progesterone and glucocorticoid receptors independently from the ubiquitin-ligase function (Imhof and McDonnell, 1996). Specificity must also be determined by some characterstic of the protein to be degraded, such as a destruction signal or domain recognized by the ubiquitin-protease system. In some cases, the stability of the protein seems to be determined by the amino acid present at the amino terminal (this has been termed the N-end rule, see Varshasky, 1997). In contrast, the signal for degradation of the large subunit of RNA polymerase II of yeast, the repeat sequence SPTSPSY, resides at the carboxy terminal (Huibregtse, et al., 1997). These are probably recognized by the WW domains an ubiquitin ligase (e.g., Wang et al., 1999). Some proteins have domains similar to ubiquitin and they have a short half life (e.g., Watkins et al., 1993). More frequently, destruction boxes (e.g., RIALGSLTD in yeast uracil permease, Galan et al., http://www.albany.edu/~abio304/text/15part1.html (16 of 19) [3/5/2003 8:21:17 PM]
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1994) appear to be responsible. These boxes are short stretches that are strongly conserved in proteins degraded by the system. One of these boxes corresponds to a stretch of 27 amino acids (Treier et al., 1994). Another sequence recognized by the system contains proline, aspartate, glutamate, serine and threonine (the PEST region; Roger et al., 1986). Removal of these sequences leads to partial stabilization. In contrast to these short recognition sequences, the signal for the mating type transcription factor α2 is the surface of the folded protein corresponding to a region of about 60 amino acids predicted to form an amphipatic helix and containing a hydrophobic face (Johnson et al., 1998). Some of the proteins degraded by the 26S proteasome and the degradation signals are listed in the first column of Table 6. What protease system degrades proteins ligated to ubiquitin? One of the ATP-dependent proteases, a 1,300 kDa complex, the so-called 26S proteasome, degrades these proteins (Hough et al., 1987). A 20S multicatalytic protease complex that has been studied independently, the proteasome, is part of the 26S proteasome. In yeast, mutational alteration of the proteasome causes accumulation of ubiquitin-protein conjugates (Heinemeyer et al., 1991). Deletion of the corresponding genes is lethal (Fujiwara et al., 1990; Heinemeyer et al., 1991), confirming its important role in the ubiquitin-protease pathway. In many cases, phosphorylation of cytoplasmic proteins has been shown to be needed for ubiquitination and degradation via the proteasomes, for example, in the case of cyclins and transcriptional regulators (e.g., Yaglom et al., 1995; Chen et al., 1995). In yeast, the degradation of integral membrane proteins also requires phosphorylation (e.g., Medintz et al., 1996). Although not demonstrated as clearly, phosphorylation also seems to be required for ubiquitination of integral membrane proteins in mammalian systems (e.g. Cenciarelli et al., 1996). In summary, it seems that two different pathways operate in proteolysis. A generalized pathway involving all proteins operates through the action of lysosomes. This pathway can be stimulated by major switches, such as nutritional step-down or insulin. The proteins broken down in this pathway are those with a long half-life. Proteins with a short half-life, including defective proteins, are broken down in an alternative pathway that involves primarily covalent binding to ubiquitin. There may be additional ubiquitin-independent pathways of degradation (McGuire et al., 1988). The ubiquitin-conjugating system has actually been implicated in many important and diverse regulatory functions (see Hershko and Ciechanover, 1998) only some of which are related to protein degradation (see Chapter 7), with possible roles in gene expression, regulation of enzyme activity, the assembly of ribosomal proteins, DNA repair, yeast sporulation, regulation of the cell cycle (see Chapter 8) and response to stress. In the initial steps of the immune response, the antigen is partially cleaved. This cleavage involves proteasomes (Michalek et al., 1993; Niedermann et al., 1995). In some cases, ubiquitination is not a signal for degradation. Certain ribosomal proteins are attached to ubiquitin at their amino terminal. In this case, ubiquitin is thought to function in the folding of the proteins (Finley et al.,
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1989). In addition, in higher eukaryotes, a portion of the histones (see Chapter 2) are monoubiquitinated and the ubiquitin, in this case, is not a degradation signal. Monoubiquitination marks a protein at the cell surface for endocytosis in both Saccharomyces cervisiae and in mammals (see Hicke, 2001). It also has a role in regulating the endocytotic machinery (see Chapter 9). In a number of cases, a special role of the ubiquitin system is indicated by indirect evidence, e.g., the synaptic development at the Drosophila neuromuscular junction was found to be a ubiquitin-dependent mechanism ( DiAntonio et al., 2001). Overexpression of the deubiquitination enzyme fat facets in Drosophila leads to an increase in synaptic boutons and branching and a breakdown in synaptic function. The results suggest a balance between positive and negative regulators of ubiquitination in the development of synapses. Ubiquitin binding domains have been identified in many proteins, although the function of these is not always clear (see Buchberger, 2002). The UBA domain of about 40 amino acid residues has been found in some proteins, some of which are thought to act as inhibitors of polyubiquitination. The ubiquitin interacting motif (UIM or LALAL-motif) of 20 residues has been found in the 20S subunit of proteasome, as well as in proteins involved in ubiquitin metabolism and receptor mediated endocytosis. Several proteins, the ubiquitin-like proteins (ULPs), have sequence similarity to ubiquitin and can also covalently attach to proteins (see Yeh et al., 2000). They require enzyme complexes equivalent to E1, E2 and E3 for their binding to target proteins. The ULPs include UCRP/ISG15, Sentrin (also called small ubiquitin-like modifier, SUMO), NEDD8 (in yeast the equivalent protein is the related to ubiquitin 1, Rub1) and Apg12. Their function is not always clear since this is a relatively new focus of research. The amino acid sequences of all the Sentrins (Sentrin-1, -2, and -3). are the same in all mammals studied and Sentrin homologues have been found in many other organisms. Sentrinization does not serve as a degradation signal but rather targets proteins to different cellular compartments (e.g., nuclear body, NPC). Sentrinization and desentrinization play a role in cell cycle progression. Sentrins conjugate mostly with nuclear or nuclear envelope proteins among these homeodomain-interacting protein kinase 2 (HIPK2), p53 and RanGAP1, a major regulator of the Ras-like GTPase Ran. Sentrinization is required for RanGAP1 localization in nucleopore complex. Ran is the small GTPase that plays an important role in both nuclear import and export (see Chapter 5). Sentrins are also conjugated to non-nuclear proteins. among these, I κ B α, the inhibitor of the transcription factor NF-κB (Desterro et al., 1998) and in addition, the glucose transporters, GLUT1 and GLUT4 (see Chapter 19). Sentrinization decreases GLUT1 and increases GLUT4. The latter is translocated from the cytoplasm to the plasma membrane following the action of insulin. The binding of SUMO-1 to transcriptional factors is thought to have a role enhancing or decreasing transcriptional activity of factors and in subcellular localization (e.g., Ross et al., 2002) The neural precursor cell-expressed developmentally regulated (Nedd8) is a ULP of 81-amino acids which conjugates to a large number of nuclear proteins, the cullins. In yeast Rub1 binds to Cdc53, a 94 kDa protein required for the G1-S progression. Two cullins conjugated to Nedd8 are components of the http://www.albany.edu/~abio304/text/15part1.html (18 of 19) [3/5/2003 8:21:17 PM]
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ubiquitin E3 ligase. Nedd8 can form polymers which are thought to target proteins to proteasomes. The protein NUB1 is a strong down-regulator of Nedd8 by recruiting Nedd8 containing complexes for proteasomal degradation (Kamitani et al., 2001). Nedd8 modification of the Cul-1 component of the a ubiquitin-ligase enzyme complex, SCF, is important for function of SCF in the ubiquitination of I κ B α. The Ubiquitin cross-reactive protein (UCRP) targets substrates to the cytoskeleton (Loeb and Haas, 1994). The role of the ubiquitin-like proteins has just begun to be explored and many more may be found. Go to Part 2 REFERENCES Search the textbook
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Back to Part 1
Proteasomes We have seen that protein degradation is an important mechanism in regulating enzyme activity. Furthermore, the degradation of proteins is required for vital cell functions such as cell division and changes in transcriptional, translational and developmental programs, and elimination of defective proteins (see Patton et al., 1998). The primary process for protein degradation is through the proteasome pathway (e.g., see Hochstrasser, 1996). In view of the very important role of proteasome in the functioning of cells, it is not surprising to find that drugs that inhibit proteasomes induce cell cycle arrest and apoptosis in cancer cell lines (Orlowski et al., 1998; Adams et al., 1999) by unknown mechanisms. The sparing of inhibitors of cell division (see Chapter 8, Section IIID) may play a role in this effect. As discussed below (Section IIIB), the degradation of mRNA is another important regulatory mechanism. The process by which degradation of RNA takes place is beginning to be understood. In part, it involves a complex, the exosome. The degradation of cytokine mRNAs containing AU-rich elements in the 3' untranslated region, appears to involve, in some way, proteasomes (Laroia et al., 1999). Some of the roles of proteasome mediated proteolysis are summarized in the second column of Table 6 (Hilt and Wolf, 1996), which lists a variety of known substrates of the 26S proteasome (column 1), their function (column 2) and their degradation signal (column 3). Table 6 Substrates and physiological role of the 26S proteasome (modified from Hilt and Wolf, 1996). Reproduced by permission.
Substrate protein
Cellular function
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Degradation signal
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Metabolic adaptation Ornithine decarboxylase
Metabolic enzyme; gluconeogenesis
?
? Fructose-1,6bisphosphatase Gcn4
Metabolic enzyme; gluconeogenesis Transcriptional activator; amino acid and purine synthesis
PEST sequences
Repression of mating type specific genes
Degradation box
Cell differentiation MAT α2 repressor
Gα
GTP-protein; signal transduction
N-end rule
Cell cycle control/cell growth Cln2
G1 cyclin; control of cdc28 kinase
PEST sequences
PEST sequences
Cln3
G1 cyclin; control of cdc28 kinase S-phase cyclin; control of cdc28 kinase
Destruction box
Clb5 Mitotic cyclin; control of cdc28 kinase Clb2
Sic1
Destruction box
CDK inhibitor; inhibition of Clb5-cdc28 complex
?
Protein kinase; meiotic arrest in oocytes
Amino terminal, 2nd amino acid
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c-Mos
Transcriptional activator; signal transduction
δ-domain
Tumor suppressor; cell cycle pause
?
Transcriptional regulator; immune and inflammatory response
?
c-Jun
p53 Stress response NFκ-B
? I-κB
Inhibitor of NF-κB
Removal of waste Canavanyl proteins Fas2 CTFR
Undefined α-subunit of fatty acid synthase
?
Ion pump; Cl- transport across plasma membrane
The proteasomes are located in the cytoplasm and the nucleus (Hügle et al., 1983, Peters et al., 1994), as shown by immunofluorescence localization (see Chapter 1) in diverse organisms and cell types. They are found in the form of 20S and 26S particles [most accurately the larger unit is 30.3S (Yoshimura et al., 1993) but not currently used in the nomenclature]. S refers to the sedimentation coefficient in the ultracentrifuge. The 20S particle is also part of the 26S proteasome. However, the 20S particle may function independently as well, although this is not established. The 20S particle is also present in complexes other than the 26S proteasome (Peters, 1994), arguing for a separate and independent role. The http://www.albany.edu/~abio304/text/15part2.html (3 of 39) [3/5/2003 8:21:34 PM]
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26S proteasomes degrade ubiquitinated proteins only, whereas the 20S proteasomes do not degrade ubiquitinated proteins but rather unfolded proteins (Wenzel and Baumeister, 1995). In vitro, the 20S proteasome has been shown to degrade only certain denatured or oxidized proteins (see Grune et al., 1998). The 20S proteasome recognizes amino acid residues that are exposed during the conformational rearrangement accompanying the oxidative denaturation, in particular, hydrophobic residues that are usually shielded. The 20S proteasome has a molecular mass of 700 kDa and is constituted of many low molecular weight components arranged in a stack of four rings, each of seven subunits. A reconstruction is shown in Fig. 5 (Hilt and Wolf, 1996). Many recent studies have been carried out with the 20S proteasome of the archaebacterium Thermoplasma acidophilum, that resembles the eukaryotic proteasome and is thought to be its ancestor. This structure has been studied with X-ray crystallography with a resolution of 3.4 Å. The complex is a hollow cylinder, 11.3 nm in diameter and 14.8 nm in length (Löwe et al., 1995). The 20S proteasome is made up of two kinds of subunits. The 7 α-subunits are probably not catalytic and can assemble into a seven membered ring. The 7 β-subunits cannot assemble by themselves but have catalytic activity. The α and β components arrange themselves in a stack of 4 rings, α on the outside and β on the inside. A similar structure was shown by X-ray diffraction of the bovine (Morimoto et al., 1995) and yeast (Groll et al., 1997) 20S proteasomes.
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Fig. 5 3D structure of the 20S proteasome derived from X-ray, (a) top view (Cα atoms only) showing the 13 A entrance to the channel, (b) view of proteasome when cut along sevenfold axis, and (c) schematic representation of cut proteasome showing the three cavities and the proteolytic sites .Reproduced from Trends in Biochemical Science, vol. 21, Hilt, W. and Wolf, D.H., Proteasomes: destruction as a programme, pp. 96-102, copyright © 1996 with permission from Elsevier Science.
The 26S proteasome of eukaryotes is formed by 20S proteolytic core, capped by one or two 19S regulatory complexes (see Tanaka, 1998; Glickman et al., 1998a,b) and has a molecular mass of 1,700 kDa. The 20S and the 19S complexes separate out when ATP is depleted, and ATP is required for their reassembly into the 26S particle. The regulatory complex, only found in eukaryotic cells, is formed by at least 17 polypeptides. The regulatory particle selects ubiquitinated substrates for translocation into the core particle. It can be dissociated into an 8 subunits subcomplex or lid, and a base that contains 6 ATPases of the AAA-ATPase (see below) family and connects the regulatory complex to the core particle
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(e.g., Glickman et al., 1998a,b). The eight subunit base activates the core particle to degrade peptides in a ubiquitin dependent manner. In order to degrade a protein (see Baumeister et al., 1998; Larsen and Finley, 1997; Gottesman et al., 1998), the 26S proteasome must be able to translocate it through a narrow channel for the protein to come in contact with the proteolytic chamber. In contrast to the bacterial proteasome, the interior of the yeast 20 S proteasome is accessible only through very narrow side entrances (Groll et al., 1997). The passage of the substrate can only take place if the protein is unfolded, a function that is chaperone-like (see below). In accordance with this view, the base alone or the whole 26S proteasome (in human and yeast 26S proteasome) were found to bind and refold a denatured protein (non-ubiquitinated citrate synthase) in an ATP dependent manner (Braun et al., 1999). The actual cleavage of the protein takes place by a N-terminal nucleophilic mechanism (see Brannigan et al., 1995). The polyubiquitin chain must be removed to allow the translocation of the unfolded substrate polypeptide into the 20S portion. The POH1 (also known as Rpn11 in yeast) subunit of the 19S complex, a Zn2+-dependent protease, is responsible for substrate deubiquitination during proteasomal degradation (other deubiquitinating enzymes (DUBs) are cysteine proteases) ( Yao and Cohen, 2002). Surprisingly the 19 S regulatory unit has been implicated in transcription. For example, two-hybrid assays and in vitro experiments have shown the binding of one of its components to a protein involved in the activation of transcription of the galactose pathway (Gal4p) ( Chang et al., 2001). In addition, yeast strains carrying alleles of two of the 19 S components exhibit elongation defects and in vitro transcription is inhibited by antibodies against one of these proteins. An elongation factor, immunoprecipitates with the 19S complex (Ferdous et al., 2001). Furthermore, the 19S complex has been shown to be recruited by the GAL promoter by the Gal4 activator upon induction by galactose (Gonzalez et al., 2002). We saw that covalent binding of ubiquitin to a protein substrate is generally needed for degradation by the 26S proteasome. Generally, the targeting requires the attachment of polyubiquitin so that the molecular weight of the ubiquitinated protein is much greater than that of the non-ubiquitinated variety. In addition, ubiquitin can also serve as a targeting signal for alternative degradation pathways. Ubiquitin targets certain proteins to lysosomes (Hicke and Riezman, 1996). Other mechanisms for targeting to the 26S proteasome must also exist because some proteins, such as ornithine decarboxylase, are degraded by the proteasome without ubiquitination (Murakami et al., 1992). Ornithine decarboxylase is targeted to the proteasome by non-covalent binding to a protein factor, the antizyme. The presence of polyubiquitin chains (Chau et al., 1989) and their targeting function was revealed relatively recently (Gregori et al., 1990). The ubiquitins are linked by an isopeptide bond (attached to the ε rather than the α amino group) between Lys48 of ubiquitin n and Gly76 of ubiquitin n+1. Mutation of Lys48 to Arg (K48R) is lethal in S. cerevisiae (Finley et al., 1994) and blocks β-galactosidase degradation in vitro (Chau et al., 1989). Furthermore, conditional expression of K48R inhibits the turnover of short-lived proteins and causes cell cycle arrest at G2/M. Although polyubiquitination seems to be the most important signal, single ubiquitin molecules can still
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serve as signals, but of lesser significance. Polyubiquitin may be the favored signal because it is bound more tightly to the proteasome, for example, octaubiquitin binds 90 times more tightly than diubiquitin (Piotrowski et al., 1997). One of the possible mechanisms of binding may involve hydrophobic patches from different ubiquitin moieties. Such a mechanism would permit regulating the degree of binding by adding or removing ubiquitin and, hence, changing the rate of degradation of a protein (see Pickart, 1997). Polyubiquitin can attach at other Lys sites. However, only Lys48 is essential (Spence et al., 1995). The polyubiquitins linked to proteins at other Lys sites have been shown to function in targeting. However, mutants (Lys to Arg) at these sites (Spence et al., 1995) do not show a major proteolytic defect, so that the ubiquitination of the Lys48 site must be considered the major targeting signal. Although proteasomes are the major agents of extralysosomal protein degradation, there have been some indications that another pathway is also present. For example, mouse lymphoma cells in culture, can become adapted to the presence of a proteasomal inhibitor (Glas et al., 1998). In fact, an additional large protease, a tripeptidyl peptidase (TPII) (Geier et al., 1999), has been found to co-purify with proteasomes. TPII is larger than the proteasome and may be able to substitute for some of the proteasome functions. A large protease complex distinct from proteasomes has also been demonstrated in the archaebacterium, Thermoplasma, the tricorn protease (Tamura et al., 1996). The tricorn protease is a complex of 720 kDa, involving six identical subunits that form a channel 8 nm in diameter. Polypeptides that are not properly folded, bind to chaperones or are ubiquitinated and degraded by proteasomes (see Wickner et al., 1999). When the two systems are insufficient to eliminate unfolded proteins, they form aggregates. A variety of prion (see Harris, 1999; Prusiner, 1998) and amyloid diseases (Martin, 1999) are associated with abnormal aggregation of proteins. Prion diseases are fatal neurodegenerative disorders of humans and animals caused by conformational conversion of a normal host glycoprotein into an infectious isoform without the intervention of nucleic acid. The amyloid diseases include a variety of disorders, such as Alzheimer's, Huntigton's, Parkinson's disease and others (see Martin, 1999). The chaperones are complexes involved in the proper folding of proteins (also discussed in Chapter 10 and in Section III below). They are represented by several families of ATP-dependent proteins that interact with a variety of proteins. Chaperonins are barrel shaped complexes organized as two stacked rings, each of seven to nine subunits (see Kessel et al., 1995). The internal chamber serves for recognition and sequestration of unfolded proteins. Other chaperones act as monomers in conjunction with a cochaperones (Bukau and Horwich, 1998). The alternative fate of misfolded proteins is to be degraded by ATP-dependent proteases, mostly the 26S proteasome complex. Chaperones recognize hydrophobic residues not usually exposed in native folded protein. As already discussed, proteasomes depend on the ubiquitin conjugating system for their selectivity. A perinuclear structure containing 20S proteasome machinery has been identified in mammalian cells in http://www.albany.edu/~abio304/text/15part2.html (7 of 39) [3/5/2003 8:21:34 PM]
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culture (Johnston et al., 1998; Wigley et al., 1999) and is particularly evident when unfolded proteins are produced in excess. (Johnston et al.), coined the term aggresome for this aggregate. Aggresome formation is accompanied by redistribution of the intermediate filament protein vimentin to form a cage surrounding a pericentriolar core of aggregated, ubiquitinated protein. Disruption of microtubules blocks the formation of aggresomes (Johnston et al., 1998). The structure is surrounded by endoplasmic reticulum and is adjacent to the Golgi (Wigley et al., 1999). It colocalizes with γ-tubulin, considered to be a centrosomal marker, and it is therefore thought to be associated with the centrosomes which may be acting as a scaffold (Wigley et al., 1999). Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins [misfolded protein is produced by transfection using vectors containing mutants of the cystic fibrosis transmembrane conductance regulator (CFTR)]. When the level of misfolded protein is high, the structure recruits the cytosolic pools of ubiquitin and proteasomal components. Degradation of membrane proteins Cell surface receptors and other integral proteins (e.g., growth factor receptors, channel proteins or transporters) that have been internalized by endocytosis can be recycled to the plasma membrane or degraded by the lysosomes (or vacuole in the case of Saccharomyces cerevisiae) (see Gruenberg and Maxfield, 1995). The choice of target, either the plasma membrane or the degradation pathway, offers a way by which the activity of the protein can be regulated. In the degradation pathway, late endosomes produce multivesicular bodies (MVB) by invagination of the surface membrane into its interior. Some of the receptors are sorted into these vesicles. Fusion of the MVB with the lysosomes delivers these receptors into the lysosomal lumen for degradation (Futter et al., 1996). The degradation of receptors is also discussed below (Section C) Ubiquitination has been found to act as a sorting signal in both the endosomal and the biosynthetic pathway of certain membrane proteins (see Hicke, 2001). In Saccharomyces cerevisiae and other eukaryotes, integral proteins have to be ubiquitinated, most generally monoubiquitinated, on specific residues in order to be taken up by endocyosis. The ubiquitin offers a 3-D internalization signal. Ubiquitination of proteins not normally internalized by this mechanism has been shown to lead to their internalization (e.g., Roth and Davis, 2000). The proteins taken up by this mechanism are degraded in lysosomes (or the vacuole in yeast) and not proteasomes (e.g., see Hicke 1999). Ubiquitination also has a role in controlling the machinery of endocytosis. For example, a growth hormone receptor (GHR) truncation mutant (which lacks ubiquitination sites) internalizes via a di-leucine motif and does not require ubiquitination to be taken up by endocytosis. However, ubiquitinating enzymes have to be present presumably to act on other proteins needed for the machinery of endocytosis (Govers et al., 1999) which may act on components of the clathrin machinery ( van Delft et al., 1997). Similarly, in Saccharomyces cerevisiae, a receptor chimera containing ubiquitin does not require ubiquitination for its internalization but requires ubiquitin-conjugating enzymes (Dunn and Hicke, 2001). http://www.albany.edu/~abio304/text/15part2.html (8 of 39) [3/5/2003 8:21:34 PM]
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Generally, the choice between recycling or the degradation pathway after internalization, depends on the ubiquitination state of the protein. The degradation requires ubiquitination ( Lee et al., 1999b). In the absence of ubiquitination the fate of the integral protein is to be recycled to the plasma membrane. In the case of the colony-stimulating factor-1 (CSF-1 receptor), a polyubiquitination is required for both endocytotic uptake and targeting to the lysosomes for degradation. In mammals, CSF-1 is involved in the survival, proliferation and differentiation of mononuclear phagocytic cells. Ubiquitination also plays a role in the fate of integral proteins packaged in the TGN. In Saccharomyces cerevisiae, the presence of the permeases at the cell surface are adjusted depending on nutrient availability. The tryptophan permease Tat2 is present constitutively and is regulated by degradation in the absence of sufficient nitrogen sources when it is transferred to the vacuole, a targeting that requires ubiquitination (Beck et al., 1999). An internal pool of Tat2 appears to be directed to the vacuole independently of the plasma membrane. The general amino acid permease Gap is induced when grown on a relatively poor nitrogen source and is targeted to the plasma membrane, where it is active in transport. When grown on a relatively rich nitrogen source, Gap is directed to the vacuole and is degraded. The transport to the cell surface requires monoubiquitination or no ubiquitination, whereas polyubiquitination targets the protein to the vacuole (Helliwell et al., 2001). In contrast to the degradation of integral membrane proteins of the plasma membrane, membrane proteins of the ER are degraded by the ubiquitin-proteasome system (see Plemper and Wolf, 1999). A member of the ubiquitin-conjugating enzyme Ubc6p of yeast has been found in the cytoplasmic face of the ER (Sommer and Jentsch, 1993). In contrast, mitochondria, chloroplasts and bacteria possess ATP-dependent proteases: the AAA-proteases which are embedded in the membranes. The AAA ATPases are also discussed in Chapter 11. AAA-proteases (see Langer, 2000) are present as large complexes of 850 kDa, made up of identical or at least very similar subunits of 70 to 80 kDa (e.g., Arlt et al., 1996; Leonhard et al., 1996). The AAAproteases have one or two transmembrane segments. In Saccharomyces cerevisiae, absence of AAAprotease function produces respiratory defects (Tzagoloff et al., 1994; Arlt et al., 1998). Apparently, the proteolytic activity is required to maintain a functional respiratory chain (e.g., Arlt et al., 1998). In humans, mutations of a mitochondrial AAA-protease, paraplegin, is responsible for a hereditary spastic paraplegia (Casari et al., 1998). The AAA-proteases degrade unfolded proteins as well as regulatory proteins (see Langer, 2000). In order to recognize proteins, AAA-proteases must have chaperone-like properties which apparently are present at the ATPase domain. The hydrolysis of ATP is essential for the degradation of substrates (e.g., Arlt et al., 1996). The possibility of a role of the AAA-proteases other than protein-degradation is also suggested by mutations in the FtsH E. coli protease which cause abnormal orientation of membrane proteins, overcome by the overproducation of chaperone proteins (Shirai et al., 1996).
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The action of the AAA-proteases appears to be regulated by an additionmal large complex of 2000 kDa, the prohibitin complex with assembles with the proteases and inhibits degradation of unassembled protein (Steglich et al., 1999) Caspases In addition to the proteolytic systems already discussed, the cysteine proteases (cysteinyl aspartate specific proteinases, caspases) play a major role in apoptosis (see Chapter 2). Apoptosis is the process of programmed death involved in development and the maintenance of multicellular organisms (see Jacobson et al., 1997, Thornberry and Lazebnik, 1998). The importance of caspases is shown by experiments with the nematode, Caenorhabditis elegans. Deletion or mutation of a single gene coding for a caspase abolishes the programmed death of 131 cells. Similarly, mice deficient in caspase-3 have defects in the apoptosis that normally accompanies the early development of mouse brain. Caspases have been found to selectively cleave proteins involved in the early stages of apoptosis (see Thornberry et al., 1997; Thornberry and Lazebnik). C. Degradation of Receptors As discussed in Chapters 6 and 7, a variety of receptors are responsible for the binding of certain hormones and growth factors. Their activation produces a cascade of events that have important results in the cell's function. The removal of receptors and ligands from the cell surface by endocytosis is part of the mechanism by which cells return to the unstimulated condition. The degradation of the protein is carried out either by lysosomes or proteasomes (see Chapter 9). One of the mechanisms for the internalization of receptors and other plasma membrane proteins depends on ubiquitination and has been discussed above.. We saw that membrane proteins of the ER are ubiquitinated and degraded by the proteasome pathway. A variety of cell membrane proteins are ubiquitinated (e.g., see Bonifacino and Weissman, 1998; Hicke, 1999), a signal for internalization into the endocytotic pathway and degradation in the lysosome (vacuole in yeast). In mammals, the lysosomal pathway is primarily responsible for the degradation, although at least one case involving proteasomes is known (see Bonifacino and Weissman, 1998, pp. 42 and 43). A typical case, the regulation of the epithelial growth factor receptors (EGFRs) can serve as an example of the targeting process. EGFRs are downregulated by the degradation mechanism. The EGFRs are sorted out by the MVB pathway where they are degraded by lysosomes or recycled to the plasma membrane (Carter and Sorkin, 1998) . After binding the ligand, the EGFRs are internalized quickly. A portion is degraded after delivery to the lysosome lumen. The marking for degradation depends on EGFR kinase, sorting signals in the EGFR tail and the ubiquitin ligase c-Cbl which supposedly polyubiquinates the EGFR (e.g., Felder et al., 1990, Kornilova et al., 1996; Yokouchi et al., 1999). Ubiquitination was found to be required for endosomal sorting and for later sorting for lysosomal degradation ( Katzmann et al., 2001). A 350 kDa complex (ESCRT-I) recognizes ubiquinated cargo and is involved on sorting it into the MVB vesicles.
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What domains recognize the ubiquitin signals? A domain of approximately 20 amino acids (the ubiquitin interacting motif, UIM) has been identified in several proteins and is suspected to act as a site for ubiquitin binding during the endocytotic process ( Hofmann and Falquest, 2001). How do ubiquitinated proteins avoid degradation by the proteasomes? In yeast, this is probably because a polyubiquitin chain at least four units long (Deveraux et al., 1994) is required for targeting to the proteasomes. In contrast, a single ubiquitin residue is sufficient to initiate endocytosis (Terrell et al., 1998). In animal cells [e.g., growth hormone (GH) and platelet-derived growth factor (PDGF) receptors], polyubiquitination of receptors does take place (Mori et al., 1992; Strous et al., 1996). However, it is not clear at this time whether the ubiquitin is attached to multiple or single lysine sites. Plasma membrane ubiquitination is positively regulated by phosphorylation. In yeast, in response to ligand binding, the phosphorylation activates both the ubiquitination and the internalization (e.g., Reneke et al., 1988; Hicke et al., 1998). In yeast, G-protein coupled receptors undergo ubiquitination dependent internalization. In mammals, a variety of tyrosine kinase or kinase linked receptors undergo ligand stimulated ubiquitination and internalization. Mammalian cells deficient in the enzyme of the ubiquitin-activating enzymes are impaired in internalization (Strous et al., 1996). Furthermore, the degradation of several receptors requiring ubiquitination is blocked by inhibitors of proteasome and lysosomal activity (Mori et al., 1995; Jeffers et al., 1997). Several observations suggest that the ubiquitination of proteins other then those being internalized play an important role. For example, the ubiquitination of the growth hormone (GH) receptor is not required for internalization. However, blocking internalization of the receptor also seems to prevent ubiquitination events (Govers et al., 1997). III. FOLDING AND QUALITY CONTROL Native proteins are in a folded configuration. Cells possess mechanisms of quality control which prevent the accumulation of defective and misfolded polypeptides (see Ma and Hendershot 2001; Ellgaard and Helenius, 2001). Inside cells, the chaperones mediate the folding of nascent proteins. The folding is coupled to ATP hydrolysis. In addition, chaperones have a role in the folding and degradation of misfolded or defective proteins. Protein misfolding and consequent aggregation can have dire consequences because misfolded polypeptides have exposed hydrophobic surfaces which interact with each other to cause aggregation. Chaperones are also discussed above in relation to the proteasomes and in Chapter 10 in relation to events occurring in the ER. Chaperones occupy a central position in the folding of proteins and in the process of degradation. Chaperones function cotranslationally on nascent chains as they emerge from ribosomes so that they are stabilized in a nonaggregated state. They can also act in the cytoplasmic or organelle environment (see http://www.albany.edu/~abio304/text/15part2.html (11 of 39) [3/5/2003 8:21:34 PM]
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Frydman, 2001). Chaperones recognize hydrophobic residues and unstructured backbone regions which are normally buried inside the native folded protein. Chaperones bind to substrates to prevent defective folding and either mediate folding or transfer the substrates to other chaperones, the chaperonins. The chaperonins (see Saibil and Ranson, 2002) are large, double ringed cylindrical complexes of about 800 kDa forming a central cavity in which a single protein chain is folded (e.g., see Frydman, 2001; Hartl and Hayer-Hartl, 2002). The multiple subunits of the chaperonins first bind to the hydrophobic regions of their substrate. Then the substrate is transferred to the central cavity where the folding takes place. Eukaryotes have group II chaperonins which contain eight orthologous subunits per ring. The chaperones Hsp70 and Hsp90 together with co-chaperones function to fold proteins in the cytoplasm (see Cyr et al., 2002, Table 1 for listing). Co-chaperones mediate the binding of denatured proteins to the chaperones. Sometimes Hsp70 and Hsp90 function sequentially to fold the same protein. The cochaperone Hop connects the two chaperones. When proteins are destined for degradation, the two chaperones bind to a set of co-chaperones that have a degradation function (see Hohfeld et al., 2001; Connell et al., 2001). Supposedly these co-chaperones bridge chaperones and components of the ubiquitinproteasome system. Short nascent polypeptides may fold without the intervention of chaperones. When acting cotranslationally, the chaperones mediate substrate binding cycles as the nascent polypeptide emerges from the ribosome. These cycles are coupled to the chaperone’s ATPase activity and involve a number of cofactor proteins. Some chaperones such as trigger factor or Hsp70 bind to the ribosome so that they can interact with the nascent chain. Longer polypeptides require the action of other chaperones. In some cases, the peptide is passed from Hsp70 to the Hsp90. Misfolded polypeptides are recognized and degraded by the proteasome system. As already indicated, the folding and the degrading pathways are thought to have some steps in common. Current thought assigns the role of partitioning between the folding and proteases to the kinetics of folding ( Wickner, 1999). However, the finding that the co-chaperone CHIP functions as an E3 complex , a ubiquitin ligase, indicates a possible regulation between these two pathways. E3 proteins are enzyme complexes responsible for ubiquitination (see above) and hence degradation via the proteasome pathway. Ubiquitination requires E1, E2 and E3 (see above) and sometimes E4 ( Koegl et al., 1999). After four or more ubiquitins are added, the protein is taken up by proteasomes and degraded. Many E3 enzymes contain the HECT domain of 350 residues. A cysteine residue accepts ubiquitin from E2. The HECT domain selects the protein to which ubiquitin is attached. Many E3 enzymes possess really interesting new gene (RING) domains or U-box domains which bind to E2 and the substrate. The RING family of E3 ubiquitin ligases are involved in the selection of misfolded proteins for degradation. Some of these have a role in quality control involving the ER. Some of the E3s recognize hydrophobic groups (e.g., doa10). Others recognize polar residues exposed in the lipid bilayer (e.g., Tul1). Another (Parkin) appears to recognize unfolded proteins. These regions serve as sorting signals for the endocytotic pathway and in
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yeast the proteins are degraded by the vacuole rather than the proteasomes. At the sites of protein synthesis such as the ER or the cytoplasm there are multiple quality control mechanisms. In the ER, the oligosaccharide chains are trimmed by glucosidases which monitor the folded state together with glycoprotein-specific chaperones such as calnexin, calreticulin and glucose transferase ( Ellgaard et al., 1999). When the folding is slow or the glyprotein is misfolded, the chain is trimmed to provide a signal for entry into the cytoplasm, followed by conjugation with ubiquitin and degradation by proteasomes (ER-associated degradation, ERAD) (see Fewell et al., 2001; Plemper and Wolf , 1999; Hohfeld et al., 2001). IV. REGULATION OF ENZYME SYNTHESIS In eukaryotes, the control of the synthesis of specific proteins may depend on a variety of regulatory mechanisms that are much more complex than those occurring in bacteria. Animal cells are thought to contain at least 1000 times more genetic information than bacteria. This genetic information is contained in the chromosomes, where DNA is combined with proteins, such as histones that may have a role in controlling transcription. The structure of the chromosome itself is likely to have an effect on its availability for transcription. The nuclear envelope may play a role in the release of mRNA and its modification after transcription. Furthermore, the mRNA used in translation is constantly being broken down, although this process takes place much more slowly in animal cells than in prokaryotes. The possible mechanisms of control in eukaryotic cells are depicted in Fig. 6 (Walker, 1977). One of the emerging threads in the control of metabolic reactions is the role of thiol-reactive proteins, such as thioredoxin. These proteins act through a variety of mechanisms including direct effects on enzyme activity, transcription (e.g., Akamatsy et al., 1997) and ubiquination (see Chapter 14). There are two special cases of regulation. Regulation in mammalian striated muscle is discussed below (see Section IIID). Neuronal plasticity is discussed in Chapter 22. A. Transcriptional Control Transcription of the appropriate genes may be repressed or activated, depending on the signal received. There are many indications that the level of certain enzymes, such as xanthine oxidase, is regulated by a transcriptional mechanism. Xanthine oxidase is an enzyme with a molecular weight of about 300 kDa and contains two flavin nucleotides. The enzyme acts in the pathway responsible for the degradation of purine rings and catalyzes the oxidation of xanthine to form uric acid, as shown in Eq. (5).
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(5) Using a spectrophotometer, the uric acid produced can be estimated by measuring the light absorbed at 292 nm, as done in the experiments represented in Fig. 7, Table 7, and Fig. 8. The optical density represented in the figures can be considered to be directly proportional to the amount of uric acid produced and is expressed per milligram of liver protein. In contrast, the results of Table 7 (Rowe and Wyngaarden, 1966) are expressed as specific activity of the enzyme, i.e., activity per milligram of purified enzyme.
Fig. 6 Transcriptional and posttranscriptional stages in the production of translatable mRNA in animal cells (Walker, 1977). Reproduced with permission from Essays in Biochemistry 13:39-69, copyright © 1977 by The Biochemical Society, London.
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directly. In in most mammals, uric acid is further degraded to produce urea. In the rat, the xanthine oxidase activity of the liver is increased by a high-protein diet (e.g., 23% protein) and decreased by a lowprotein diet (e.g., 8%). The effect of diet is shown in Fig. 7. Rats normally fed a 23% protein diet are switched at time zero to an 8% diet (first arrow). After 14 days on the low-protein diet, the animals are again placed on the high-protein diet (second arrow), and a subsequent increase in xanthine oxidase is observed. In Figs. 7 and 8, the xanthine oxidase activity is expressed in terms of optical density change (in relative units). The values represent the amount of uric acid produced in the test tube assay in relative units. As shown in Fig. 7 (first part of the curve), switching to a low-protein diet causes a 10-fold drop in activity. The return to a high-protein diet dramatically increases the level of the enzyme after a short lag. The enzyme activity after reintroduction of the high-protein diet is also shown in Fig. 8 (Rowe and Wyngaarden, 1966), curve 1. The results of Table 7 (Rowe and Wyngaarden, 1966) show that the increase in enzyme activity does not correspond to activation of preexisting molecules. Rats were injected with [14C]leucine 6 h before being sacrificed. Items 1 and 2 represent control animals that were maintained on 23% protein for 14 days, and items 3 and 4, represent results obtained with rats on an 8% protein diet. Items 5 and 6 represent animals treated as in items 1 and 4 but subsequently fed the high-protein diet for 12 h. The activity per milligram of enzyme (specific activity of the enzyme, column a) is the same regardless of physiological condition. Activation would be expected to increase the enzyme activity per milligram of enzyme. The incorporation of label into the enzyme molecule (column b) is the same for the control animals and the animals maintained on an 8% protein diet, although the xanthine oxidase activity is low. Hence, the decrease in the activity of xanthine oxidase probably corresponds to a higher rate of breakdown. In animals for which the low-protein diet was replaced after 14 days by 23% protein, there is an increase in radioactivity that roughly corresponds to the five-fold increase in enzymatic activity shown in Fig. 7. Therefore, the increase in enzyme activity appears to be the result of increased synthesis of the enzyme.
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Fig. 7 Induction of xanthine oxidase in rat liver. Data from Rowe and Wyngaarden (1966), expressed per milligram of liver protein. Reproduced by permission.
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Fig. 8 Effects of inhibitors of protein synthesis or RNA in the induction of xanthine oxidase. Data from Rowe and Wyngaarden (1966), expressed per milligram of liver protein. Reproduced by permission.
The results shown in Fig. 8 confirm that the increase in enzyme activity is the result of an increased enzyme synthesis. The controls, originally maintained on a diet containing 8% protein, are transferred to a 23% protein diet at 0 time. They exhibit an increase in xanthine oxidase activity as previously shown (curve 1). Puromycin (curve 3), 5-fluorouracil (curve 2), or actinomycin D (not shown) block the increase. Puromycin blocks protein synthesis, whereas 5-fluorouracil and actinomycin D block RNA synthesis. The control is therefore transcriptional. Similar results are obtained for the induction of rat liver serine dehydratase (Jost et al., 1968). Many other types of controls are likely to involve a mechanism at the level of transcription. For example, the synthesis by guinea pig peritoneal cells of one of the serum proteins associated with the inflammatory response (protein C4) seems to depend on a factor that switches on the production of C4. The response can be inhibited by actinomycin D (Cahoun and Hatfield, 1975). Similarly, purine biosynthesis in hepatoma cells in tissue culture is repressed by adenine (Martin and Owen, 1972). The derepression is http://www.albany.edu/~abio304/text/15part2.html (17 of 39) [3/5/2003 8:21:34 PM]
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sensitive to both actinomycin D and cycloheximide (the latter also blocks protein synthesis). Since the amount of mRNA coding for tryptophan pyrrolase parallels the amount of enzyme induced, the control is probably transcriptional (Schutz et al., 1975). A number of hormonal controls are thought to be exerted at the transcriptional level and some examples are shown in Table 8. Enzyme production is not always affected by the mechanisms controlling transcription. In some cases, the control is probably at a posttranscriptional level, as also shown in this table. An increase in specific mRNA in response to a hormone has been clearly demonstrated in the induction of yolk proteins by estrogen in rooster liver. DNA synthesized by reverse transcriptase (cDNA) from purified specific mRNA can recognize the mRNA in hybridization tests, where nucleic acid strands combine by virtue of their complementarity (see Chapter 1). The mRNA used as a template is not difficult to isolate by standard procedures, provided that it is present in the tissue in sufficient amounts. In vitro, translation systems can confirm the nature of the mRNA isolated. After production of the cDNA, the template RNA is removed by degradation in alkali, and DNA polymerase is used to replicate the missing complementary strand of the DNA duplex. The duplex inserted into a vector, either a bacterial plasmid or a phage, can reproduce multiple copies or clones in a bacterial host. Hybridization of the cDNA bound to a filter can then be used to recognize the appropriate mRNA. Table 7 Effect of Diet of Specific Enzyme activity and 14C-Labelled Amino Acid Incorporation in Xanthine Oxidase
Group
[optical density min-1(mg enzyme)-1]
Enzyme specific radioactivity [counts min-1 (mg protein)1]
2000
170
3260
195
2000
158
3180
200
Specific activity
Control (23% protein diet) 1 2 8% Protein diet 3 4
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8% Protein diet for 14 daysshifted to 23% protein diet for 12 h 2100
778
3245
900
5 6
Reproduced with permission from P.B. Rowe and J.B. Wygaarden, J. Biol. Chem. 241: 5571-5576. Copyright © 1966 American Society of Biological Chemists, Inc.
Administration of estrogen induces the production of egg yolk protein. Figure 9 (Wiskocil et al., 1980) shows various specific mRNAs as a function of time in the liver of roosters after a single injection of estrogen. The mRNA for serum albumin remains relatively unchanged (curve 1). However, the mRNAs for the yolk proteins apoVLDLII (curve 2) and vitellogenin (curve 3) are induced. Interestingly, after the initial synthesis they are also rapidly degraded suggesting that both transcription and degradation of the mRNA play a regulatory role.
Fig. 9 Levels of apoVLDLII mRNA, vitellogenin mRNA,and serum albumin mRNA during 10 days after http://www.albany.edu/~abio304/text/15part2.html (19 of 39) [3/5/2003 8:21:34 PM]
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primary stimulation with 17-estradiol. Absolute levels of serum albumin mRNA (
), apoVLDLII mRNA
( ), and vitellogenin mRNA ( ) after injection of hormone. The data are expressed as molecules of mRNA per nuclear equivalent of DNA. The theoretical accumulation curves (--) were calculated from data of Wiskocil et al., with permission, Proceedings of the National Academy of Sciences, 77:4474-4478, 1980.
Table 8 Hormonal Control of Macromolecular Synthesisa
Evidence for level of control
Hormone
Experimental system or target organ
Protein Hormones Growth Rat tissue (liver muscle and others) Insulin
Peptide hormones ACTH TSH LH Thyroxine
Steroid Androgen
Muscle
Adrenals Thyroid Ovaries Rat and amphibian liver
Accessory sex tissue
Uterus Estrogen
Transcriptional
RNA ↑(including mRNA), RNA polymerase ↑
Translational
In vitro stimulation of protein synthesis b
RNA ↑, RNA polymerase ↑
Actinomysin D inability to block some actions, RNA and ribosome interaction aidedb
Not clear RNA ↑ Not clear RNA ↑, including mRNA
b b b
RNA ↑, RNA polymerase ↑, ribosomes ↑ RNA ↑, actinomycin D inhibition, increased template activity of chromatin, appropriate RNA mimics the affect of hormone, RNA polymerase ↑
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Amino acid incorporation before increased transcription and with administration to cell-free systemb b
b
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Chick oviduct
Corticosteroids
Embryonic chick retina
Rat kidney cortex Kidney cortex Chick embryo Retina, liver
New class of RNA (hybridization) RNA polymerase ↑ Inhibitors of RNA or protein synthesis block effects, new mRNA appears for one induced enzyme Appearance of new mRNA RNA ↑ Specific RNA ↑ Increased template activity, hormone blocks mRNA synthesis Actinomycin D inhibition of hemoglobin synthesis RNA ↑ in chromosomal loci Actinomysin D inhibition of avidin synthesis, RNA polymerase ↑, new RNA made (hybridization)
b
New mRNA appears for one induced enzymec,d,e,
f f b b
g
In vitro simulation of cytoplasmic extract b
Ecdysone Progesterone
Thymus nuclei Erythoid cells
Calliphoralarvae
Chick oviduct
a Upward
arrow indicates increases B.W., Trans. N.Y. Acad. Scie. 31:478-503 (1969) cMoscona, A.A. et al., Proc. Natl. Acad. Sci. USA 61: 160-167 (1968) dReif-Lehrer, L. and Amos, H., Biochem. J. 106: 425-430 (1968) eSchwartz, R.J., Nature, New Biol. 237: 121-125 (1972) fCongote, L.F. and Trachewsky, D., Biochem. Biophys. Res. Comm.. 46: 957-971 (1972) gAbraham, A.D. and Sekeris, C.E., Biochem. Biophys. Res. Comm. 47: 562 (1971) bO'Malley,
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b
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Transcriptional control mechanisms seem to play a prominent role in the regulation of enzyme synthesis. As outlined in Fig. 6, a number of regulative events may occur after transcription. Several experiments indicate that the specific degradation of mRNA is significantly involved in the regulation (Styles et al., 1976). Conceivably, the rate of synthesis of an enzyme could also be regulated by changing the rate at which the translational events take place. The molecular mechanics of translation are discussed in Chapter 3, section III and some examples at the molecular level of posttranscriptional regulation in Chapter 3, Section IVB). The induction of tyrosine transaminase in rat liver or rat liver tumor cells (HTC cells) by adrenal steroid hormones or their analogs (e.g., dexamethasone phosphate), suggests that the control is exerted after the formation of the appropriate mRNA.
(6) The transamination of tyrosine is represented in Eq.(6). In this reaction, the amino group of the amino acid is transferred to α-ketoglutaric acid with the formation of β-hydroxyphenyl pyruvate and glutamic acid. The β-hydroxyphenyl pyruvate can be broken down by several oxidative steps that eventually lead to the formation of acetoacetate, which is metabolized by the mitochondria after the appropriate activation reaction. The regulation of tyrosine transaminase may shed some light on the physiological mode of action of the adrenal steroid hormones, as mentioned for tryptophan pyrrolase. The experiment illustrated in Fig. 10 (Thompson et al., 1966) shows that induction of the enzyme in HTC cells is triggered by the presence of dexamethasone phosphate. The inducer brings about a 20-fold increase in enzyme activity after a lag period of 1.5 to 2h. The induction occurs in the presence of actinomycin D (Tomkins et al., 1969). Immunological precipitation (as described in Fig. 1 for the tryptophan pyrrolase assay) demonstrates that the induction corresponds to actual formation of the enzyme and not to activation of a preexisting molecule (Granner et al., 1968). The degradation of the enzyme, which normally takes 3 to 7 h, seems to be unchanged by administration of the drug (Auricchio et al., 1969; Martin et al., 1969).
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Fig. 10 Kinetics of induction of tyrosine transaminase activity in HTC cells at 37C. ESA refers to tyrosine transaminase specific activity. Reproduced with permission from E. B. Thompson, et al., Proceedings of the National Academy of Sciences, 56:296-303, 1966.
Fig. 11 Model to explain the observed results in the regulation of tyrosine transaminase production (see text).
The data presently available permit the formulation of a model, shown in Fig. 11, which has some similarities to that presented for the lac operon of E. coli. However, it also has a number of significant differences. The enzyme is coded by gene GS and the repressor by GR (Fig. 11). Both genes are transcribed to produce the corresponding mRNAs (mRNAE and mRNAR, respectively) in steps 1a and 1b of Fig. 11. The two mRNAs can be translated to produce enzyme (E) or repressor (R) (steps 2a and 2b). http://www.albany.edu/~abio304/text/15part2.html (23 of 39) [3/5/2003 8:21:34 PM]
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The repressor acts by binding to the mRNA (reaction 5), thereby making it unavailable for production of the enzyme. In this model, the inducer (adrenal steroid) can complex with a protein repressor, thus removing its inhibitory ability to bind to the mRNA coding for the tyrosine transaminase (step 7). Normally, the mRNA repressor combination is degraded more rapidly than the free mRNA. The model assumes that the repressor and the mRNA corresponding to the repressor are labile (reactions 3b and 4b in Fig. 11). These features have been proposed on the basis of several experiments. The addition of cycloheximide to the HTC cells inhibits protein synthesis by about 97%. Naturally, this technique blocks induction, since the enzyme cannot be formed ( mutt and Kipnis, 1972). However, on washing the cells and adding the inducer (dexamethasone phosphate), the system is again capable of responding. If the inducer is added after washing, without pretreatment with cycloheximide, induction occurs after a lag period of 1.5 to 2 h (Fig. 12a curve 2) (Peterkofsky and Tomkins, 1968). The washing itself has no effect on the induction, as shown in curve 1. After the cycloheximide is washed off in the presence of inducer, the cells synthesize the enzyme (Fig. 12b). When the inducer is added after washing, the customary lag period occurs (curve 2). However, when the inducer and cycloheximide are present from the beginning, the synthesis of the enzyme no longer involves a lag period; it begins without delay (curve 4). In fact, synthesis occurs when the inducer, present in the original incubation medium together with cycloheximide, is washed away and is not reintroduced (curve 1). The cycloheximide alone has no significant effect on induction (curve 3). The accumulation of the capacity to produce the enzyme in the absence of protein synthesis suggests that it is the result of the accumulation of mRNA.
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Fig. 12 Level of tyrosine transaminase (TTA) activity after preincubation of HTC cells with or without various additions. Cells were preincubated for 1.5 h, washed with medium, and reincubated. (a) Curve 1 ( ), no preincubation addition, no reincubation addition; curve 2 (
), no preincubation addition, Dex addition at reincubation. (b) Curve 1 (
addition, no reincubation addition; curve 2 (
), preincubation CH + Dex
), preincubation CH addition, Dex addition reincubation;
curve 3 ( ), preincubation CH addition, no reincubation addition, curve 4 ( ), preincubation CH + Dex addition, Dex addition reincubation. Dex, dexamethasone phosphate; CH, cycloheximide. Reproduced with permission from B. Peterkofsky and G. M. Tomkins, Proceedings of the National Academy of Sciences, 60:222-228, 1968.
Fig. 13 Degradation of casein mRNA. Both control (curve 1) and experimental (in the presence of prolactin, curve 2) runs were in the presence of insulin and hydrocortisone. In the absence of prolactin, the half-life corresponds to 1.1 h, and in its presence to 28.5 h. Reproduced with permission from Guyette et al. (1979); copyright © 1979 by Cell Press.
A role of mRNA degradation in posttranscriptional regulation has been shown directly in a number of cases. Chapter 3 discusses the control of tubulin production by the level of tubulin itself. This mechanism operates through the degradation of its mRNA. The mRNA of breast cells is also regulated primarily by controlling the rate of its degradation. Casein is the major protein in milk secreted by breast cells. The secretion is hormonally controlled in part by prolactin. Cultured breast cells respond to the presence of prolactin by increasing the casein mRNA 100-fold. However, the nuclei of breast cells can increase the synthesis of casein mRNA only three-fold in response to prolactin -- apparently, the major effect is reduction of the degradation of mRNA. Results of an experiment showing this are presented in Fig. 13 (Guyette et al., 1979).
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In this experiment, cultured breast cells were pulse-chased with [3H]-labeled uridine in the absence (curve 1) or presence of prolactin (curve 2). The casein mRNA (ordinate) was estimated from the radioactivity hybridized to the corresponding cDNA probes (seeChapter 1). The points correspond to cell samples withdrawn at different times. The results show that prolactin blocks the degradation of casein mRNA. Various hormones alter the half-life of specific mRNAs, indicating that this control mechanism is common. Some of these hormones and the corresponding mRNA half-lives are shown in Table 9 (Shapiro and Brock, 1985). Table 9 Biological Systems That Exhibit Regulation of mRNA Stability
mRNA Half-lifea mRNA
Tissue
Regulatory signal
+Effector
-Effector
Vitellogenin
Xenopus liver
Estrogen
+E: 500 h
-E: 16 h
Vitellogenin
Rooster liver
Estrogen
+E: ~24 h
-E: <3 h
Ovalbumin, conalbumin
Hen oviduct
Estrogen, progesterone
+E: ~24 h
-E: 2-5 h
Casein
Rat mammary gland
Prolactin
+Pro: 92h
-Pro: 5h
Prostatic steroidbinding protein
Rat ventral prostate
Androgen
∆t1/2 ~30x
Lactate dehydrogenase, A subunit
Rat C6 glioma cells
cAMP, isoprotenerol, dibutyl cAMP
+Ipt: 2.5 h
-Ipt: 45 min
β-Interferon
Human fibroblasts
Poly(I.C)+cyclo
(IC)+ChX or (IC)+N.V., t1/2>12 h
(IC) t1/2<30 min
heximide or Newcastle virus Histones
HeLa cells
DNA replication
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Histones
Yeast
DNA replication
During replication ~15 min after ~5 min
Adenovirus 1A (9S), 1B (14S)
HeLa cells
Early/late infection
Late, 60-100 min; early, 6-10 min
L3 (ribosomal protein)
Yeast
mRNA overproduction
∆t1/2~2x
γ integrase (int)
γ infected E. coli
Early/late infection (terminator read through)
∆t1/2~10x
aAbbreviations:
E: estrogen; pro: prolactin; Ipt: isoprotenerol; IC: poly(IC); ChX: cycloheximide; NV: Newcastle virus.
Reproduced by permission from: D.J. Shapiro and M.l. Brock, Biochem. Action of Hormones 12 :139-172 (1985).
Obviously, the regulation of mRNA degradation must have a prominent role in gene expression. Some insight into this aspect is provided by studies carried out with Saccharomyces cerevisiae (see Herrick et al., 1990; Wang et al., 2002) which reveal that mRNA of housekeeping genes (i.e., those involved in the day to day tasks for survival of cells) have fairly invariant and prolonged life-spans. In contrast, the mRNAs whose half-lives differ in response to external signals have much shorter life-spans, as we have already seen (e.g., the case of casein mRNA shown in Fig.23). In the study of Wang et al., the rate of decay of mRNA was studied in a massive survey after blocking mRNA production by thermal inactivation of a temperature-sensitive RNA polymerase II. The individual mRNAs were recognized with DNA microarrays representing over four thousand genes (Wang et al., 2002). The half-lives were found to be variable, in the range of 3 more than 90 minutes. The decay rates of mRNAs of proteins present in stoichiometric complexes were very close. Physiological function was found to be most closely related to mRNA decay rates. Not surprisingly, malfunctions in the mechanisms that regulate mRNA stability have been implicated in disease (see Chen and Shuyu, 1995; Conne et al., 2000). What is responsible for the degradation of the RNAs? Two major pathways have been found. However, the rate of degradation of mRNA is determined by the mRNA molecules themselves and proceeds at the same rate irrespective of pathway. Specific sequences which affect the decay rates of mRNA have been identified in yeast by deletions and point mutations. Instability elements occur throughout the length of the transcripts. There is also evidence that the degradation may depend on the translational process.
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The major pathway of mRNA degradation in Saccharomyces cerevisiae first shortens the 3' poly(A) tail. This is followed by removal of the cap at the 5' end, followed by 5' to 3' degradation (see Caponigro and Parker, 1996). Sm-like (Lsm) proteins related to the Sm proteins, play a major role in decapping, whereas the Sm-proteins are active in splicing (e.g., Stevens and Abelson, 1999). Lsm and Sm proteins contain a common sequence (the Sm sequence). Mutations in seven Lsm proteins in yeast, block mRNA decapping. These proteins also co-precipitate with the mRNA decapping enzyme (Dcp1), a decapping activator and mRNA. The Lsm complex that functions in mRNA decapping is different from the U6-associated Lsm complex suggesting that Lsm proteins form distinct complexes affecting different mRNA-reactions (Tharun et al., 2000). A complex of 3'-to-5' exonucleases, the exosome, has been found to have a role in either processing RNAs to shorter forms or degrading them (e.g., see van Hoof and Parker, 1999). The exosomes are needed for maturation of 5.8S rRNA (Mitchell et al., 1996, Mitchell et al., 1997) that requires the removal of 140 nucleotides from the 3' end of an intermediate. In addition, the complex is active in the degradation of mRNA (Jacobs et al., 1998). Exosomes are present in the nucleus and in the cytoplasm (e.g., Allmang et al., 1999a; Zanchin and Goldfarb, 1999). In Saccharomyces cerevisiae, the exosome is formed by a minimum of 10 proteins (Allmang et al., 1999a). The nuclear exosome has an extra subunit. The molecular weight of the complex suggests that each subunit is represented once in the 300-400 kDa complex (Mitchell et al., 1997). Each subunit is thought to have 3'-to-5' exoribonuclease activity. Six of these are phosphorylytic enzymes: they attack phosphate during RNA hydrolysis, resulting in the production of nucleotides 5' diphosphates. Each of the subunits are essential for function, possibly because of a failure to assemble if one of them is missing: the inactivation of any one of them produces a failure of exosome function (e.g., Jacobs et al., 1998; Allmang et al., 1999a and b). It is also possible that each subunit carries out a specific step in the degradation (see van Hoof and Parker, 1999). Similar complexes are present in other eukaryotes, including humans. The system has been shown to operate under conditions in which the deadenylation-decapping mechanism is blocked. In addition, the 3'-to-5' mechanism has been shown to be present while 5'-to-3' degradation is proceeding. In yeast, the exosome pathway also requires the product of three other genes: SKI2, SKI3 and SKI8 (Jacobs et al., 1998). These may be involved in the assembly of the exosome or may modify the mRNA to facilitate interaction between the RNA molecules and the exosomes. Since Saccharomyces cerevisiae strains lacking both the 3'-to-5' and the 5'-to-3' RNA-degradation pathways cannot survive, it is likely that the two are the major RNA-degradation pathways of the cells. In yeast, unspliced pre-mRNAs are degraded in the nucleus in the 3'-to-5' direction by the exosome and in the 5' to 3' direction by an exonuclease (Rat1p), with the exosome pathway predominating (BousquetAntonelli et al., 2000). Defective pre-mRNAs are degraded. In addition, the degradation of normal premRNA takes place in competition with splicing and is physiologically regulated, for example, it is increased by glucose. Inhibition of pre-mRNA degradation increases pre-mRNAs and consequently spliced mRNAs. Therefore this pathway appears to be part of the machinery regulating transcription. Similar mechanisms may be present in mammalian cells since it has been observed that defective prehttp://www.albany.edu/~abio304/text/15part2.html (28 of 39) [3/5/2003 8:21:35 PM]
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mRNA are degraded mostly in the nucleus (see Hentze and Kulozik, 1999) A close regulatory interaction between transcription and degradation is also suggested by the finding that in yeast the mRNA deadenylases Crc4p and Caf1p bind to transcription factors (e.g., Chang et al., 1999; Tucker et al., 2001). In fact, the corresponding genes (CCR4 and CAF1) have been shown to affect the initiation of transcription of some genes . In vertebrates, similar mechanisms are thought to be operating. Deadenylation is one of the processes involved in mRNA degradation. A poly(A)-specific deadenylating nuclease (poly(A) ribonuclease) is involved in mammals and Xenopus (e.g., Martinez et al., 2000). Decapping is likely to be the next step since decapped mRNA intermediates have been isolated from mouse liver cells (Couttet et al., 1997). It is not clear however whether the rest of the mRNA is degraded by a 5'-to-3' or a 3'-to-5' exoribonuclease. In general, the poly(A) tails play a major role in mRNA degradation. The tails block mRNA decay by binding to the poly(A)-binding protein (PABP) (Bernstein et al., 1989). In addition, PABP binds to the initiation translation factor eIF4G which complexes to the cap-binding protein eIF4E which promotes translation (Gingras et al., 1998). These complexes block access to the deadenylation and decapping enzymes. The involvement of proteins active in the process of translation also suggest a role of translation on mRNA stability. In fact, inhibition of translation initiation destabilizes mRNA ( Schwartz and Parker, 1999), whereas inhibiting translational elongation favors mRNA stabilization ( Beelman and Parker, 1994) Cytokines, proto-oncogene and transcription factors mRNAs are rapidly degraded (see Chen and Shyu, 1995), a process requiring AU-rich elements (AREs) in the 3' untranslated region (3'UTR). Exactly how this happens is a matter of conjecture. However, the degradation depends on the binding of the AU-rich region by a AU-binding protein, AUF1 (also known as heterogeneous ribonucleoprotein D (hnRNP D) (Laroia et al., 1999). AUF1 complexes with heat shock proteins hsc70-hsp70, translation initiation factor eIF4G and poly(A) binding protein. eIF4G is displaced from AUF1 and then AUF1 is ubiquitinated. The AUF1 is degraded by proteasomes. Interference with any of these processes interferes with the degradation. The ARE-binding proteins also determine their subcellular localization and translation of the mRNA. Other sequence elements with similar functions are also known and some of these are located in the 5'UTRs (see Wilusz et al., 2001). Some proteins that shuttle between the nucleus and the cytoplasm have been found to have a role in the localization of the mRNA (which in many cases is translated at a specific cytoplasmic target site), its translation and its turnover (see Shyu and Wilkinson, 2000). Possibly the best understood role is that of the hnRNPs, which are also involved in a variety of functions such as transcriptional regulation, maintenance of telomere length, alternative pre-RNA splicing and processing of the 3' end (see Krecic and Swanson, 1999) (see also Chapter 5). hnRNPs have been found to accompany mRNAs as they exit the nucleus and to shuttle between the http://www.albany.edu/~abio304/text/15part2.html (29 of 39) [3/5/2003 8:21:35 PM]
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nucleus and the cytoplasm. They control the localization of the mRNA in the cytoplasm, its translation and its turnover (see Shyu and Wilkison, 2000). For example, the localization of the mRNA for myelin basic protein (MBP) depends of hnRNP A2 as shown, for example, when antisense oligonucleotides (see Chapter 1) complementary to hnRNP A2 mRNA, inhibited the efflux of MBP mRNA and a reporter RNA with an A2 binding site (Munro et al., 1999). Since many proteins are translated only after their mRNA has arrived at the intended target, it is not surprising that hnRNP A2 also has a role in the regulation of translation (Kwon et al., 1999). In one case, hnRNP was found to be involved in translational silencing. hnRNP K and E1 were found to block the translation of 15-lipoxygenase (an enzyme involved in mitochondrial membrane breakdown during erythroid cell differentiation) by inhibiting the assembly of 80S ribosomes on the mRNA coding for the oxygenase (Ostareck et al., 1997). Nonsense-mediated decay (NMD) eliminates faulty transcripts with premature termination codons (see Hentze and Kulozik, 1999). An involvement of an hnRNP-like protein (HRP1) is shown by the fact that a mutation in HRP1 stabilizes nonsense-containing mRNAs and abolishes its binding to the defective mRNA (Gonzalez et al., 2000). At least in yeast, NMD depends on a downstream sequence element (DSE), and an hnRNP-like factor, Hrp1. Apparently, when DSE is located within normal coding sequences the ribosome blocks the interaction between Hrp1 and DSE. However, with a prematurely terminated translation the DSE is in the untranslated region and the binding of Hrp1 triggers the degradation events ( Gonzalez et al., 2000) (see Wilusz et al., 2001). C. Regulation in Skeletal Muscle Mammalian skeletal muscle has to adapt to changing conditions, such as exercise, by undergoing changes in the kind of myosin present in its fibers and in the metabolic pathway favored. Skeletal muscle fibers differ in their speed of contraction from very slow (type 1) to very fast (type 2B) depending on the type of myosin heavy chain present (see Schiaffino et al., 1989; Bottinelli et al., 1994). There are four major kinds of myosin heavy chains. In contrast, endurance of a muscle fiber does not depend on the myosin component but on the availability of ATP. Type 2B fibers have a high level of glycolytic enzymes and can provide ATP rapidly from glycogen. However, the supply of glycogen is limited. Type 1 fibers depend mostly on oxidative-phosphorylation, which produces ATP more slowly. With increased stimulation, muscle fibers can go from one phenotype to another, for example, from type 2B to type 1 (see Gorza et al., 1988; Windisch et al., 1998). What mediates these changes? Several myogenic transcription factors are known to regulate muscle gene expression (see Molkentin and Olson, 1996) and may respond to electrical activity. One of these factors is myogenin. The overexpression of myogenin in transgenic mice (see Chapter 1) (Hughes et al., 1999) produced as much as three fold elevation of the oxidative-phosphorylation levels of fast fibers, accompanied by a decrease in glycolytic enzymes. In this particular study, there was no alteration in the myosin types.
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V. Circadian Rhythms Daily rhythms, the circadian rhythms, are characteristic of all living matter (see Pittendrigh, 1993; Takahashi, 1995). They have three basic properties that include a self-sustained oscillation with a period of approximately 24 hours in an organism's activity. The activity can be expressed in an animal's behavior or an organism's physiology or biochemistry. The oscillations are intrinsic and persist even under constant conditions. The cycle is synchronized (entrained) (see below) in both period and phase by environmental cycles of light or temperature. The circadian rhythm compensates the period length of the rhythms for temperature changes. The role of circadian rhythms in human physiology, disease and the efficacy of drugs is well recognized (see Kraft and Martin, 1995). A. Clock mechanisms The general components of circadian systems that have been shown to be universal, center on a circadian pacemaker or clock that generates the oscillations. Input pathways convey environmental information for entraining (i.e. setting) the pacemaker (see below), while efferent pathways communicate the rhythms to the rest of the organism. Although the pacemakers may have common elements in different organisms, the input and the efferent pathways differ significantly from organism to organism. A circadian oscillator is a structure which expresses self-sustained oscillation. A pacemaker is a circadian oscillator that drives a cyclic process. The circadian oscillators are cell based. This is not only clear from unicellular organisms but also has been shown in primary cell cultures of a single cell type. Furthermore, in the case of the marine mollusc, Bulla, cultured, isolated single cells express circadian rhythms (Michel et al., 1993). The oscillatory system of the suprachiasmatic nucleus (SCN) of mammals has entire clock elements in individual neurons (Welsh et al., 1995; Liu et al., 1997c). In mammals, the pacemaker has been shown to be localized in the brain in the SCN of the hypothalamus. For example, surgical removal of the SCN in hamsters eliminates rhythmicity. Neuronal grafts from other animals reestablish the rhythm that corresponds to that of the donor (Ralph et al., 1990). Although the SCN is recognized as the main center responsible for rhythmicity, there are other independent centers. It has become increasingly apparent that clock mechanisms function independently in a variety of tissues and can be independently regulated (that is entrained, see below) by different signals. In the golden hamster, a circadian oscillator that operates independently from the SCN has been found in the retina (Tosini and Menaker, 1996). The mouse oscillator gene, Clock, is expressed in a variety of tissues examined (King et al., 1997; Stockan et al., 2001). Similarly, the per1 gene, also involved in rhythmicity (see below), is expressed in a variety of tissues in the rat . In the latter experiments, the per1 promoter was linked to a luciferase reporter. The activation of of per1 could be monitored by recording light emission of the luciferase system in vitro ( Yamazaki et al., 2000). When the feeding was restricted to a limited time each day, the rhythmicity in the SCN remained entrained by the light-dark cycle. However, liver cells were entrained rapidly to the feeding schedule (Stokkan et al., 2001). In addition, in the vascular system, the clock mechanism is regulated by retinoic acid via the appropriate nuclear receptors http://www.albany.edu/~abio304/text/15part2.html (31 of 39) [3/5/2003 8:21:35 PM]
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(see Chapter 7) (McNamara et al., 2001) and a clock mechanism in the mammalian forebrain (Reick et al., 2001) is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors, supposedly in response to metabolic conditions and may constitute a system of intracellular redox sensors. (Rutter et al., 2001). These results indicate that a peripheral clock can behave quite independently of the SCN. The presence of several oscillators may be a general biological phenomenon in multicellular organisms. In Drosophila melanogaster, independent oscillators are present throughout the fly's tissues (Plautz et al., 1997). In mammals, the neurons of the SCN secrete neuropeptides with circadian rhythmicity. The SCN also signals the pineal gland to secrete melatonin (see Foulkes et al., 1997). Several inputs impinge on the SCN, including visual clues from the retina which in mammals entrain circadian rhythms to the light-dark cycle (see below). Molecular components of circadian oscillators (see Dunlap, 1996) must: (a) show a rhythm in activity or amount corresponding to the overall rhythm, (b) be affected by mutational changes such that some basic property of the clock is also changed, (c) have some sort of feed-back regulation at the molecular level, (d) respond to abrupt changes in the component by resetting the clock, (e) stop the clock when clamped at any level, and (f) reset the phase of the component within one cycle with changes in light-dark cycle (reflecting the entrainment properties of the clock). Macromolecular synthesis is inextricably involved in circadian rhythms and is required for the functioning of the circadian pacemaker, as well as for the input and the efferent pathways. The dependence of the oscillator on protein synthesis is shown by the fact that single-gene mutations affect the circadian period. In addition, pulses of inhibitors of protein synthesis applied at critical periods cause phase shifts. The effects of inhibitors of RNA synthesis also cause similar phase shifts. The application of inhibitors for long periods stops the pacemaker entirely. Similarly, entrainment depends on transcriptionally controlled protein synthesis. This is shown by the fact that light stimuli induce the expression of transcription factors at the site of the oscillator (such as the SCN in mammals). The need for expression of certain genes for entrainment has been shown using antisense oligonucleotides (Wollnick et al., 1994). These presumably block gene expression by hybridizing to mRNA (see Chapter 1). In mammals, the DNA-arrays technique (see Chapter 1) has allowed recognizing the expression of 650 genes involved in circadian rhythm in the SCN and the liver. Approximately 650 transcripts were implicated. Only 28 were found to be common to both tissues ( Panda et al., 2002), indicating that the two are regulated independently Many of the proteins involved in the oscillations are similar in various organisms, although their role may be quite different. In order to distinguish the genes or proteins, those of Drosophila are preceded by the letter d, those of mice by m and hdenotes the human variety. In Drosophila (see Hall, 1995), the circadian rhythm depends on two genes, the period, per gene (Konopka and Benzer, 1971) and the timeless, tim gene. Nonsense mutations in per, result in loss of http://www.albany.edu/~abio304/text/15part2.html (32 of 39) [3/5/2003 8:21:35 PM]
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locomotor rhythm and single amino acid replacements alter the length of the cycle (see Takashi, 1995). The PER protein is controlled transcriptionally and by degradation, as witnessed by the short half-life of its mRNA (Hardin et al., 1992). Mutations of the tim gene block the oscillations in per expression. Similarly, the cyclic expression of tim is affected by per mutants (see Reppert and Sauman, 1995). As suggested by the mutual dependence of their expression, the TIM protein binds the PER protein to form a dimer, which is active in cyclicity. PER contains a domain referred to as the PAS domain needed for interaction between two proteins (Huang et al., 1993) that is characteristic of clock proteins. However, the PAS domain is not present in TIM (see Reppert and Sauman, 1995). In Drosophila present evidence agrees with the diagram represented in Fig. 14. The positive transcriptional regulation is from dCLOCK (dCLK) and dCYCLE (dCYC also called dBMAL1) (coded by the genes dclk and dcyc). dCLK and BMAL1 have basic helix-loop-helix ( bHLH)and PAS domains. The bHLH domains bind to a DNA sequence (referred to as an E box). The PAS domain is needed for dimerization (see Chapter 6). The two proteins heterodimerize and the complex binds to E box enhancers in the promoter region of the genes [(1)in Fig. 14](Hao et al., 1997; Allada et al., 1998; Darlington et al., 1998; Rutila et al., 1998)and activate the transcription of genes coding for proteins required for function (e.g., in eclosion behavior and locomotion) (2) and the clock genes per and tim (3). The RNA and protein of these genes oscillate and the cycling depends on dimerization and entry of the PER and TIM proteins(4) into the nucleus (5). PER and TIM function as negative regulators of their own transcription, forming a feedback loop (6). In the nucleus, these proteins inhibit transcription of the corresponding genes by interfering with CLK-BMAL1-mediated transcription (Darlington et al., 1998; Lee, 1999)). Thus, dCLK drives the expression of per and tim, and in turn PER and TIM inhibit dCLK's activity and close the feedback loop. The return to dCLK's activity requires the decrease in concentration of PER. Therefore, the degradation of PER is as necessary as its initial expression. The proteasome inhibitors lactacystin, a microbial metabolite ( Fenteany et al., 1995) and MG115, a synthetic peptide aldehyde inhibitor (Rock et al., 1994), were applied to the central nervous system of Drosophila larvae and found to inhibit the degradation of TIM accompanying light pulses (used to modify the timing of the clock, see below) (Naidoo et al., 1999). These results implicate the proteasome in clock function. Other findings have added a complexity to this simple picture. The transcription of another gene, vri, cycles in phase with per and tim (Blau and Young, 1999) and is directly regulated by the transcription factors dCLK and dCYC. vri-less mutants block per and tim expression. When the vri cycle is interfered with, per and tim expression is suppressed and long-period behavioral rhythms and arrhythmicity take place, suggesting the vri is an essential part of the Drosophila clock. Oscillations in the absence of light require the kinase DOUBLE-TIME (DBT) which controls the phosphorylation and stability of PER. Without DBT, PER proteins accumulate and the clock is stopped (Price et al., 1998; Kloss et al., 1998). Genetics have been applied to mammalian circadian systems only recently (see Dunlap, 1999). In the SCN, the basic elements determining the circadian rhythm are conserved in mammals [PER, TIM, CLK and CYC (BMAL)]. The NPAS2 gene encodes an analog of Clock. which expressed differently in various tissues (e.g., Reick et al., 2001). However, there are three per genes expressed in the SCN of http://www.albany.edu/~abio304/text/15part2.html (33 of 39) [3/5/2003 8:21:35 PM]
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mammals that are differentially regulated by light (e.g., Shearman et al, 1997; Takumi et al., 1998) (per3 is expressed independently of light). As in Drosophila, in mammals, CLK/BMAL1 also drive transcription of mper (Jin et al., 1999). The mouse PER proteins have a PAS domain and a bHLH domain (Albrecht et al., 1997; Antoch et al., 1997). The bHLH domain is absent in the Drosophila PER. mper3 knockout mice have a normal clock (Shearman et al., 2000) whereas a mper2 mutants have been found to have a shortened circadian period with loss of rhythmicity in the dark (Zheng et al., 1999). Null mutations in the mper1 gene produces a shorter circadian period with less precision. Mice deficient in both mper1 and mper2 display no circadian rhythms. mPER2 acts at the transcriptional level. In contrast mPER1 regulates mPER2 at the posttrascriptional level. mPER1 and mPER2 regulate clock-controlled genes independently (Zheng et al., 2001). In contrast to the behavior in Drosophila (Section B, below), the cryptochrome genes (mcry1 and mcry2) are essential for circadian behavioral rhythms (van der Horst et al., 1999; Vitaterna et al., 1999) (see below). They are also transcriptionally controlled by CLOCKBMAL1 heterodimers (e.g., Jin et al., 1999). mPER and mCRY feed-back inhibit the transcription mediated by CLOCK/BAML1 (e.g., Kume et al., 1999). The role of mtim is unknown (see Field et al., 2000) but does not seem to be involved in the oscillations, although mTIM inhibits the CLK.BMAL1 transcriptional activation (e.g., Jin et al., 1999; Kume et al. 1999). In mammals, PER-TIM complexes are not detected (Kume et al., 1999). In mammals, the casein kinase I ε (cki ε) gene corresponds to the dbt gene of Drosophila (Lowrey et al., 2000). tau is a mutant of ckiε with reduced phosphorylating ability. The casein kinase lε enzyme (CKI), a serine/threonine kinase, binds and phosphorylates mammalian PER (Vielhaber et al., 2000). Supposedly, when the kinase phosphorylates PER1, it masks the nuclear localization sequence (NLS) and PER1 remains in the cytoplasm. Furthermore, the phosphorylation of PER decreases its stability (Price et al, 1998). With a defective kinase (e.g., in tau mutants), the PER moves to the nucleus more rapidly with the consequent early repression of the transcription of per genes. A mutation in human per2 (hper2) has ben shown to speed up the circadian clock in people suffering from familial advanced sleep phase syndrome (Toh et al., 2001). In individuals with this syndrome, a serine was replaced by glycine in the casein kinase I ε (CKI ε) binding region of hPER2, which causes hypophosphorylation of the protein. In the fungus Neurospora, the frequency, frq gene is a major part of the oscillator (see Luo et al., 1998). Expression of frq at the wrong time interrupts the rhythm. The mRNA from frq produces two distinct proteins by alternative initiation of mRNA translation (Liu et al., 1997b). The choice of protein depends on temperature. In addition, several phosphorylated versions of these two proteins may act as distinct entities (Garceau et al., 1997). The FRQ protein has properties of transcription factors, such as a nuclear localization sequence (NLS) (see Chapter 5) and a DNA binding domain. A negative loop is also present in Neurospora: FRQ negatively regulates the level of its own transcription. The proteins encoded by the white collar 1 and white collar 2 genes favor the transcription of frq (see Crosthwaite et al., 1997). The FRQ protein depresses the level of frq transcription, probably by interfering with the activation of the transcription by White collar 1 and White collar 2 protein.
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Fig. 14 Diagram representing the feedback control of circadian rhythmicity in Drosophila. The proteins coded by clock and bmal continuously activate the transcription of per or tim (1). The mRNA from these genes code for the proteins responsible for activity and eclosion (2) and in addition for proteins (3) that upon phosphorylation dimerize (4) are translocated into the nucleus (5) and block the action of the activators (6).
B. Entrainment Entrainment sets the clock in response to external clues. The major factor determining entrainment in plants and animals is light, in particular blue light (of a wavelength of 400-500 nm). Therefore, pigments capable of absorbing this wave-length range have been looked for some time. Some of these are cryptochromes (CRYs), pterin/flavin containing proteins (e.g., see Cashmore et al., 1999). CRY receives the light information and transmits it to the core oscillator in plants (Somers et al., 1998) and Drosophila (Stanewsky et al., 1998; Emery et al., 1998; Egan et al., 1999). However, in mammals the nature of photoreceptor molecules in entrainment is still obscure (see below). The plant, Arabidopsis, has two cryptochrome genes and five genes for phytochrome photoreceptors (see Quail et al., 1995). Phytochrome protein is a protein dimer of 120 kDa each. The prosthetic group of phytochrome is an open chain tetrapyrrole attached to the protein as a cysteine thioester. It absorbs light mostly in the red and also to some extent in the blue range. Entrainment in Arabidopsis has an input from both kinds of photoreceptors (Somers et al., 1998). Similarly in Drosophila, CRY and rhodopsin also
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function in entrainment (see Stanewsky et al., 1998). Redundancy in photoreceptors is also found in nonmammalian vertebrates (see Yoshikawa and Oishi, 1998) and, as discussed below, is likely to be the case in rodents. In Drosophila embryonic cells in culture (Ceriani et al., 1999), CRY was shown to block the function of PER-TIM heteromeric complexes in a light dependent fashion and to block the degradation of TIM. CRY and TIM are part of the same complex. After absorption of light, PER-TIM and CRY are found in the nucleus. The CRYs bind to the corresponding PER proteins and translocate each from cytoplasm to nucleus. The interaction between the light-activated CRY and TIM, blocks the negative feedback effect of the PER-TIM complex (shown diagramatically in Fig. 14). In the peripheral tissues of Drosophila, CRY has also been implicated in the clock mechanism independently from photoreceptor function. A CRY mutation has been found that does not affect the circadian oscillator function of the central circadian pacemaker neurons but makes the peripheral circadian oscillators arrhythmic (Krishnan et al., 2001). Note that in mice, a role of CRY1 and CRY 2 in the clock is also likely (see below). As in other systems, entrainment of the SCN clock in mammals depends mostly on light, with the primary effect exerted by light exposure during the "night" phase of the spontaneous rhythm. When the animal is maintained in continuous darkness, light exposure during the early "night" (that is the portion of the spontaneous oscillation corresponding to early night) causes phase delays, whereas late "night" exposure causes advancements (De Coursey, 1960). The information is conveyed to the clock from the retina by the retinal projections of the retinohypothalamic tract (e.g., Johnson et al., 1988) and is mediated by the neurotransmitter glutamate (e.g., see Ding et al., 1994). The study of entrainment has provided a number of insights on the process (see Ding et al., 1994). The direct application of glutamate to the SCN brain slices induces light-like clock resetting (Ding et al., 1994). The data indicate that resetting of the clock, following light stimulation, first activates glutamate receptors by glutamate originating from the retinal projections. This is then followed by Ca2+ influx in the cytoplasm and subsequent activation of nitric oxide synthase. NO may stimulate guanylyl cyclase (see Chapter 7). The increase in cGMP activates cGMP-dependent protein kinase (PKG) (see Chapter 7). Activation of the cGMP pathways advances the clock at night, but not during the day (e.g., Liu et al, 1997a). Injection of a PKG specific inhibitor was found to block light induced advances in glutamateinduced effect at late night (e.g., Weber et al., 1995). The early "night" effect is not blocked by the PKG inhibitor, but the effect apparently depends on intracellular Ca2+ stores (see Chapter 7) and the Ca2+ release mediated by the ryanodine receptors (Ding, 1998). The results indicate that there are two divergent pathways for the early and late "night" light exposure. The light-induced resetting at late "night" involves a pathway where cGMP advances the clock. The early "night" light effect follows a distinct pathway where Ca2+ release delays the clock. In mammals, the entrainment of the SCN must transmit some sort of signal to the peripheral tissues under its control (see Rusak and Zucker, 1979). Since light signals entrain the clock, in mammals the stimulus is likely to come through the eyes as might
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be expected. With some exceptions, rodents and humans without eyes are unable to set the clock (see Foster, 1998). However, rods and cones are not involved. A gene that destroys retinal rod and cone cells in mice was unable to stop the clock, as seen from running behavior (Freedman et al., 1999) or the secretion of melatonin (Lucas et al., 1999). Therefore, under these conditions, other photoreceptors are suspected as responsible for entrainment, and the cryptochromes are natural candidates in view of their role in other organisms. CRY1 and CRY2 were found to be expressed in the ganglion cells of the retina (Miyamoto and Sancar, 1998; Thresher et al., 1998). In addition, CRY1 is expressed at a high level in the SCN and oscillates in this tissue in a circadian manner. In mutants lacking either CRY1 or CRY2 (van der Host et al., 1999), the free-running periodicity of locomotor function was accelerated or delayed respectively. These data indicate antagonistic clock-adjusting functions, which are essential for circadian rhythmicity. When both proteins were absent, under dark-dark conditions the, free running rhythmicity was entirely lost indicating that CRY1 and CRY2 must have an important function in the clock mechanism itself. However, under light-dark conditions, the double mutants exhibited a 24 hours rhythm, demonstrating that a light input is still present and therefore a pigment other than CRY1 or CRY2 must be involved. In mice (Kume et al., 1999), CRY1 and CRY2 bind to the PER proteins and translocate them from cytoplasm to nucleus. The PER and CRY proteins appear to inhibit the transcriptional complex differentially. Either CRY1 or CRY2 alone block CLK.BMAL mediated transcription. Present data indicates that in mice, CRYs play a role in the negative feed-back loop of the clock mechanism. However, a role in light-entrainment is also possible as shown by some of the studies already discussed. In mammals, molecular clues to a possible role of photoreceptor molecules in light-entrainment would require a study of events taking place in the ganglion cells of the retina. To our knowledge, these have not been carried out. As already discussed, in mammals peripheral tissues can express circadian rhythms independently of the SCN. Not surprisingly, entrainment can also occur in a tissue specific manner. Liver cells can be entrained to a feeding schedule (Stockan et al., 2001). In addition, in vascular cells, the binding to ligand activated retinoic acid receptors (RAR α and RXR α) bind to the protein NPAS2 or CLOCK and thereby negatively regulate the CLOCK/MOP4:BMAL1 induced transcription and act as a negative-feedback loop (McNamara et al., 2001) (see Fig. 14). In addition, at least in neural tissues, the DNA-binding activity of the Clock/BMAL1 and NPAS2/BMAL1 is regulated by the redox state of nicotinamide adenine dinucleotide (NAD) cofactors in a purified system. The reduced forms of the redox cofactors, NAD(H) and NADP(H), strongly enhance DNA binding of the Clock/BMAL1 and NPAS2/BMAL1, whereas this binding is inhibited by the oxidized (Rutter et al., 2001) (see Fig. 14). The situation is quite different in zebrafish. Zebrafish tissues in vivo or culture show endogenous oscillations (Whitmore et al., 1998). The peripheral organ clocks have been found to be set by light-dark cycles either in culture or in derived cell lines in culture (Whitmore et al., 2000). The resetting of circadian clocks due to temperature changes is also an important factor in entrainment (e.g., see Pittendrigh, 1960). High temperatures elicit responses similar to light and low temperatures elicit those elicited by darkness (e.g., Zimmerman et al., 1968). Levels of FRQ protein were measured in
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Neurospora at various temperatures (Liu et al., 1998). The amount of FRQ oscillates at higher concentrations when the temperature is higher. However, the level of frq-mRNA remains unchanged, indicating that the effect is posttranscriptional. The same concentration of FRQ corresponds to different times at different temperatures. Therefore, temperature shifts result in shifts in clock time without immediate synthesis of FRQ. SUGGESTED READING Protein and RNA Degradation Baumeister, W., Walz, J., Zuhl, F. and Seemuller, E. (1998) The proteasome: paradigm of a selfcompartmentalizing protease, Cell 92:367-380 (Medline) Caponigro, G. and Parker, R. (1996) Mechanisms and control of mRNA turnover in Saccharomyces cerevisiae, Microbiol. Rev. 60: 233-249. (Medline) Ciechanover, A., Orian, A., and Schwartz, A.L. (2000) Ubiquitin-mediated proteolysis: biological regulation via destruction, BioEssays 22:442-451. (MedLine) Hargrove, J. L. and Schmidt, F. H. (1989) The role of mRNA and protein stability in gene expression, FASEB J. 3:2360-2370.(Medline) Olson, T. C. and Dice, J. F. (1989) Regulation of protein degradation rates in eukaryotes, Curr. Opin. Cell Biol. 1:1194-1200.(Medline) Control of Protein Synthesis and Folding Bukau, B. and Horwich, A.L. (1998) The Hsp70 and Hsp60 chaperone machines, Cell 92:351-366. (MedLine) Frydman, J. (2001) Folding of newly translated proteins in vivo: the role of molecular chaperones, Annu. Rev. Biochem. 70:603-647. (MedLine) Merrick, W.C. (1992) Mechanism and regulation of eukaryotic protein synthesis, Microbiol. Rev. 56:291315.(Medline) Circadian Rhythms Cashmore, A.R., Jarillo, J.A., Wu, Y.J. and Liu, D. (1999) Cryptochromes: blue light receptors for plants and animals, Science 284:760-765. (Medline)
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Dunlap, J.C. (1999) Molecular bases of circadian clocks, Cell 96:271-290. (Medline) King, D.P. and Takahashi, J.S. (2000) Molecular genetics of circadian rhythms in mammals, Annu. Rev. Neurosci. 23:713-742. (MedLine) Pittendrigh, C.S. (1993) Temporal organization: reflections of a Darwinian clock-watcher, Annu. Rev. Physiol. 55:16-54. (Medline) Reppert, S. (1998) A clockwork explosion! Neuron 21:1-4. (Medline) Takahashi, J.S. (1995) Molecular neurobiology and genetics of circadian rhythms in mammals, Annu. Rev. Neurosci. 18:531-553. (Medline) REFERENCES Search the textbook
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Chapter 15: References
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Wollnik, F., Brysch, W., Uhlmann, E., Gillardon, F., Bravo, R. and Zimmermann, M., Schlingensiepen, K.H. and Herdegen, T.(1995) Block of c-Fos and JunB expression by antisense oligonucleotides inhibits light-induced phase shifts of the mammalian circadian clock, Eur. J. Neurosci. 7:388-393.(Medline) Woodside, K. H. and Mortimore, G. E. (1972) Suppression of protein turnover by amino acids in the perfused rat liver, J. Biol. Chem. 247:6474-6481.(Medline) Yaglom, J., Linskens, M.M.H.K., Sadis, S., Rubin, D.M. Fuchter, B. and Finley, D. (1995) p34cdr28mediated control of Cln3 cyclin degradation, Mol. Cell. Biol. 15:731-741.(Medline) Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block, G.D., Sakaki, Y., Menaker, M. and Tei, H. (2000) Resetting central and peripheral circadian oscillators in transgenic rats, Science 288:682-685. (MedLine) Yao, T. and Cohen, R.E. (2002) A cryptic protease couples deubiquitination and degradation by the proteasome, Nature 419:403-407. (MedLine) Yeh, E. T. H., Gong, L., and Kamitani, T. (2000) Ubiquitin-like proteins: new wines in new bottles Gene 248, 1-14. (MedLine) Yokouchi, M., Kondo, T., Houghton, A., Bartkiewicz, M., Horne, W.C., Zhang, H., Yoshimura, A. and Baron, R. (1999) Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7, J. Biol. Chem.274:31707-31712. (MedLine) Yoshimura, T., Kameyama, K., Takagi, T., Ikai, A., Tokunaga, F., Koide, T., Tanahashi, N., Tamura, T., Cejka, Z., Baumeister, W., et al (1993) Molecular characterization of the "26S" proteasome complex from rat liver J. Struct. Biol. 111:200-211.(Medline) Zanchin, N.I. and Goldfarb, D.S. (1999) The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA, Nucleic Acids Res. 27:1283-1288.(Medline) Zheng, B., Larkin, D.W., Albrecht, U., Sun, Z.S., Sage, M., Eichele, G., Lee, C.C. and Bradley, A. (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock., Nature 400:169173. (MedLine) Zheng, B., Albrecht, U., Kaasik, K., Sage, M., Lu, W., Vaishnav, S., Li, Q., Sun, Z.S., Eichele, G., Bradley, A. and Lee, C.C. (2001) Nonredundant roles of the mPer1 and mPer2 genes in the mammalian circadian clock, Cell 105:683-694. (MedLine> Zimmerman, W.F., Pittendrigh, C.S. and Pavlidis, T. (1968) Temperature compensation of the circadian http://www.albany.edu/~abio304/ref/ref15.html (27 of 28) [3/5/2003 8:22:01 PM]
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oscillation in Drosophila pseudoobscura and its entrainment by temperature cycles, J. Insect Physiol. 14:669-684.(Medline) Zylka, M.J., Shearman, L.P., Weaver, D.R. and Reppert, S.M. (1998) Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain, Neuron 20:1103-1110.(Medline)
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16. Oxidative Phosphorylation and Mitochondrial Organization I. General Considerations A. Photosynthesis and Oxidative Phosphorylation B. Oxidation Without Phosphorylation C. Anaerobic Mitochondria II. Structural Organization of Mitochondria III. Biochemical Organization of Mitochondria A. The Electron Transport Chain B. The Complexes C. Proton Pumping
Suggested Reading Web Resources References Back to List of Chapters I. GENERAL CONSIDERATIONS A. Photosynthesis and Oxidative Phosphorylation In green plants and in photosynthetic bacteria, energy is generated by photosynthetic reactions. In most other cells, the energy is generated primarily by oxidative phosphorylation. The products of photosynthesis in plants, fuel oxidative phosphorylation in animals. The role of these two groups of reactions is so fundamental and their mechanisms are so intriguing that a good deal of effort has gone into their study. Although photosynthesis and oxidative phosphorylation are formally very different, they share many features. Differences and similarities are summarized in Table 1. Another metabolic pattern that occurs in some bacteria is chemolithotrophy, i.e., the oxidation of reduced inorganic substrates, which occupies an important biological niche (see Wood, 1988; Ehrlich, 1995) but is not discussed further in this book. In oxidative phosphorylation, the oxidation of substrates takes place by a process significantly different from that of nonbiological systems. The latter involves oxygen or other oxidants directly and results in the release of energy in the form of heat. In contrast, the oxidative reactions of the cell proceed in steps, each progressively dissipating part of the energy as heat and, in some of these steps, part of the energy is used to phosphorylate ADP. Oxygen is involved only in the terminal reaction. The discrete steps of http://www.albany.edu/~abio304/text/16part1.html (1 of 15) [3/5/2003 8:22:40 PM]
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oxidative phosphorylation may be regarded as the passage of electrons through the electron transport chain until finally oxygen itself accepts the electrons and water is formed. In photosynthesis, light is the source of energy. The photosynthetic pigments, chlorophyll and the accessory pigments, absorb the radiant energy. The excited chlorophyll of the reaction centers transfers an electron to the primary acceptor and then to the electron transport system. In both oxidative phosphorylation and photosynthetic reactions, the reactions involved in the passage of electrons - i.e., the reactions of the electron transport chain - are associated with the internal membranes of specialized organelles or structures. Oxidative phosphorylation takes place in mitochondrial or in bacterial membranes. Similarly, photosynthesis takes place in the choloroplasts of green plants and in the membranes of bacteria. Table 1 Energy Capturing Systems of Cells
Oxidative phosphorylation systems
Characteristics
Photosynthesizing systems
Source of energy
Entirely or in part energy from sunlight
Oxidizable substrates
Location in cells
Membrane structure: chloroplasts or chromatophores
Membrane structure: mitochondria or mesosomes
Biochemical organization
Electron transport systems involving cytochromes
Electron transport systems involving cytochromes
Primary form of chemical energy trapping
Synthesis of ATP, reduction of NADP
Synthesis of ATP
Overall results
a. Release of o2 or other oxidized component (S or H2SO4)
O2 uptake CO2release
b. Fixation of CO2 or some other C source
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General pattern of system
Light →photosynthetic reaction involving chlorophyll ↓ e- ↑ electron transport steps ↑e- H2O → O2 photosynthetic reaction involving chlorophyll
Substrate → oxidized substrate ↓ eelectron transport stepd ↓ eO2 → H2O
B. Oxidation Without Phosphorylation In brown fat, a number of substrates can be oxidized in mitochondria without phosphorylation (Nedergard and Cannon, 1984). In nonshivering thermogenesis, the mitochondria of brown adipose tissue oxidize substrates, but the energy is dissipated as heat to warm the animal. This takes place by the uncoupling of oxidation from phosphorylation by a special mechanism (Flatmark and Pederson, 1975). Three uncoupling proteins have been found (UCPs) and these are the subject of discussion in Chapter 14. Other oxidative reactions that do not produce ATP take place elsewhere in the cell. The P450 cytochromes, a family of hemoproteins, (see Guengerich, 1992; Coon et al., 1992), oxidize a variety of important chemicals including drugs, carcinogens, steroids, pesticides, hydrocarbon and endogenous compounds, such as fatty acids, steroids, acetone, vitamins, and many others. Accordingly, they are significant in understanding pharmacology and human disease. In mammals, the enzymes are present in the endoplasmic reticulum membranes and to a lesser extent in mitochondria. Their ubiquitous presence in other organisms, including prokaryotes such as bacteria, is not only of general interest but has facilitated research on characterizing the system. The P450 enzymes are coded by a gene superfamily; 150 of these enzymes have been identified so far. The compounds oxidized by P450 induce the expression of the P450 genes. The activation of the genes is mediated by nuclear hormone receptors (see Chapter 7), some of them the so-called orphan receptors (receptor for a yet unknown ligand) (see Waxman, 1999). Mice without a nuclear hormone receptor cannot induce the P450 genes (Lee, 1995; Wei et al., 2000, Xie et al., 2000). Some of the isoforms of P450 may be fairly specific, but others, such as those in the hepatic endoplasmic reticulum, have been estimated to catalyze about 250,000 different reactions involving foreign substances. Newly created chemicals synthesized in the laboratory are likely to become substrates. P450 enzymes are generally but not always monoxygenases; that is, the steps involved in the oxidation of a substrate (e.g., RH, where R represents an organic compound) involve one oxygen atom (to form ROH). Studies of the P450 system should make it possible to screen drugs with liver microsomal preparations or reconstituted P450 systems which would permit predictions of metabolic stability and toxicity. Similarly, http://www.albany.edu/~abio304/text/16part1.html (3 of 15) [3/5/2003 8:22:40 PM]
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it may be possible to place the P450 systems in the appropriate microorganisms using recombinant DNA techniques (see Chapter 1,IIA) to dispose of toxic compounds in the environment. The desaturation of fatty acids, which requires O2 and NADPH, and their β-oxidation (i.e., the oxidation at the β-carbon atom) is carried out in the endoplasmic reticulum by the P450 system. The function of the β-oxidation is unknown. Production of the endoplasmic system and P450 can be induced by exposure of the animal to foreign substances. The peroxisomal oxidative system does not involve cytochromes (Huang et al., 1981). The enzymes of this system are contained in vesicular cellular elements, the peroxisomes, which are enclosed by a single membrane. Peroxisomes (see Titorenko and Rachubinski, 2001) are very heterogeneous organelles which originate from a common precursor. They differ in composition, morphology and enzymatic activity depending on cell type as well as metabolic and environmental condition. Peroxisomes range in size from 0.1 µm to 1 µm. They are related to the glyoxysomes of plants (see below) and glycosomes of trypanasomes. Together these organelles form a family of microbodies. The peroxisomal proteins are coded by nuclear genes and are generally synthesized in the cytoplasm. 23 proteins are required for peroxisome assembly. These proteins are encoded by the PEX genes (see Subramani et al., 2000). Mutations in 11 of these genes cause lethal disorders in humans (see Gould and Valle, 2000). The proteins are generated by free polysomes and then posttranslationally transferred to peroxisomes (see Tabak et al., 1999). The various components are imported in steps to form the mature peroxisome (see Titorenko and Rachubinski, 2001). In addition, peroxisomes undergo division when cells divide or when there is an increase in enzymes induced by external signals ( Marshall et al., 1996). For example, hypolipidemic drugs and plasticizers produce a proliferation of these organelles (see Lock et al., 1989). The nuclear hormone receptor peroxisome proliferator activated receptors (PPARs) (see Schoonjans et al., 1996 and Chapter 7) are involved in peroxisomal proliferation in response to a variety of ligands. In mammals, the peroxisomal matrix is granular and, at least in rat liver, contains a crystalline or polytubular structure made of urate oxidase. In mammalian cells, peroxisomes contain enzymes capable of carrying out the β-oxidation of fatty acids (see below). Shortened fatty acids are exported from peroxisomes to mitochondria (see van den Bosch et al., 1992). In S. cervisiae, fatty acids are exclusively oxidized in peroxisomes (see Erdmann et al., 1997) indicating that acetylCoA and NADH are produced there. The peroxisome system can oxidize a variety of substances in two steps. In one step, H2O2 is formed from oxygen using reducing equivalents from the substrate, and substances such as urate, D- and L-amino acids, L--hydroxy acids and glyoxylate are oxidized. In the second step, the H2O2 is used as an oxidant; phenols, nitrites, ethanol, methanol and formate can be oxidized. The system can also operate to oxidize NADH indirectly. The glycolate oxidized by the peroxisomes to form glyoxylate can be regenerated in a reaction that uses NADH as a source of reducing equivalents. Catalytic amounts of these two metabolites operating in oxidation-reduction cycles can oxidize NADH indefinitely. In plants, a similar cycle is thought to be responsible for photorespiration (see below).
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The purpose of these oxidative reactions is not entirely clear. In mammalian liver or intestine, where peroxisomes are also present, they may be important in the regulation of the concentration of metabolites. The oxidation of NADH by the peroxisomes may be significant in the regulation of glycolysis that requires the regeneration of NAD+ for its continuous operation. NADH oxidation may also play a role in the prevention of high levels of O2 which might be damaging. The enzyme system capable of β-oxidation is likely to function in the breakdown of the fatty acids, which are poorly processed by mitochondria, and may play a significant role when the need for fatty acid breakdown is high (Hashimoto, 1982; Mannaerts and Debeer, 1982; Osmundsen, 1982). However, the peroxisomes are most likely to act in concert with mitochondria, since they are not capable of processing short-chain fatty acids. In higher plants, glyoxysomes are found in the oil-rich tissues of seeds (Huang et al., 1983). In these tissues, they are exclusively responsible for β-oxidation of fatty acids and they contain glyoxylate cycle enzymes. The glyoxylate cycle can convert 2 mol of acetyl-CoA to 1 mol of succinate, thereby permitting the synthesis of sugars in conjunction with reactions occurring in the cytoplasm and in mitochondria. Leaf peroxisomes (Huang et al., 1983) are involved in photorespiration, in which they oxidize reducing equivalents formed by photosynthesis in conjunction with reactions taking place in the cytoplasm and the mitochondria. The complex reactions can function in the carbon reduction pathway of the chloroplasts or in the oxidative pathway, depending on the competition between oxygen and CO2. Oxygen favors the oxidation of ribulose-1,5-biphosphate in the oxidative pathway, whereas CO2 favors the carboxylation of ribulose-1,5-biphosphate with the formation of phosphoglycerate. The precise physiological role of photorespiration is not known, but the process appears to be essential. Mutants lacking the system (Sommerville and Ogren, 1980) do not survive in the presence of oxygen. Without the photorespiratory system, oxygen and light cause a loss of photosynthetic capacity, which suggests that photorespiration is involved in maintaining a low oxygen concentration. Photorespiration may also be required to consume excess photosynthetically produced reducing equivalents. This chapter focuses primarily on the electron transport system of mitochondria. Sections II and III describe the structural and biochemical organization of mitochondria. Chapter 18 presents in more detail electron transport and its coupling to ATP synthesis and ion transport. C. Anaerobic Mitochondria Several kinds of mitochondria exist in of eukaryotes, including protists and metazoans (e.g., gold fish!), that deal with electrons resulting from carbohydrate oxidation in the absence of sufficient oxygen. The electron pathways produce ATP (see Tielens et al., 2002). These mitochondria use as terminal electron
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acceptors nitrite, nitric oxide, succinate and other metabolites. The anaerobic mitochondria may use electron acceptors present in the medium (e.g., NO3-) or some generated by metabolism (e.g., fumarate). The hydrogenosome is another organelle related to mitochondria which is present in anaerobic protists and produces H2 without intervention of electron carriers (e.g., see Hackstein et al., 1999; Van der Giezen et al., 2002). II. STRUCTURAL ORGANIZATION OF MITOCHONDRIA The protein complexes which constitute the machinery of oxidative phosphorylation are organized in the mitochondria in the inner mitochondrial membrane. In bacteria, they are present in infoldings of the plasma membrane. Mitochondria, seen with the electron microscope, are distinctive. Their appearance varies with the tissue and the preparative technique. The mitochondria appear either spherical or tubular, depending on the tissue. The mitochondria from fibroblasts are long (as long as 46 µm). Two membranes enclose the mitochondrial matrix: an outer membrane and an inner membrane. The inner mitochondrial membrane lining the mitochondrial lumen forms deep folds called cristae. These folds have been thought to be lamellar, however, more recent technology shows them to be pleiomorphic. EM tomographic reconstruction of the mitochondrial structure has yielded a wealth of detail (see e.g., Frey and Mannella, 2000) and has shown that the morphology of the cristae depends on the tissue and physiological state. In liver and fibroblasts, three dimensional reconstructions from high resolution scanning electron microscopy (HRESEM) (see Chapter 1) show them to be predominantly tubular and approximately 30 nm in diameter (Lea et al., 1994). In some cristae the tubular elements merge, some forming flattened lamellae (e.g., in neurons). Those of cold adapted brown fat adipocytes were lamellar. In muscle, they were found to be both lamellar and tubular. Fig. 1 shows a electron micrograph of a mitochondrion from an hepatocyte (Lea at al., 1994). The view also illustrates the close association of some mitochondria to the endoplasmic reticulum (see below). The cristae connect to the intramembrane space by a tubular structure that have been referred to as peculi crista (Daems and Wisse, 1966) or crista junctions (Perkins and Frey, 2000), generally 30 to 40 µm in diameter. Despite the narrowness of the crista junctions (and the presence of an outer membrane), the surface of the inner membrane appears to be available to external solutes of low molecular weight as shown by permeabilty studies of isolated mitochondria (Tedeschi, 1959 as corrected by Garlid and Beavis, 1985).Where inner and outer membrane make contact (the so-called contact sites) show a structure 14 nm in length and 14 nm in diameter (see Frey and Mannella, 2000). It has been suggested that these sites represent macromolecule assemblies involving channels.
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Fig. 1 Stereo pair scanning electronmicrograph of (A) a portion of a human liver cell showing several mitochondria (bar corresponds to 880 nm) and (B) a mitochondrion (bar corresponds to 494 nm). S stands for smooth endoplasmic reticulum, R for rough enoplasmic reticulum and M for mitochondrion. From Lea et al., 1994, reproduced by permission.
Variations in the volume of the internal mitochondrial lumen would increase or decrease the separation between the internal and external membranes. There is evidence that these structural alterations actually take place during changes in metabolic state (Hackenbrock, 1966). The matrix is expanded in the absence of phosphorylation (the orthodox configuration) and condensed when phosphorylating (the condensed configuration). It has been suggested that these changes in the volume of the lumen are osmotic in nature (Anagnosti and Tedeschi, 1970; Izzard and Tedeschi, 1970). http://www.albany.edu/~abio304/text/16part1.html (7 of 15) [3/5/2003 8:22:40 PM]
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3-D reconstructions (Mannella et al., 1994) show that the spaces between cristae, tubular when in the orthodox configuration, become sac-like in the condensed configuration. The cristae are connected to the outside medium and to each other by tubes 20 nm in diameter. In negatively stained preparations (Fig. 2) (Parson, 1963), the mitochondrial membranes have the appearance of tubes. The inner surfaces of the internal membranes are studded with particles 8 nm in diameter, which are attached to the membranes by a stalk. At times, this arrangement has been observed with conventional electron microscopy (Ashhurst, 1965; Schneider et al., 1972). These particles are not always visible after the usual preparatory procedures, perhaps due to their lability. They do not appear with negative staining after conventional fixation. In submitochondrial preparations or reconstituted systems, these granular structures are correlated with the presence of F1, the ATP synthase.
Fig. 2 (a) Negatively stained mitochondria spread on potassium phosphotungstate. (b and c) Subunits associated with the inner membrane or cristae. The particles are shown in more detail in the inset (in which the contrast is reversed). Abbreviations: om, outer membrane; c, a crista; g, an intramitochondrial granule; ims, a particle; p, projection of outer membrane; j, branching of crista. Reproduced with permission from Parson, D.F. (1963) Mitochondrial structure: two types of subunits on negatively stained mitochondrial membranes. Science, 140:985-988, copyright ©1963 American Association for the Advancement of Science.
Freeze-fracture, also used in the study of membranes, is entirely different from the two electron microscopic methods already discussed. In this method, a very small piece of tissue is rapidly frozen at http://www.albany.edu/~abio304/text/16part1.html (8 of 15) [3/5/2003 8:22:40 PM]
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very low temperature. The preparation is then fractured by using a chilled microhammer. The fractured preparation is etched by sublimating the water to a depth of a few tens of nanometers. A carbon or a platinum and carbon layer is then condensed onto the exposed surface to produce a replica, which is then viewed with the electron microscope. The fracture is thought to expose the inner hydrophobic central surface of the unit membrane. Observations suggest that the inner hydrophobic face of the inner membrane has many particles ranging in diameter between 10 and 15 nm which probably correspond to intrinsic proteins, i.e., proteins embedded in the lipid framework (Wrigglesworth et al., 1970). Anatomically, ER and mitochondria are frequently in close contact (e.g., Perkins et al., 1997) and are likely to interact. Electron microscopic tomography of mitochondria (see Chapter 1) in situ, show mitochondria in clusters with stacks of the ER forming extended structures (Mannella et al., 1998). The structural relationship of the two has been studied in living HeLa cells (Rizzuto et al., 1998) using specifically targeted green fluorescent proteins with different spectral characteristics (see Chapter 1) one to identify mitochondria and the other ER. Many close contacts were observed between the ER and mitochondria. Furthermore, in situ, mitochondria formed a closely associated dynamic network. Not surprisingly, mitochondria and ER interact in Ca2+ exchanges (see Chapter 7) and in biochemical reactions. Glycosylphosphatidylinositols (GPIs) biosynthetic reactions involve the sequential addition of monosaccharides, fatty acid, and phosphoethanolamine(s) to phosphatidylinositol (PI) that take place in the ER. The subcellular fractions of homogenates of mammalian cells (Vidugiriene et al., 1999) revealed that some of the reactions take place in a sub-compartment of the ER associated with mitochondria. The relationship between mitochondria and ER membranes is likely to be regulated. In digitoninpermeabilized Madin-Derby canine kidney (MDCK) cells, rat liver cytosol stimulates the dissociation of the two at low Ca2+, whereas high concentrations (higher than 1 µM) favor association (Wang et al., 2000). III. BIOCHEMICAL ORGANIZATION OF MITOCHONDRIA Although the major function of mitochondria is in oxidative phosphorylation other roles are also known. For example, mitochondria have been found to have a significant role in programmed cell death (see Chapter 2). Substrate oxidation coupled to phosphorylation involves three kinds of reaction sequences: reactions in which the substrates themselves are oxidized (e.g., in the tricarboxylic acid cycle), reactions in which the electrons are transported through the cytochrome chain, and reactions involved in the coupling of the electron transport to ATP synthesis or ion transport. The three kinds of reactions can actually be dissociated by isolating functional complexes or by lysing the mitochondria. Some of the enzymes concerned directly with the oxidation of substrates can be isolated by relatively simple procedures. They are thought to be either loosely bound to the membrane or dissolved in the mitochondrial lumen. The isolation of succinate dehydrogenase, α-glycerophosphate http://www.albany.edu/~abio304/text/16part1.html (9 of 15) [3/5/2003 8:22:40 PM]
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dehydrogenase, and the complexes involved in the electron transport requires disruption of the membranes, since these enzymes are either firmly attached or part of the lipoprotein framework of the inner mitochondrial membrane. Part of the ATP synthase complex (the Fo portion) requires disruption of the lipoprotein framework, and part (the F1, portion) can be removed by simpler disruptive procedures. Interactions between the matrix enzymes suggest that they may be associated in physiologically significant complexes. The measured rates in a pathway are in excess of the rate calculated from the known intramitochondrial concentrations of substrates and enzymes free in solution (e.g., Halper and Srere, 1977). Furthermore, enzymes that catalyze consecutive reactions in a metabolic pathway tend to associate with each other (Beckman and Kanarek, 1981), and at least some of them are capable of being bound to the inner mitochondrial membrane (Sumegi and Srere, 1984). In intact mitochondria or submitochondrial vesicles (i.e., vesicles derived from mitochondria), electron transport may be coupled to or uncoupled from phosphorylation. Except for the evolution of heat in mitochondria of brown fat cells, mitochondria are involved in oxidative phosphorylation. Mitochondria capable of phosphorylation may be uncoupled by the addition of chemicals, so-called uncouplers, such as 2,4-dinitrophenol. In this condition, they continue to oxidize substrates, frequently at an increased rate, but without producing ATP. Other chemicals have other distinct effects. Some of them block the flow of electrons at specific sites without substantially affecting the coupling. In contrast, oligomycin and aurovertin block ATP synthesis and hydrolysis without directly interfering with coupling or electron transport. Uncouplers and inhibitors have been used extensively in studies of the mechanisms of electron transport and phosphorylation. The points at which some of the inhibitors block the respiratory chain are discussed later. A. The Electron Transport Chain The metabolic breakdown of carbohydrates and fatty acids eventually results in the production of reduced equivalents and acetyl-CoA. The formation of acetyl-CoA, further oxidation of the acetyl moiety through the enzymes of the tricarboxylic acid cycle and the oxidation of reducing equivalents through the electron transport chain, take place in the mitochondria. Some of the amino acids are also metabolized in reactions in which they form acetyl-CoA, although the carbon skeleton of other amino acids may enter the catabolic pathway through the tricarboxylic acid cycle or the glycolytic pathway (see Fig. 3). This chapter primarily addresses the reactions of the electron transport chain: a series of oxidative-reductive steps in which each reduced component is in turn oxidized by the one that follows. In the last oxidative step, the reducing equivalents are directly oxidized by oxygen. In essence, the electrons are passed from one component of the electron transport chain to the one that follows. The reactions can be represented as shown below. NADH + FP + H+→ NAD+ + FPH2 (1) FPH2 + 2 cyt b3+ → FP + 2H+ + 2 cyt b2+ (2) http://www.albany.edu/~abio304/text/16part1.html (10 of 15) [3/5/2003 8:22:40 PM]
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2 cyt b2+ + 2cyt c3+ → 2 cyt c2+ + 2 cyt b3+ (3) Here FP is flavoprotein and cyt represents a is cytochrome. The final reaction of the series involves oxygen: 2 cyt a32+ + ½O2→ 2 cyt a33+ + 2 cyt b3+ (4)
Fig. 3 Diagram of metabolic reactions show the role of reactions taking place in mitochondria (in the solid line boxes).
More schematically, the reactions of Eqs. (1) to (4) can be represented as a single equation: NAD → FP→ 2 cyt b→ 2 cyt c ...→ cyt a3→ O2 (5) In this representation, the arrows indicate the direction of the electron flow. Many of the components of the electron transport chain have been known and intensely studied for a number of years. To evaluate the role of each of these components in oxidative phosphorylation, it is first necessary to know whether they are actually functioning in the redox reaction chain, and then to http://www.albany.edu/~abio304/text/16part1.html (11 of 15) [3/5/2003 8:22:40 PM]
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determine their order in the chain. The electron transport chain includes NAD, the cytochromes, flavoproteins, coenzyme Q (CoQ), and nonheme iron. Copper is involved in oxidase activity (e.g., via cytochrome-c oxidase) in a way that is still unclear. There is some evidence that one of the coppers in cytochrome-c oxidase is an electron carrier and the other is involved in ligand binding. The flavoproteins are specific proteins containing flavin groups (riboflavin-5'-phosphate or flavin-adenine dinucleotide). The cytochromes contain as a redox component Fe3+ (or Fe2+) in the form of iron porphyrins. CoQ (ubiquinone) is a lipid soluble quinone. Some of the known components of the cytochrome chain and their probable position in the electron transport chain, are discussed in more detail below (see Figs. 6, 7, and 10). Oxidation and reduction of the cytochrome system are reflected in changes in the light absorbed, as shown in Fig. 4 (Chance and Williams, 1956). The solid line in Fig. 4 represents the difference in absorption spectra between mitochondria in the presence of substrate under anaerobic conditions (more reduced) and under aerobic conditions (more oxidized). The various peaks correspond to the components indicated. Curve 1 pertains to the present discussion; curve 2 is discussed later. The redox changes of some of the components of the respiratory chain have also been studied with electron paramagnetic resonance (EPR), also called electron spin resonance (ESR). This technique has been very useful in the study of the electron transport, particularly at low temperatures (e.g., 13K). Electrons usually occur in pairs, and the paired electrons have opposed spins. Unpaired electrons can be present either in free radicals or in compounds such as the electron transport components. The unpaired electrons respond to changes in the magnetic field, and the interaction can be detected by changes in the absorption of microwaves. In EPR, generally the magnetic field is varied and the transmitted radiation is amplified, recorded, and displayed, most commonly as the derivative of the absorbed radiation in relative units. The relationship between the magnetic field and the absorption is dependent on the constant g or g factor, following the relationship of Eq. (6). In this equation, h is Planck's constant; β is the Bohr magneton, also a constant; and H and ν are the strength of the magnetic field and the frequency of the microwave radiation, respectively. For an electron without any disturbances, g = 2.00232. However, other charged groups may disturb this pattern so that the g value is different, the usual case in biological samples. hν = gβH (6) Since the frequency is generally maintained constant, the position of resonance in the spectrum (i.e., the absorption of the radiation) can be indicated by the magnitude of either H or g. In biological studies it is frequently indicated by the g value. Figure 5 shows the parameters of the EPR spectrum of complex I from beef heart mitochondria (see Section III,B) at 11oK (obtained with cold helium gas). The proportion of the components reduced under various conditions can provide information about the organization of the electron transport chain. The order in which the cytochromes function should be deducible from the order in which they are oxidized when O2 is suddenly introduced into the system. If the mitochondria are first exposed to a nitrogen atmosphere, the cytochromes become entirely reduced; http://www.albany.edu/~abio304/text/16part1.html (12 of 15) [3/5/2003 8:22:40 PM]
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the electrons originating from the substrate no longer have a terminal acceptor. When O2 is introduced, the components closer to O2 should be oxidized first. The results indicate that cytochrome a is oxidized first, followed by cyt c, cyt b and flavoprotein.
Fig. 4 Difference spectra of the respiratory carriers in rat liver mitochondria. The solid line represents absorbancy changes brought about by the presence of substrate under anaerobic conditions. The dashed line represents the change produced by substrate in the presence of antimycin A. The results are measured as differences from the absorption under aerobic conditions. Reproduced by permission from B. Chance and G. R. Williams (1956), Advances in Enzymology, vol. 17, p. 74, Copyright ©1956 by John Wiley & Sons, Inc. Data from The Journal of Biological Chemistry.
Selective blocks can be introduced at various points in the chain. The components on the NADH side of the block, no longer connected to an electron acceptor, will become more reduced. The components on the oxygen side of the block will be unable to receive electrons and will become more oxidized. The location of a block of this kind, where the redox state of two neighboring components sharply differs, is known as a crossover point. Specific reactions in the sequence can be blocked by means of inhibitors. Addition of the inhibitor antimycin to a metabolizing suspension blocks the oxidation. Changes in the http://www.albany.edu/~abio304/text/16part1.html (13 of 15) [3/5/2003 8:22:40 PM]
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absorption spectrum induced by the presence of antimycin are shown by the dashed line (curve 2) of Fig. 4, where it is clear that cytochromes c and a are fully oxidized, whereas cytochrome b, flavoprotein and pyridine nucleotides remain reduced. Thus, cytochromes c and a are situated on the oxygen side of the block. Similar experiments can be carried out with other inhibitors, such as amytal, which blocks between the pyridine nucleotide-linked flavoproteins and CoQ. A number of others, including British antilewisite (BAL), leave only cytochrome b, flavoprotein and pyridine nucleotide in reduced forms. In contrast, in the presence of substrate and cyanide, all carriers remain reduced. Cyanide is known to react with cyt a3; thus cytochrome a3 can be considered the terminal component of the chain, in line with its well-known reactivity with CO and the competition for binding between CO and O2. We can conclude that, as far as this experimental approach can be carried, the findings are entirely in agreement with the order shown in Figs. 6 and 7. Figure 6 summarizes the steps in the cytochrome chain. Figure 7 incorporates the iron sulfur protein detected with EPR techniques (Ohnishi and Salerno, 1982). Any functional component of the electron transport chain must respond rapidly to account for the rates observed under limiting conditions, such as sudden admission of O2 or ADP, withdrawal of ADP, or inhibition of electron transport with an appropriate inhibitor (e.g., see Klingenberg and Kroger, 1967).
Fig. 5 Parameters of the electron paramagnetic resonance spectrum of beef heart complex I at 12 mW, 9.49 MHz, at 11oK. (Courtesy of John Salerno.) The centers, g values, and redox potentials are as follows:
Center
g
g
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g
Eh(mV)
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(2Fe, 2S)
(4Fe, 4S)
N-la
2.03
1.94
1.91
-400
b
2.03
1.94
1.91
-250
N-2
2.05
1.93
1.93
-20
N-3
2.04
1.93
1.86
-250
N-4
2.10
1.93
1.88
-250
Go to Part 2 REFERENCES Search the textbook
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Back to Part 1
B. The Complexes From the very first studies of mitochondria, biochemists have attempted to break down the system into simpler fractions more amenable to elucidating the mechanisms of the reactions. The close association of the electron transport components with the mitochondrial membrane structure has made this type of analysis difficult. The citric acid cycle enzymes (e.g., isocitrate dehydrogenase, malate dehydrogenase, fumarase, condensing enzyme, and aconitase) and the enzymes for fatty acid oxidations (fatty acid synthase and thiokinases) can be brought into solution by relatively mild treatments of rat liver or beef heart mitochondria. The so-called electron transport particles (ETPs), vesicles prepared from mitochondria by sonication or mechanical disruption, can carry out all electron transport reactions from either NADH or succinate. Some lipoprotein complexes extracted from mitochondria or ETPs can carry out electron transport, but they are unable to phosphorylate. Four such complexes corresponding to portions of the electron transport chain have been isolated (see Hatefi, 1985). The reconstitution of such a system is represented in Fig. 8. The complexes do not function together without the addition of CoQ and cyt c, which are therefore presumed to have been lost during the preparation and to operate normally in the spans shown in the figure. Further fractionation of this preparation is possible with a number of disruptive procedures. As seen previously, the inner mitochondrial membranes are encrusted with particles attached to the membranes by stalks. Stalks, particles and the corresponding piece of membrane (the so-called base piece or Fo) constitute the basic unit of phosphorylation. The particles (termed F1) have ATPase activity and by all indications normally function as an ATP synthase.
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Fig. 6 Diagrammatic presentation of the respiratory chain. Roman numerals indicate probable sites of phosphorylation. Wavy arrows indicate probable sites of action of inhibitors.
Fig. 7 Respiratory chain redox components present in the inner mitochondrial membrane. Fe-S clusters associated with NADH-UQ and succinate-UQ reductase segments are designated with suffixes N-x and Sx, respectively. Qs, Qn, and Qc are protein-associated pools of ubiquinone in succinate-UQ NADH-UQ, and ubiquinol-cytochrome c reductase segments, respectively, which can be distinguished from the bulk ubiquinone pool. Reproduced by permission from Ohnishi and Salerno (1982). Iron-sulfur clusters in the mitochondria electron transport chain, in Iron Sulfur Proteins ed. T.G. Spiro, p.288, Copyright ©1982 by John Wiley and & Sons, Inc. http://www.albany.edu/~abio304/text/16part2.html (2 of 16) [3/5/2003 8:22:48 PM]
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Fig. 8 Diagrammatic representation of the sections of the electron transport chain that can be isolated.
The stalk piece is necessary for the attachment to the membrane (MacLennan and Asai, 1968). The presence of enzymes and enzyme complexes fixed to a membrane may also have other implications. Since the position of each enzyme molecule is critical, components with a structural role may be significant in the functioning of the assembly. This may be one of the reasons why the functioning of the cytochrome chain depends on the presence of lipid components. This dependence is illustrated by the experiment represented in Table 2 (Fleischer et al., 1967). As shown by sample a in the table, mitochondria can readily oxidize succinate with the concomitant reduction of cytochrome c (columns 3-5). These reactions are essentially abolished by the removal of lipids or CoQ (ubiquinone) from the system by acetone extractions (column 3, samples b-d). The reactions are readily reestablished by adding the missing components (samples b, c, and d, column 5; or sample d, column 4). This reactivation can be accounted for by the addition of CoQ to the systems containing some phospholipid (samples b and d), but where the amount of phospholipid is low, addition of CoQ by itself is insufficient to reestablish activity (samples c and e, column 4). Thus, phospholipid components are
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essential for the maintenance of the enzymatic activity. Apparently, phospholipid is required by the electron transport chain in all three of the spans tested (from succinate to CoQ, from reduced CoQ to cytochrome c, and from reduced cytochrome c to oxygen) (Green and Fleischer, 1963). Although the lipid components of the membrane may play a role by holding the various enzymes and electron carriers in appropriate positions, it is also conceivable that they help to maintain them in their appropriate conformation. In Chapter 4, we saw that many domains of integral proteins are accessible from only one phase. Similar information is available for mitochondrial complexes. Fig. 9 (Capaldi et al., 1987; Ohnishi, 1987; Ragan, 1987; Weiss, 1987) summarizes the orientation of the four electron transport complexes of mitochondria in relation to the mitochondrial inner membrane. The information comes from a variety of experimental approaches including: electron microscopy of two-dimensional crystals (see Chapter 1), the use of photoactive hydrophobic probes to label the portions in contact with the lipid components, chemical cross-linking, which provides information about the spatial relationships between the polypeptides of a complex, and the predictions of hydropathy profiles. The complexes that make up the electron transport chain and the ATP synthase complex can be separated (Hatefi, 1985) by appropriate techniques (see Figs. 8 and 10) and can be recombined with reconstitution of activity. These complexes are thought to represent enzyme assemblies present as independent units in the native membrane. Studies of the mobility of the four complexes,and in addition cytochrome c and ubiquinone by a photobleaching and recovery technique similar to the one described in Chapter 4, suggest that all these components are free to move in the inner mitochondrial membrane (Hackenbrock et al., 1986). The kinetics of the appropriate redox reactions are consistent with diffusional control of electron transport (Hackenbrock et al., 1986). We would expect ubiquinone (lipid soluble) and cytochrome c (water soluble), which have high diffusion coefficients and link the electron transport between the various complexes, to play an important role as shuttles in these exchanges. In Chapter 17, we will see that plastoquinone (lipid soluble) and plastocyanin (water soluble) play a similar role in chloroplast electron transport. The idea that the components of the electron transport chain are free to move in the plane of the membrane is supported by other observations. For example, when some of the electron transport chains are partially blocked with CO, many of them are nevertheless oxidized, but at a lower rate (Chance et al., 1970). From this observation it may be postulated that the electron transport chains are interconnected in some way. However, since the interaction is relatively slow, the possibility of diffusional movement in the plane of the membrane deserves attention. A summary of the complexes, associated components. and their stoichiometry is represented in Fig. 10 (Hatefi, 1985). Table 2 Phospholipid Content and Enzymic Activity of Beef Heart Mitochondria
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Succinate cytochrome reductasea activity after addition of (1)
(2)
(3)
(4)
(5)
Nothing
CoQ
CoQ + MPL
Sample
Treatment
P (µg/mg protien)
a. Mitochondria
None
16.8
0.59
0.52
0.53
b."Neutral lipiddepleted" mitochondria
4% water in acetone
14.5
0.02
0.84
1.00
c. "Lipid-deficient" mitochondria
10% water in acetone
3.7
0.01
0.08
0.97
10.5
0.07
0.77
1.03
2.2
0.01
0.01
0.39
c + MPL e. "Lipid-free" mitochondria
10% water in acetone + NH3
From Fleischer et al., (©1967) reproduced from The Journal of General Physiology, 1967, vol 32:193-208, by copyright permission of the Rockefeller University Press. a
micromoles cytochrome reduced per min per mg of protein at 30oC; CoQ, coenzyme Q; MPL, mitochondrial phospholipid.
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Fig. 9 Representation of the structure of the various electron transport complexes, their components and their arrangement in relation to the mitochondrial inner membrane. From left to right: NADH-ubiquinone reductase (complex I) (Ragan, 1987), succinate-ubiquinone oxidoreductase (complex II) (Ohnishi, 1987), ubiquinone-cytochrome-c reductase (complex III) (Weiss, et al., 1987) and cytochrome-c oxidase (Complex IV) (Capaldi et al., 1987). Reproduced by permission.
The transfer of electrons within a complex may involve adjacent amino acids, such as tryptophan, that are capable of transferring electrons, or it may take place through a distance by the phenomenon of electron tunneling. Electron tunneling is thought to be involved in a variety of electron transfers in mitochondria. In part, this is suggested by the evidence for short distances between the active centers (Salerno and Ohnishi, 1979). Its likelihood is indicated by the events of photosynthetic electron transport. Cytochrome oxidation in photosynthetic bacteria is independent of temperature at very low temperatures (4-lOO oK), and this is also the case for the transfer of electrons from the primary electron acceptor back to the oxidized reaction center (DeVault, 1979). A summary of our present understanding of the biochemical organization of the mitochondrial complexes is shown in Fig. 10 (Hatefi, 1985). In this diagram, the top line represents the redox potentials of the components. The corresponding mitochondrial electron transport complexes are indicated below this line. The numbers in parentheses indicate the stoichiometry of the complexes in the intact mitochondrion compared to complex I (which is taken as unity). The wavy lines indicate the site at which the listed inhibitors act. As shown, the electron transport reactions of each complex are thought to produce a translocation of protons from the mitochondrial matrix (H+m) to the cytoplasmic phase (H+c) resulting in an electrochemical proton gradient (indicated by µ+H) The translocation of protons in complex V (the FoF1, complex) in the opposite direction generates ATP from ADP and Pi. This process is discussed in more detail in Chapter 18.
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Fig. 10 Profile of the mitochondrial electron transport-oxidative phosphorylation system showing the wellcharacterized components of complexes I, II, III, IV, and V. DCCD stands for N,N'dicyclohexylcarbodiimidole and TTFA for thenoyltrifluoroacetone. The abbreviations in complex V represent various peptides or peptide assemblies (Hatefi, 1985). Reproduced, with permission, from the Annual Review of Biochemistry, vol. 54, Copyright ©1985 by Annual Reviews Inc.
C. Proton Pumping The link between the electron transport reactions and phosphorylation presumably is the production of an electrochemical gradient for H+. In mitochondria, the electron transport chain pumps H+s out of the mitochondrial matrix; in thylakoid vesicle, it pumps into the vesicle's interior. The passage of H+ in the direction of the gradient and involving the ATP synthase is coupled to the synthesis of ATP. The question of how protons are pumped by the electron transport chain is therefore of prime importance. The previous section has shown that electrons are transferred from NADH to O2 through three large
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complexes: complex I, NADH-ubiquinone oxidoreductase; complex III, ubiquinol-ferricytochrome c oxidoreductase; and complex IV, ferrocytochrome c-oxygen oxidoreductase. The most probable stoichiometry is a minimum of 4 H+ per electron pair at each coupling site (e.g., Reynafarje et al., 1976). How do the three complexes of the electron transport chain function as H+ pumps? The mechanisms are not clear, however some of the details are beginning to emerge. The NADH-ubiquinone oxidoreductase has been shown to produce at least 4 H+ per electron pair transferred through the complex (see Weiss et al., 1991). The redox potential gaps predict that the energy transduction would occur between FMN and the isopotential clusters N-1, N-3 and N-4, and between these and the N-2 cluster. Weiss et al. (1991) proposed that the reoxidation of FMNH2 is linked to the translocation of 2 H+ to the external face of the inner mitochondrial membrane. From the isopotential FeS cluster, the electrons might be transferred to an hypothesized internal quinone near the outer face of the inner mitochondrial membrane. Quinone reduction would produce an uptake of 2H+ on the matrix side and a release of 2H+ at the external face. Cluster N-2 would then transfer the two electrons across the membrane to reduce ubiquinone. Two additional H+ are taken up in the matrix side. This model would account for the 4H+ per electron pair found experimentally. This model is represented in Fig.11
(Weiss et al., 1991). Fig. 11 Model of the operation of NADH-ubiquinone oxidoreductase. From Weiss et al., 1991. Reproduced by permission.
Much of our knowledge of complex III, the ubiquinol-cytochrome c oxidoreductase (the bc1 complex) comes from the bacterial bc1 systems which catalyze the same reactions as the eukaryotic equivalents, but with less subunits. Use of the bacterial systems also permits the use of genetic techniques. An additional advantage of the use of bacteria rests on the presence of alternative pathways that allow
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metabolism despite a malfunctioning bc1 complex. Furthermore, in bacteria the components of the complex are coded by the conventional genetic apparatus, whereas in eukaryotes cytochrome b is coded by mitochondrial DNA (see Gennis et al., 1993).
Fig. 12 Protonmotive Q-cycle of the mitochondrial bc1 complex. See text. The open bars indicate a block by inhibitors of electron transport, ISP stands for FeS protein. The broken lines indicate the movement. From Trumpower (1990a) reproduced by permission.
In the so-called protonmotive Q-cycle pathway, electrons are transferred from ubiquinol to cytochrome c in a branched pathway known as the protonmotive Q cycle (Fig.12, Trumpower, 1990a) (see Trumpower, 1990b). In this process, one molecule of ubiquinol is oxidized and two molecules of cytochrome c1 are reduced. This generates four protons on the outer side of the mitochondrial inner membrane and consumes two protons on the inside. The process occurs in two phases. In the first, one electron is removed from ubiquinol via FeS protein (ISP) and cytochrome c1. The resulting ubisemiquinone dissociates to the anion and one H+ (step 1). This step generates two H+ at the outer surface (one from the reduction and the other from the ionization). The second electron from the ubisemiquinone, reduces cytochrome b-566 (bL)(step 2) and is transferred to b-560 (bH)(step 3). Ubiquinone at the inner surface is then reduced to the ubisemiquinone anion by accepting an electron from b-560 (step 4) at the inner mitochondrial surface. In the second phase, one additional ubiquinol molecule is oxidized in the same way (step 1) with the production of 2H+ at the outer surface and one electron following the cytochrome c pathway. Through step 2 and 3, Cytochrome b-560 transfers one electron to the ubisemiquinone anion at the inner surface (formed in the previous phase). The reduction of the ubisemiquinone anion to ubiquinol consumes 2 H+ (step 5 and 6). For one complete cycle (involving both phases) two molecules of ubiquinol are oxidized to ubiquinone, http://www.albany.edu/~abio304/text/16part2.html (9 of 16) [3/5/2003 8:22:49 PM]
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one molecule of ubiquinol is formed by reduction of one of these two quinones, two molecules of cytochrome c are reduced, and 4 H+ are formed at the outer surface. 4 H+ are produced per electron pair. The FeS protein, cyt c1 and the b cytochromes go through two redox turnovers. Alternatively, b-560 reduces ubiquinone to ubisemiquinone anion and ubisemiquinone to ubiquinol. As represented in the diagram, cyt b must traverse the membrane, FeS protein and cyt c1 must be at the outer portion of the membrane. All of the presently available observations on electron transport and proton translocation are consistent with this model. As already suggested, the functioning of complex III is very dependent on the geometry of the system. The complex is a dimer. Each monomer is a complex of 11 polypeptides and 250 kDa in molecular weight which contains b heme, c1 heme and an iron sulfur protein (ISP) (the Rieske protein) in a 2:1:1 stoichiometry. The arrangement of the subunits in relation to the inner mitochondrial membrane were defined by biochemical studies (e.g., Gonzalez-Halphen et al., 1988) that included proteinase digestion, the use of lipid soluble probes and crosslinking studies. The latter allowed detecting the location of the various subunits in relation to each. The position of the various players is that suggested in Fig. 13 (Smith, 1998). The transmembrane region spans 38 Å and contains most of cytochrome b. The largest portion of the complex (about 75 Å) extends into the mitochondrial matrix. A large part of cytochrome c1 and ISP represent a projection of 38 Å into the intermembrane space (that is in the cytoplasmic side). Crystallographic studies provided more information. The initial study (Xia et al., 1997) of 80% of the complex with a resolution of 2.9 Å, gave the general features of the complex, including the positions of the metal centers. Zhang et al. (1998) reported a 3 Å resolution structure of 9 of the subunits with complete tracing of the extrinsic domains of cytochrome c1 and the Rieske subunits. Iwata et al. (1998) report two new crystal forms of the bovine cytochrome bc1 complex. The position of the hemes bL and bH and c1 are invariant. The external domain of the Rieske Fe2S2 center moves. In one position, it is close to QP, allowing for the reduction of the ISP. In another, it is close to the heme of cytochrome c1, permitting its oxidation (Zhang et al., 1998; Iwata et al., 1998; Kim et al., 1998). In one conformation, the histidines that protrude from the ISP bind the cytochrome b in close proximity to the QP. Release from this binding is controlled by the redox state of QP. Oxidation releases the histidines from the cytochrome b binding (Zhang et al., 1998; Kim et al., 1998) and allows them to move close to the heme
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of cytochrome c1. Fig. 13 Structure of cytochrome bc1. The hollow between its two monomers allow the shuttling of ubiquinol/ubiquinone to and from the complex and betwen Qp and QN. The Rieske extrinsic domain (represented by the plug) also shuttles between a site near Qp (the socket) and cytochrome c1. The passage of each electron from Qp through Rieske and cytochrome c1 to cytochrome c displaces 2 H+. Reproduced with permission from Smith J.L., Science 281:58-59. Copyright ©1998 American Association for the Advancement of Science. http://www.sciencemag.org
The presence as a dimer is required for the bc1 complex to carry out its biochemical reactions. This is indicated by the location of the ISP intrinsic and extrinsic domains in opposite monomers and the significance of this arrangement in relation to the Q cycle, which explains how the transfer of two electrons through the complex can give rise to 4 H+ (shown in Fig. 13). The major role in the cycle is played by hemes bL and bH and the adjacent ubiquinol/ubiquinone sites (QP and QN), all attached to the transmembrane domain of cytochrome b. In the Q cycle, QP is at the site of bifurcation in the pathway of electron transport. One electron supposedly goes to the ISP extrinsic domain and the other is cycled through the b hemes to return to QN. These processes require a specific geometry. The ubiquinone species must be free to move in and out of the bc1 complex. In addition there must be a rapid exchange between QP and QN. These needs are taken care of by the arrangement of the bc1 dimer. Together, two cytochrome b proteins form a space accessible from the bilayers with an opening of 25 Å. The opening allows rapid exchanges of the very lipid soluble Qs. The transmembrane helices of the cytochrome bc1 form the walls and its external domains form the floor and ceiling. QP and QN are present in the walls, each in different monomers of the composite complex.
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The structure of cytochrome c-oxidase has been elucidated at high resolution on both bacteria (Iwata et al., 1995) and mammals (Tsukihara et al., 1995). 4H+ are produced per electron pair transferred through the complex. At least when reconstituted in liposomes, cytochrome c oxidase translocates two protons during its oxidation and two during its re-reduction (Verkhovsky et al., 1999). The electrons are thought to follow a linear path: c CuA a3-CuB. The bacterial complex is formed by only four subunits, whereas the eukaryotic complex has thirteen. This discussion will generally refer to the simpler system. The bacterial subunits I, II and III are common to all members of the heme-copper oxidase family. The arrangement is represented in Fig.14 (Gennis and Ferguson-Miller, 1996). Cytochrome c oxidase has four metal redox centers which are contained in subunits I and II. One redox center (CuA) is in subunit II. Heme a and the heme a3-CuB bimetallic center are in subunit I and they are at approximately the same level: 15 Å below the outer surface of bacteria. Heme a is ligated to two histidines, heme a3 to a single histidine and CuB to three. The interaction with oxygen occurs at the heme a3-CuB site which is deep in the protein. It follows that there must be pathways (referred to as channels) for the passage of the protons from or to the appropriate surface and the redox centers of the molecule. Water molecules and amino acid side chains act as proton wires (see Nagle et al., 1980; Rammelsberg et al., 1998). These routes that could function as conduits for the protons have been implicated by site-directed mutagenesis (e.g., Thomas et al., 1993; Fetter et al., 1995) . One of the channels involved in the uptake of protons from the mitochondrial interior (the D channel) is lined with polar residues (e.g., Ostermeier et al., 1997) and a chain of hydrogen-bonded water (e.g., see Riistama et al., 1997; Hofacker and Schulten, 1998). The D channel ends at a hydrophobic cavity containing water molecules. Other channels are less certain. The actual pumping mechanism is still a mystery. Some models propose a displacement of the histidines which ligate the heme a3-CuB center and undergo cyclic protonation-deprotonation to produce a unidirectional proton flow (Morgan et al., 1994). A possible mechanism for pumping protons by cytochrome c oxidase, might involve movement of an acidic group of the protein which would be exposed alternatively to the two faces of the inner mitochondrial membrane depending on the redox state of the heme a3-CuB sites. To function as a pump, the movement would have to be accompanied by a change in pK. This mechanism has been proposed based on crystallographic data from bovine heart cytochrome c oxidase (Yoshikawa et al., 1998). The results of this study are consistent with a movement of an aspartate residue (Asp51). With the reduction of the enzyme the aspartate becomes accessible to the external phase, a change accompanied by a decrease in pK . The results also suggest an acidified tyrosine residue as a source of protons for the oxygen reduction. These events require only minor changes in structure that can be deduced by comparing the crystal structure in various states. In this model, in the oxidized state, Asp51 is connected to the inner surface by a network that includes a http://www.albany.edu/~abio304/text/16part2.html (12 of 16) [3/5/2003 8:22:49 PM]
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peptide unit, hydrogen bonds, a cavity and a water path. The network interacts with heme a3. Upon reduction, Asp51 loses its accessibily to the matrix phase and is accessible from the intramembrane space. Studies of another redox system, that of ferredoxin I of Azotobacter vinelandii lend support to this kind of model. Ferredoxin is a small protein with a buried 3Fe-3S cluster. A single long-range electroncoupled proton transfer occurs in ferredoxin I. Reduction of the Fe-S cluster brings in a proton from the water. In contrast, re-oxidation requires a proton release to the water. The proton is directed through the protein matrix by movement of the side-chain of an aspartate (Asp 15) close to the surface. The pK of the aspartate changes in response to the electrostatic charge of Fe-S cluster (Chen et al., 2000).
Fig. 14 Model of bacterial cytochrome c oxidase based on the crystal structure and site directed mutagenesis, showing predicted pathways and key residues. Reproduced from Gennis and FergusonMiller (1996) by permission. (Available in the BioMedNet library at: http://biomednet.com/cbiology/cub)
Presumably, the main function of mitochondrial electron transport is the production of ATP from ADP and Pi. One of the functions of photosynthesis is to support electron transport coupled to the phosphorylation of ADP. Therefore, the similarity between the two systems is considerable, so the oxidative and photophosphorylative reactions are best discussed together. For this reason, various aspects of photosynthesis are presented in Chapter 17, whereas the phosphorylative reactions of the mitochondrial and chloroplast electron transport chains are treated together in Chapter 18. SUGGESTED READING Overall view Capaldi, R.A. (2000) The changing face of mitochondrial research, Trends Biochem. Sci. 25:212-214. http://www.albany.edu/~abio304/text/16part2.html (13 of 16) [3/5/2003 8:22:49 PM]
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(MedLine) Saraste, M.(1999) Oxidative phosphorylation at the fin de siècle, Science 283:1488-1493. (Medline) Wallace, D.C. (1999) Mitochondrial diseases in man and mice, Science 283:1482-1488. (Medline) Other references Cramer, A.W. and Knaff, D.B. (1990) Energy Transduction in Biological Membranes, Chapters 3,4 and 5. Springer-Verlag, New York. Gennis, R.B. and Ferguson-Miller, S. (1996) Protein structure; Proton-pumping oxidases, Curr. Biol. 6:36-38. (Medline) Nicholls, D. (1984) Mechanisms of energy transduction. In Bioenergetics (Ernster, L., ed.), pp. 29-48. Elsevier, New York. Tzagoloff, A. (1982) Mitochondria, Chapters 2-5. Plenum, New York. Wikstrom, M. and Saraste, M. (1984) The mitochondrial respiratory chain. In Bioenergetics (Ernster, L., ed.), pp. 49-94. Elsevier, New York. Alternative References Harold, F.M. (1986) The Vital Force: A Study Bioenergetics, Chapter 7, pp. 197-250. W.H. Freeman, New York. Nicholls, D.G. and Ferguson, S.J. (1992) Bioenergetics 2, Academic Press, Chapter 1-8. Structure of mitochondria Frey, T.G. and Mannella, C.A. (2000) The internal structure of mitochondria, Trends Biochem. Sci. 25:319-324. (MedLine) Special Aspects Capaldi, R.A., Takamiya, S., Zhang, Y. -Z., Gonzalez-Halphen, D. and Yanamura, W. (1987) Structure of cytochrome-c oxidase, Curr. Top. Bioenerg. 15:91-108. Ehrlich, H. (1995) Geomicrobiology, 3rd ed., Dekker, New York.
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Friedrich, T., Steinmüller, K. and Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts, FEBS Lett. 367:107. (Medline) Hackenbrock, C.R., Chazotte, B. and Gupte, S.S. (1986) The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport. J. Bioenerg. Biomembr. 18:331368. (Medline) Hatefi, Y., Ragan, I. and Galante, Y.M. (1985) The enzymes and the enzyme complexes of the mitochondrial oxidative phosphorylation system. In The Enzymes of Biological Membranes, 2d ed., Vol. 4, pp. 1-70 (Martonosi, A.N., ed.). Plenum, New York. Jones, C.W. (1988) Membrane associated energy conservation in bacteria: a general introduction. In Bacterial Energy Transduction (Anthony, C., ed.), pp. 1-82. Academic Press, New York. Kadenbach, B., Kuhn-Nentwig, I. and Buge, U. (1987) Evolution of a regulatory enzyme: cytochromec oxidase (complex IV). Curr. Top. Bioenerg. 15:114-151. Mitchell, P. (1987) Respiratory chain systems in theory and practice, in Advances in Membrane Biochemistry and Bioenergetics (Kim, C.H., Tedeschi, H., Diwan, J.J. and Salerno, J.C., eds.), pp. 25-52. Plenum, New York. Moynagh, P.N. (1995) Contact sites and transport in mitochondria, Essays in Biochemistry 30: 1-14. (Medline) Ohnishi, T. (1987) Structure of the succinate-ubiquinone oxido-reductase (complex II), Curr. Top. Bioenerg. 15:37-66. Ragan, C.I. (1987) Structure of NADH-ubiquinone reductase (complex I), Curr. Top. Bioenerg. 15:1136. Rutter, G.A. and Rizzuto, R. (2000) Regulation of mitochondrial metabolism by ER Ca2+ release: an intimate connection, Trends Biochem. Sci. 25:215-221. (MedLine) Senior, A.E. (1988) ATP synthesis by oxidative phosphorylation, Physiol. Rev. 68:177-231. Senior, A.E. (1990) The proton translocating ATPase of Escherichia coli, Annu. Rev. Biophys. Chem. 19:7-41.(Medline) Tolbert, N.E. (1981) Metabolic pathways in peroxisomes and glyoxysomes, Annu. Rev. Biopys. Chem. 50:133-158.(Medline)
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16. Oxidative Phosphorylation and Mitochondrial Organization
Weiss, H. (1987) Structure of mitochondrial ubiquinol-cytochrome c reductase (complex III), Curr. Top. Bioenerg. 15:67-90. Wikström, M. (1998) Proton translocation by bacteriorhodopsin and heme-copper oxidases, Curr. Opin. Struct. Biol. 8:480-4888. (MedLine) Yoshida, M., Muneyuki, E. and Hisabori, T. (2001) ATP synthase - a marvellous rotary engine of the cell, Nature. Rev. Mol. Cell Biol. 2:669-677. (MedLine) WEB RESOURCES Diwan, J.J., Electron transfer chain, http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/redox.htm REFERENCES Search the textbook
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Chapter 16: References
Back to Chapter 16
REFERENCES Anagnosti, E. and Tedeschi, H. (1970) Mechanism of low-amplitude orthophosphate-induced swelling in isolated mitochondria, J. Cell Biol. 47:520-525. Ashhurst, D.E. (1965) Mitochondrial particles seen in sections, J. Cell Biol. 244:497-499. Beckman, S. and Kanarek, L. (1981) Demonstration of physical interaction between consecutive enzymes of the citric acid cycle and the aspartate-malate shuttle, Eur. J. Biochem. 117:527-535. Capaldi, R.A., Takamiya, S., Zhang, Y.-Z., Gonzalez-Halphen, D. and Yanamura, W. (1987) Structure of cytochrome-c oxidase,Curr. Top. Binenerg. 15:91-113. Chance, B. and Williams, G.R. (1956) The respiratory chain and oxidative phosphorylation, Adv. Enzymol. 17:65-134. Chance, B., Erecinska, M. and Wagner, M. (1970) Mitochondrial responses to carbon monoxide toxicity, Ann.N.Y. Acad. Sci. 174:193-204.(Medline) Chen, K., Hirst, J., Camba, R., Bonagura, C.A., Stout, C.D., Burgess, B.K. and Armstrong, F.A. (2000) Atomically defined mechanism for proton transfer to a buried redox centre in a protein, Nature 405:814817. Coon, M.J., Ding, X., Pernecky, S.J. and Vaz, A.D.N. (1992) Cytochrome P450: progress and predictions, FASEB J. 6:669-673.(Medline) Daems, W.T. and Wisse, E. (1966) Shape and attachment of the cristae mitochondriales in mouse hepatic cell mitochondria, J. Ultrastruct. Res. 16:123-140. (MedLine) DeVault, D. (1979) Introduction to biological aspects. In Tunneling in Biological Systems (Chance, B., DeVault,D.C., Fraunfelder, H., Marcus, R.A., Schrieffer, J.R. and Sutin, N., eds.), pp. 303-316. Academic Press, New York. Ehrlich, H. (1995) Geomicrobiology, 3rd ed., Dekker, New York.
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Chapter 16: References
Erdmann, R., Veenhuis, M. and Kunau, W.-H. (1997) Peroxysomes: organelles at the crossroads, Trends Cell. Biol. 7:400-407. Fetter, J.R., Qian, J., Shapleigh, J., Thoams , J.W., Garcia-Horsman, A., Schmidt, E., Hosler, J.P., Babcock, G., Gennis, R.B. and Ferguson-Miller, S, (1995) Possible proton relay pathway in cytochrome c oxidase, Proc. Natl. Acad. Sci. USA 92:1604-1608.(Medline) Flatmark, T. and Pederson, J.I. (1975) Brown adipose tissue mitochondria, Biochim. Biophys. Acta 416:53-103.(Medline) Fleischer, S., Fleischer, B. and Stoekenius, W. (1967) Fine structure of lipid depleted mitochondria, J. Cell Biol. 32:193-208. Frey, T.G. and Mannella, C.A. (2000) The internal structure of mitochondria, Trends Biochem. Sci. 25:319-324. (MedLine) Friedrich, T., Steinmüller, K. and Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts, FEBS Lett. 367:107.(Medline) Garlid, K.D. and Beavis, A.D. (1985) Swelling and contraction of the mitochondrial matrix. II. Quantitative application of the light scattering technique to solute transport across the inner membrane, J.Biol.Chem. 260:13434-13441. (MedLine) Gennis, R.B. and Ferguson-Miller, S. (1996) Protein structure; Proton-pumping oxidases, Curr. Biol. 6:36-38.(Medline) Gennis, R.B., Barquera, B., Hacker, B., Van Doren, S.R., Arnaud, SE., Crofts, A.R., Davidson, E., Gray, K.A. and Daldal, F. (1993) The bc1 complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus, J. Bioenerg. Biomembr. 25:195-209.(Medline) Goldenberg, H. (1982) Plasma membrane redox activities, Bichim. Biophys. Acta 694:203-223.(Medline) Gonzalez-Halphen, D., Lindorfer, M.A. and Capaldi, R.A. (1988) Subunit arrangement in beef heart complex III, Biochemistry 27:7021-7031.(Medline) Gould, S.J. and Valle, D. (2000) Peroxisome biogenesis disorders: genetics and cell biology, Trends Genet. 16:340-345. (MedLine) Green, D.E. and Fleischer, S. (1963) The role of lipids in mitochondrial electron transfer and oxidative phosphorylation, Biochim. Biophys. Acta 70:554-581.
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Chapter 16: References
Green, D.E., Allman, D.W., Bachmann, E., Baum, H., Kopaczyk, K., Korman, E.F., Lipton, S., MacLennan,D.H., McConnell, D.G., Perdue, J.F., Rieske, J.S. and Tzagoloff, A. (1967) Formation of membranes by repeating units, Arch. Biochem. Biophys. 119:312-325.(Medline) Guengerich, F. P. (1992) Cytochrome P450: advances and prospects, FASEB J. 6, 667-668.(Medline) Hackenbrock, C.R. (1966) Ultrastructural bases for metabolically linked mechanical activity in mitochondria. I. Reversible ultrastructural changes with change in metabolic steady state in isolated liver mitochondria, J. Cell Biol. 30:269-297.(Medline) Hackenbrock, C.R., Chazotte, B. and Gupte, S.S. (1986) The random collision model and a critical assessment of diffusion and collision in mitochondrial electron transport, J. Bioenerg. Biomembr. 18:331368.(Medline) Hackstein, J.H., Akhmanova, A., Boxma, B., Harhangi, H.R. and Voncken, F.G. (1999) Hydrogenosomes: eukaryotic adaptations to anaerobic environments, Trends Microbiol. 7:441-447. (MedLine) Hall, J.D. and Crane, F.L. (1970) An intracristal structure in beef heart mitochondria, Exp. Cell Res. 62:480-483.(Medline) Halper L.A. and Srere, P.A. (1977) Interaction of citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethelene glycol, Arch. Biochem. Biophys. 184:529-534.(Medline) Hashimoto, T. (1982) Individual peroxisomal β-oxidation enzymes, Ann. N.Y. Acad. Sci. 386:512.(Medline) Hatefi, Y. (1985) The mitochondrial electron transport and oxidative phosphorylation system, Annu. Rev. Biochem. 54:1015-1069.(Medline) Hofacker, I. and Schulten, K. (1998) Oxygen and proton pathways in cytochrome c oxidase, Proteins 30:100-107. (MedLine) Huang, A.H.C., Trelease, R.N. and Moore, T.S., Jr. (1983) Plant Peroxisomes, Chapter 4, pp. 87-155. Academic Press, New York. Iwata, S., Ostermeieer, C., Ludwig, B. and Michel, H. (1995) Structure at 2.8 resolution of cytochorme c oxidase from Paracoccus denitrificans, Nature 376:660-669.(Medline) Iwata, S., Lee, J.W., Okada, K., Lee, J.K. Iwata, M., Rasmussen, N., Link, T.A., Ramaswamy, S. and http://www.albany.edu/~abio304/ref/ref16.html (3 of 8) [3/5/2003 8:23:00 PM]
Chapter 16: References
Jap, B.K. (1998) Complete structure of the 11-subunit bovine mitochondrial cyrochrome b1 complex, Science 281:64-71.(Medline) Izzard, S. and Tedeschi, H. (1970) Ion transport underlying metabolically controlled volume changes of isolated mitochondria, Proc. Natl. Acad. Sci. U.S.A. 67:702-709.(Medline) Kim, H., Xia, D., Yu, C.A., Xia, J.Z., Kachurin, A.M., Zhang, L., Yu, L. and Deisenhofer, J. (1998) Inhibitor binding changes domain mobility in the iron-sulfur protein of the mitochondrial bc1 complex from bovine heart, Proc. Natl. Acad. Sci. USA 95:8026-8033.(Medline) Kimura, Y., Vassylyev, D.G., Miyazawa, A., Kidera, A., Matsushima, M., Mitsuoka, K., Murata, K., Hirai, T. and Fujiyoshi, Y. (1997) Surface of bacteriorhodopsin revealed by high-resolution electron crystallography, Nature 389:206-211. (MedLine) Klingenberg, M. and Kroger, A. (1967) On the role of ubiquinone in the respiratory chain. In Biochemistry of Mitochondria (Slater, E.C., Kaniuga, Z. and Wojtczak, L., eds.), pp. 11-27. Academic Press, New York. Lea, P.J., Temkin, R.J., Freeman, K.B., Mitchell, G.A. and Robinson, B.H. (1994) Variations in mitochondrial ultrastructure and dynamics observed by high resolution scanning electron microscopy (HRSEM), Micr. Res. and Tech. 27:269-277.(Medline) Lee, S.S.T., Pineau, T., Drago, J., Lee, E.J., Owens, J.W., Kroetz, D.L., Fernandez-Salguero, P.M., Westphal, H. and Gonzalez, F.J. (1995) Targeted disruption of the alpha isoform of the peroxisome proliferator-activated receptor gene in mice results in abolishment of the pleiotropic effects of peroxisome proliferators, Mol. Cell. Biol. 15:3012-3022. (MedLine) Lock, E.A., Mitchell, A.M. and Elcombe, C.R. (1989) Biochemical mechanisms of induction of hepatic peroxisome proliferation, Annu. Rev. Pharmacol. Toxicol. 29:145-163. (MedLine) MacLennan, D.H. and Asai, J. (1968) Studies on the mitochondrial adenosine triphospbatase system. V. Localizationof the oligomycin-sensitivity conferring protein, Biochem. Biophys. Res. Commun. 33:441447.(Medline) Mannaerts, G.P. and Debeer, L.J. (1982) Mitochondrial and peroxisomal beta-oxidation of fatty acids in rat liver, Ann. N.Y. Acad. Sci. 386:30-38.(Medline) Mannella, C.A., Marko, M., Penczek, P., Barnard, D. and Frank, J. (1994) The internal compartmentation of rat-liver mitochondria : tomographic study using the high-voltage transmission electronmiscocope, Micr. Res. and Tech. 27:278-283.(Medline)
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Chapter 16: References
Mannella, C.A., Buttle, K., Rath, B.K. and Marko, M. (1998) Electron microscopic tomography of ratliver mitochondria and their interaction with the endoplasmic reticulum, Biofactors 8:225-228.(Medline) Marshall, P.A., Dyer, J.M., Quick, M.E. and Goodman, J.M. (1996) Redox-sensitive homodimerization of Pex11p: a proposed mechanism to regulate peroxisomal division, J. Cell Biol. 135:123-137. (MedLine) Morgan, J.E., Verkhovsky. M. I. and Wikström, M. (1994) The histidine cycle; a new model for proton translocation in the respiratory heme-copper oxidases, J. Bioenerg, Biomembr. 26:599-608.(Medline) Nagle, J.F., Mille, M. and Morowitz, H.J. (1980) Theory of hydrogen bonded chains in bioenergetics, J. Chem. Phys. 72:3959-3971. Nedergard, J. and Cannon, B. (1984) Thermogenic mitochondria. In Bioenergetics (Ernster, L., ed.), pp. 291-314. Elsevier, New York. Ohnishi, T. (1987) Structure of the succinate-ubiquinone oxidoreductase (complex II), Curr. Top. Bioenerg. 15:37-66. Ohnishi, T. and Salerno, J.C. (1982) Iron-sulfur clusters in the mitochondrial electron-transport chain. In Iron Sulphur Proteins (Spiro, T.G., ed.), pp. 285-327. Wiley, New York. Osmundsen, H. (1982) Peroxysomal β-oxidation of long fatty acids: effects of high fat diet, Ann. N.Y. Acad. Sci. 386:12-27.(Medline) Ostermeier, C., Harrenga, A., Ermler, U. and Michel, H. (1997) Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody FV fragment, Proc. Natl. Acad. Sci. USA 94:10547-10553. (MedLine) Parson, D.F. (1963) Mitochondrial structure: two types of subunits on negatively stained mitochondrial membranes, Science 140:985-988. Perkins, G.A. and Frey ,T.G. (2000) Recent structural insight into mitochondria gained by microscopy, Micron 31:97-111. (MedLine) Perkins, G., Renken, C., Martone, M.E., Young, S.J., Ellisman, M. and Frey, T. (1997) Electron tomography of neuronal mitochondria: three-dimensional structure and organization of cristae and membrane contacts, J. Struct. Biol. 119:260-272. (MedLine> Ragan, C.I. (1987) Structure of NADH-ubiquinone reductase (complex I), Curr. Top. Bioenerg. 15:1-36.
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Chapter 16: References
Rammelsberg, R., Huhn, G., Lubben, M. and Gerwert, K. (1998) Bacteriorhodopsin's intramolecular proton-release pathway consists of a hydrogen-bonded network, Biochemistry 37:5001-5009. (MedLine) Reynafarje, B., Brand, M.D. and Lehninger, A.L. (1976) Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements, J. Biol. Chem. 251:7442-7451.(Medline) Riistama, S., Hummer, G., Puustinen, A., Dyer, R.B., Woodruff, W.H. and Wikström M. (1997) Bound water in the proton translocation mechanism of the haem-copper oxidases, FEBS Lett. 414:275-280. (MedLine) Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz, L.M., Tuft, R.A. and Pozzan, T. (1998) Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses, Science 280:1763-1776.(Medline) Roux, B., Nina, M., Pomes, R. and Smith, J.C. (1996) Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study, Biophys. J. 71:670-681. (MedLine) Salerno, J.C., and Ohnishi, T. (1979) Electron transport in the succinate ubiquinone segment of the respiratory chain. In Tunneling in Biological Systems (Chance, B., DeVault, D.C., Fraunfelder, H., Marcus, B.A., Schrieffer, J.R. and Sutin, N., eds.), pp. 473-482. Academic Press, New York. Schneider, D.L., Kagawa, Y. and Racker, E. (1972) Chemical modification of the inner mitochondrial membrane, J. Biol. Chem. 247:4074-4079.(Medline) Schoonjans, K., Staels, B. and Auwerx, J. (1996) The peroxisome proliferator activated receptors (PPARS) and their effects on lipid metabolism and adipocyte differentiation, Biochim. Biophys. Acta. 1302:93-109. (Medline) Smith, J.L. (1998) Secret life of cytochrome bc1, Science 281:58-9. (MedLine) Sommerville, C.R. and Ogren, W.L. (1980) Photorespiration mutants in Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase, Proc. Natl. Acad. Sci. U.S.A. 77:2684-2687. Subramani, S., Koller, A. and Snyder, W.B. (2000) Import of peroxisomal matrix and membrane proteins, Annu. Rev. Biochem. 69:399-418. (MedLine) Sumegi, B. and Srere, A. (1984) Complex I binds several mitochondria NAD-coupled dehydrogenases, J. Biol. Chem. 259:15040-15045.(Medline)
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Chapter 16: References
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17. Photosynthesis
17. Photosynthesis I. The Photosynthetic Membranes II. The Events of Photosynthesis: Light and Dark Reactions III. Photooxidation A. Organization of the Reaction Centers B. Cyclic Photophosphorylation of Bacteria C. Production of Reducing Equivalents in Bacteria D. The Special Case of Bacteriorhodopsin IV. The Two Photochemical Systems of Chloroplasts V. Components of the Chloroplast Photosynthetic Systems A. PSII B. PSI C. The Cytochrome b6f Complex VI. VII. VIII. IX.
The Light-Harvesting System Coordination Between PSI and PSII Protection from Photo-oxidation Photosignalling Web resources Suggested Reading References Back to List of Chapters
With few exceptions, such as the chemolithotrophic bacteria, the ultimate source of most of the energy used by biological systems is sunlight. Consequently, almost all organisms depend directly or indirectly on the conversion of radiant energy to chemical free energy. The heterotrophs, which do not carry out photosynthesis, degrade complex molecules provided by other organisms. The overall process of photosynthesis is frequently represented as shown in Eq. (1). CO2 + 2H2A + light → [CHOH] + H2O + 2A (1) In green plants, the A of equation (1) corresponds to oxygen, which originates from water as shown by the equation. This has been demonstrated with 18O-labeled water (Stemler and Radmer, 1975). In some bacteria, the A of equation (1) corresponds to S [Eq. (2)] and even S and H2O [Eq. (3)].
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17. Photosynthesis
CO2 + 2H2S + light → (CHOH) + H2O+ 2S (2) 3CO2 + 2S + 5H2O + light → 3(CHOH) + 2H2SO4 (3) In bacteria, the source of carbon may be not CO2 but rather an organic molecule, and the final product may be not a carbohydrate but some other reduced organic compound. Several of the known schemes are summarized in Table 1 (Stanier, 1961). Table 1 Characteristics of Photosynthetic Organisms
Green plants
Cyanobacteria
Green sulfur bacteria
Purple bacteria
Source of reducing power
H2O
H2O
H2S, reduced organic compounds
H2S, reduced inorganic & organic compounds
Photosynthetic O2 evolution
Yes
Yes
No
No
Principle source of carbon
CO2
CO2
CO2
CO2 or organic compounds
Relation to oxygen
Aerobic
Aerobic
Strictly anaerobic
Strictly or faculatively anaerobic
Site
Chloroplast
Membrane-like structures
Chlorosomes
Internal membranes (chromatophores)
The absorption of light quanta and the subsequent reactions that convert radiant energy to chemical energy are included in what we call photosynthesis. The chlorophylls (sometimes abbreviated in this discussion as Chl) are the most abundant pigments, but many others (such as carotenoids and, in some algae, phycobilins) are also present. Many of these pigment molecules absorb light and funnel the energy http://www.albany.edu/~abio304/text/17part1.html (2 of 21) [5/5/2002 6:53:20 PM]
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to the reaction centers, which are involved more directly in energy transduction. The characteristics of some of the pigments are shown in Fig. 1. Chlorophylls a and b are the predominant types of Chl in plants and cyanobacteria (Fig. 1a). Purple photosynthetic bacteria contain bacteriochlorophyll (BChl), either a or b, whereas green bacteria have a third type of BChl. Chlorophyll and pheophytins, shown in Fig. 1a, resemble protoporphyrin IX, the prosthetic group of hemoglobin and the c-type cytochromes. In contrast to those proteins, in Chl, Fe is replaced by Mg2+ and, in pheophytins by 2H+. Fig. 1b shows βcarotene, a major carotene in many plants, and spheroidine, a bacterial carotenoid. The phycobilins, many of which are poorly characterized, are not shown; they are proteins containing tetrapyrroles similar to the bile pigments in animals. Phycobilins are present in large complexes, the phycobilisomes of cyanobacteria (blue-green algae) and red algae. The phycobilisomes absorb light over a wide spectral region. All the reactions involved in light absorption, transfer of energy, electron transport, and phosphorylation are associated with membranes. Not surprisingly, the latter two resemble the processes in oxidative phosphorylation discussed in Chapter 16. I. THE PHOTOSYNTHETIC MEMBRANES The chloroplasts present in eukaryotes are discrete structures enclosed by membranes. They may be spiral-shaped, cup-shaped, star-shaped, or irregular. In higher plants and bryophytes, they are generally shaped like saucers and are approximately 5 to 10 µm in diameter. The light-driven reactions, as well as the biochemical pathways involved in the fixation of CO2, occur in chloroplasts. Fig. 2 (Staehelin, 1986) shows a chloroplast of a red alga. A thin section of chloroplast of a higher plant as seen with the electron microscope is shown in Fig. 3 (Park, 1966). Fig. 4 illustrates the probable arrangement of a chloroplast similar to that of Fig. 3. The membrane enclosing the organelles is double. The internal membrane system of flattened vesicles or sacs, the thylakoids, is embedded in the chloroplast interior, the stroma. These vesicles may be closely apposed in a combination of shorter and longer lamellae. These composite structures form stacks, the grana. The grana stacks are interconnected by lamellae (the stroma lamellae), so at least some of the thylakoid inner spaces are continuous with those in other stacks. Parts of the thylakoid surface are apposed to other thylakoid surfaces and other parts, such as the stroma lamellae, are exposed to the chloroplast interior. Although the general features remain the same, the details may vary considerably, even within the same cell. The thylakoids have been shown to contain the chlorophyll as well as the light harvesting complexes (LHCs), the reaction centers (RCs), and the components of the electron transport chain, which will be discussed later. The soluble material contains the enzymes responsible for carbon dioxide fixation and the biochemical synthetic pathways. The significance of structural details, such as the stacking, is not clear, since mutants lacking grana are functional (Goodenough et al., 1969; Goodenough and Staelin, 1971).
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Fig. 1 Structures of photosynthetic pigments. (a) Chlorophylls & pheophytins.
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Fig. 1 Structures of Photosynthetic pigments (Continued). (b) Carotenoids.
Fig. 2 Section through a chloroplast of the red alga Spermothamnion tuneri. The phycobilosomes are attached to the thylakoids (T). EM, Envelope membrane; GDT, girdle thylakoid. Bar corresponds to 769 nm. Reproduced by permission from Staehelin (1986).
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Fig. 3 Thin section of KMnO4-fixed Spinacea oleracea chloroplast, bar corresponds to 769 nm.
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Reproduced by permission from Park (1966).
Fig. 4 Chloroplast thylakoid membrane architecture viewed in two and in three dimensions. This drawing of a sectioned chloroplast is intended to aid in visualizing the relationship between the appearance of thylakoid membranes in two dimensions with that in three dimensions. From D. R. Ort (1986), Encyclopedia of Plant Physiology, with permission. Copyright ©1986 Springer-Verlag.
In bacteria (Fig. 5) (Sprague and Varga, 1986), the morphology of the photosynthetic membranes varies. The cell membrane may be directly involved, or complex invaginations of the cell membrane or special vesicles, such as the chlorosomes, may be present. Vesicles prepared from photosynthetic bacteria, the chromatophores, have been found to be active in photosynthesis.
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Fig. 5 Morphologies observed in the anoxygenic photosynthetic bacteria for housing the photosynthetic apparatus. The green bacteria contain chlorosomes attached to the inner side of the cytoplasmic membrane (CM). Most purple bacteria elaborate an extensive membrane system within the cytoplasm for the photosynthetic components. A few "simple" bacteria contain only a single CM, which contains the pigments and proteins necessary for photosynthetic growth. From S. G. Sprague and A. R. Varga (1986), Encyclopedia of Plant Physiology, with permission. Copyright ©1986 Springer-Verlag.
II. THE EVENTS OF PHOTOSYNTHESIS: LIGHT AND DARK REACTIONS
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In photosynthesis, pigments present in specialized complexes, the antennae, or light harvesting complexes (LHCs) absorb most of the light. Absorption of photons of incident visible or ultraviolet light produces a change in the electron orbitals of the absorbing molecules (Sauer, 1986). The electrons generally occupy molecular orbitals in the lowest possible energy state, where they are present in one pair per orbital. In the excited state produced by the photon absorption, one of the electrons is moved to an orbital other than the ground state to produce a singlet excited state. The energy of the photon is converted into an orbital electronic energy of the excited state. The excitation is then transferred from pigment to pigment by resonance energy transfer over distances as long as 8 to 10 nm. Energy can also be transferred in this way from PSII to PSI. The energy transferred to the RC eventually results in the oxidation of a pigment. P680 and P700 in green plants and P870 in some bacteria (see below) lose one electron to initiate the events of electron transport (Sauer, 1986) Conceivably, photosynthesis could involve separate processes: one set of processes involving the trapping of radiant energy, electron transport and the production of reducing equivalents, and the other set involving the fixation of CO2. Such a dichotomy would be similar to the events of oxidative phosphorylation, where the enzymes of the tricarboxylic acid cycle can be considered separate from those of electron transport and oxidative phosphorylation. The information presently available is in agreement with this view. The reactions responsible for the fixation of CO2 or some other carbon donor clearly belong to a separate system from those involved in the capture of light quanta. These so-called dark reactions do not require light. They can take place if reduced NADPH and ATP are present or if the system containing oxidized NADP+ and ADP is exposed to the light. The simplest way to test this proposition is to incubate the photosynthetic system in the dark in the presence of 14CO2 (or, for bacteria, some other 14C compound) and other required components after illumination. The data from this kind of experiment are shown in Table 2 (Trebst et al., 1958). In sample 1, the mixture containing chloroplast membranes (presumably thylakoids), NADP+ and ADP was exposed to light. After removal of the chloroplast membranes, the 14CO was introduced into the system in the dark. The counts shown represent CO assimilated. For 2 2 sample 2, the procedure was identical except that ADP and NADP+ were left out. The amount of CO2 fixed is very low and probably reflects the presence of a small residual amount of ATP and NADPH. Sample 3 represents the complete system exposed to light with both 14CO2 and chloroplast membranes present. Sample 4 corresponds to the complete system maintained in the dark. It is clear that considerable 14CO fixation occurs in the dark as long as energy has been supplied to the system in the form of ATP 2 and reducing equivalents. Evidently, once the energy is stored in a biochemically utilizable form, neither light nor the photosynthetic machinery of the chloroplast is needed. Table 2 CO2 Fixation by a Chlorophyll-Free Extract and a Complete Chloroplast System from Spinacha
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Treatment
14CO
2
fixed
(counts/min x 103) 1. Chlorophyll-free extract, dark (NADP and ADP present)
134
2. Chlorophyll-free extract, dark (no NADP and no ADP)
9
3. Complete chloroplast system, light
200
4. Complete chloroplast system, dark
20
Trebst et al. (1958). Reproduced with permission from Nature 182: 352-355, copyright ©1958 Macmillan Magazines Limited.
Similar experiments have been carried out with bacterial systems, for instance, with extracts of the purple sulfur bacteria Chromatium, which incorporate [14C]acetate (Table 3) (Losada et al., 1960). As long as ATP (item 2) or light (item 5) is present, the preparation incorporates [14C]acetate. Hexokinase and glucose together (items 4 and 7) largely eliminate the incorporation, probably by decreasing the amount of ATP available. In this case ATP alone suffices, since the reducing equivalents can be produced from ATP hydrolysis by running electron transport in reverse (see Section III,C). Table 3 Equivalence of ATP and Light in the Assimilation of [14C]Acetate by Cell-Free Preparations of Chromatium
Treatment
14C
fixed
(counts/min) x 103 1. Dark, Control
27
2. Dark. ATP
180
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3. Dark, ATP, hexokinase 4. Dark, ATP, hexokinase and glucose
186 6
5. Light, control
414
6. Light, hexokinase
348
7. Light, hexokinase, glucose
20
Losada et al. (1960). Reproduced by permission from Nature 186:753-760 copyright ©1960 by Macmillan Magazines Limited.
Conceivably, the biochemical pathway followed may depend on whether light is present. However, the radioautographic paper chromatographs of Fig. 6a and b (Trebst et al., 1958), obtained with the reaction products of experiments such as those of Table 2, show that this is not the case. The results show that the intermediates accumulating under the two sets of conditions (i.e., in the dark, after illumination or under continuous illumination) are the same. The principles of paper chromatography used to separate these molecules are discussed in the legend of the figure. The results just discussed show that the light-requiring reactions per se are not involved in carbon fixation, except to supply chemical free energy in the form of NADPH and ATP. Seen from this point of view, photosynthesis is concerned directly only with trapping radiant energy and converting it into biologically utilizable forms: reducing equivalents and a phosphate of high group-transfer potential. In different organisms, photosynthesis differs in detail. However, the light-requiring processes correspond to the production of ATP and reducing equivalents. The subsequent biochemical reactions can take place in the dark. III. PHOTOOXIDATlON The sections that follow are concerned with the events that result in the production of ATP and reducing equivalents during photosynthesis. After the primary photochemical event -- the light-energized oxidation of Chl in the RCs -- the return of the excited electrons through electron transport carriers could provide enough energy for the synthesis of ATP from ADP and Pi by a process analogous to that taking place in mitochondria. Similarly, these electrons could be used to reduce electron carriers with the eventual production of NADPH. http://www.albany.edu/~abio304/text/17part1.html (11 of 21) [5/5/2002 6:53:20 PM]
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Let us begin with the photooxidation of chlorophyll. When Chl or BChl are oxidized, one electron is removed, not from the metal as in the case of cytochromes (in this case Mg2+), but from the aromatic electron system of the molecule. The spin of the remaining electron is delocalized over the whole electron system.
Fig. 6 Tracings of radioautographs of chromatograms showing (a) products of photosynthetic 14CO2 assimilation by illuminated spinach chloroplasts and (b) products of dark 14CO2 assimilation by chlorophyll-free extract of chloroplasts. In paper chromatography, a mixture is placed at one end of the chromatographic paper. The solution moves by capillary action and it is submitted to minute partition steps between the solvent and the wet cellulose fibers of the paper. The migration of various compounds on the paper and in a particular medium will depend on their individual solubility in the solvent. The location of the individual compounds can be seen by means of reactions generating colored compounds or, in the case of radioactive compounds, by autoradiography. Ideally, each spot is unique and corresponds to a single compound. In the chromatographs, the compounds have migrated in one direction in one particular solvent system (phenol:water) and in the other direction, at right angles from the first, in another solvent system (butanol:acetic acid). Separation with such a technique depends on the solubility of the compounds in the different solvents, resulting in increased separation. The position of the compounds was recorded by placing the paper on photographic paper. From Trebst et al. (1958). Reproduced with permission from Nature, 182:351-355, copyright ©1958 Macmillan Magazines Limited.
The photooxidation can be studied and detected by electron paramagnetic resonance (EPR) and also by light absorption measurements. For the latter, the experimental design and the apparatus used for spectrophotometric measurements in photosynthetic systems are illustrated in Fig. 7 (Clayton, 1980). In this representation, the monitoring beam provides the light needed to measure changes in absorption. The beam is weak and ideally has no effect on the pigments and therefore none on the absorption spectrum. The detector, which measures the light transmitted through the sample, is shielded in some way from stimulation by the excitatory beam. Since excitatory and monitoring beams are generally at different wavelengths, this can be done with light filters.
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Fig. 7 System for measuring light-induced absorbance changes and the quantum efficiency of a photochemical process. From Clayton (1980). Copyright ©1980 by Cambridge University Press, reproduced with the permission of Cambridge University Press.
Various experiments have demonstrated that the primary photosynthetic event is the oxidation of chlorophyll by light. A mutant of the purple photosynthetic bacteria, Rhodobacterium sphaeroides, that lacks carotenoids, was used in the experiment of Fig. 8 (Clayton, 1980). Fig. 8a represents the absorption spectrum of the isolated RCs. The pigments responsible for the peaks are indicated. Fig. 8b represents the change in optical density brought about by illumination, which occurs primarily in the region of absorption of BChl. The effect is an oxidation, as shown by the inhibition of the effect with a reducing compound (Fig. 8c).
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Fig. 8 (a) Absorption spectrum and (b and c) difference spectra of light-induced absorbance changes of reaction centers isolated from carotenoidless mutant R. sphaeroides. (b) Reaction centers alone. (c) Reaction centers plus an electron donor to prevent the accumulation of oxidized bacteriochlorophyll during illumination. Changes due to oxidation of the donor have been discounted. From Clayton (1980). Copyright ©1980 by Cambridge University Press, reproduced with the permission of Cambridge University Press.
The chlorophylls that are photooxidized have been named after the wavelength, in nanometers, of the major absorbance decrease produced by the photooxidation, in this case, P870 (P stands for pigment). In chloroplasts, two such reactive complexes have been detected spectrophotometrically: P700 and P680, corresponding to the two photosystems (I and II) discussed below. In bacteria, two BChl a or b molecules, depending on the bacteria, appear to be involved in the photooxidation, since the remaining unpaired electron is delocalized over two BChl molecules. Two Chl a molecules are also involved in the chloroplasts. A. Organization of the Reaction Centers As indicated in Section II in the native photosynthetic systems, light is absorbed by LHCs (see below), constituted of Chls, carotenoids and bilins, to produce excited electronic states. The antennae then http://www.albany.edu/~abio304/text/17part1.html (14 of 21) [5/5/2002 6:53:20 PM]
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transfer the excitation to the reaction center (RC) (see Allen and Williams, 1998; Heathcote et al., 2002). The chlorophylls that undergo photooxidation are complexed to the RC. Type I RCs have 4 FeS clusters as electron acceptors, whereas Type II have pheophytin and quinones. Photosystems I and II (PSI and PSII) (see below) include Type I and Type II RCs respectively. The RCs from two purple photosynthetic bacteria have provided detailed information of the complexes. These features are common to all RCs (e.g., see Fyfe and Jones 2000) and include Chl or BChl dimers as the primary electron donors, the arrangement of the cofactors carrying electrons across the membrane in two branches traversing the bilayer oriented by an axis of symmetry perpendicular to the plane of the membrane (Fig. 9) (see Allen et al., 1987). The purple bacteria RCs contain 4 molecules of BChl a, 2 of bacteriopheophythin a, 2 of ubiquinone (QA and QB), 1 non-heme Fe and 1 carotenoid. The Fe is thought to play a structural role, and the carotenoid is thought to have a photoprotective role. These redox cofactors are held at the interface between L and M polypeptides forming a hydropohobic environment. Each of the polyptides have 5 transmembrane α helices. An additional polypeptide (H) is on top of the cytoplasmic faces of the L and M polypeptides. L, M, and H are present in 1:1:1 stoichiometry in most bacteria studied, and their molecular masses are 32, 34, and 29 kDa respectively. The H polypeptide is present only in some bacteria and can be removed without affecting function. Two of the Bchl molecules have been termed BA and BB. The two other BChl molecules are packed close together; and their optical absorption changes with illumination identify them as P870. Fig. 9 shows the arrangements of the prosthetic groups of the center as identified by the threedimensional reconstruction of x-ray crystallography (Deisenhofer et al., 1984, 1985). The four hemes are in the cytochrome subunit; the other components shown are in the L-M complex.
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Fig. 9 Arrangement of the prosthetic groups in the Rhodopseundomonas viridis reaction center. QB is shown at the site identified by Deisenhofer et al. (1984) but the orientation of the quinone in this site is drawn arbitrarily; the exact orientation of QB in the crystal structure has not been described. The four hemes at the top are in the cytochrome subunit; the other components are in the L-M complex as indicated by the subscripts. The plane normal to the chromatophore membrane is approximately vertical and the periplasmic side of the complex is at the top. The L and M proteins traverse the membrane, with the periplasmic side represented at the top and the cytoplasmic side at the bottom. The orientation of the quinone in this diagram is arbitrary. Reproduced by permission from W.W. Parson, Photosynthesis, pp.4362. Copyright ©1987 Elsevier Science Publishers, Amsterdam.
The arrangement of plant, cyanobacterial and algal Type II RCs appear to be similar to that of the purple bacteria. In these cases the D1 and D2 polypeptides, each with 5 transmembrane helices, are in positions equivalent to those of the L and M polypeptides. Similarly, the redox cofactors are also similar. In this case, they are constituted by 4 Chl a, 2 pheophytins and 2 plastoquinones. However, unlike the RC of purple bacteria, the D1 and D2 polypeptides bind 1 Chl α each. This Chl might act to transmit energy from the antenna to the RC. As in purple bacteria, the cofactors also are arranged as two transmembrane sections. Two light antennae constituted of proteins CP43 and CP47 are present in close proximity. They correspond to 6 transmembrane α helices containing 12 -14 Chl α molecules. The acceptor side of the purple bacteria and the PSII reaction center are very similar. The electron http://www.albany.edu/~abio304/text/17part1.html (16 of 21) [5/5/2002 6:53:20 PM]
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transfer across the membrane is carried out from the primary donor to QAquinone followed by the formation of quinol QB. However, the redox events on the donor side are quite different. In purple bacteria the electron missing in the photooxidized P870+ is replaced from an electron from a cytochrome. In contrast, in PSII RC, P680+ receives an electron from the oxygen evolving complex (OEC) (see below) with the production of O2. The OEC is thought to involve a cluster of 4 Mn as well as Ca2+ and Cl ions (see Nugent, 2001; Rutherford and Faller, 2001). Oxygenic photosynthetic organisms have two photosystems, PSI and PSII and these function in tandem (see Section IV). The PSI RC are larger than those of PSII. The RC of PS1 is thought to be present as a trimer; each monomer consisting of 12 polypeptides (see Jordan et al., 2001). Each PSI RC monomer contains 96 Chls a, 22 carotenoid, 4 lipids, 3 FeS clusters and 2 phylloquinones. The redox cofactors are enclosed in 5 carboxy-terminal portion of each PsaA and PsaB polypeptides which traverse the bilayer. The amino-terminal portion contribute 6 α transmembrane helices and connect to antenna domains present along the core domain. The transmembrane sector contains 6 Chl a, and 2 phylloquinones (QK) present in two branches similar to Type II RCs. The P700 primary donor is a heterodimer of Chl and an epimer Chl a (e.g., see Maeda et al., 1992; Webber and Lubitz, 2001). The acceptors are A, A0and A1. The two branches end at the FxFe-S center. The electrons are then tranferred to ferredoxin. Epimerization changes the conformation of the group attached to C13 of the Chl and allows the incorporation of Chl a’ to the PsA side. In contrast to Type II complexes the phylloquinones of the PSI RC, transfer electrons to the single redox acceptors of the FeS centers. The question remains of which branch of the redox carriers is responsible for the transmembrane transfer of electrons from P700 and the FeS centers and the phylloquinones. Present evidence suggests that the transfer is over both branches (see Heathcote et al., 2002). Heliobacteria and green sulfur bacteria have an arrangement similar to the Type I components. However, the RCs have a homodimeric core (similar to PsaC) and a small peptide, not the PsaA-PsaB heterodimer. The polypeptide composition of the Type I complexes are simpler than those of PSI. Similarly to the PSI RC, in green sulfur bacteria the homodimeric RCs possess epimers of Bchl a and in heliobacteria Bchl g. It is likely that electrons move in both branches of the RC and the primary electron acceptor Ao is a Chl (not a Bchl). Electron transfers within the RC are likely to occur through tunneling pathways (Balabin and Onuchic, 2000) where constructive and destructive interference can take place (see Betts et al., 1992). The pathways were previously defined from X-ray diffraction data collected at cryogenic temperature from crystals of the RC in the dark or under illumination (Stowell et al., 1997). The electron transfer is coupled through interactions with the surrounding medium which is affected by the thermal motion. The electron transfer from donor to acceptor can take place only if the two are energetically matched. This match will occur occasionally due to the thermal motion of the surrounding protein matrix and will depend on constructive interference.
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B. Cyclic Photophosphorylation of Bacteria When RCs of isolated bacteria are excited with a very short flash, a transient P870 excited single state is formed. This state decays in picoseconds, forming a P870+BPh- radical pair. Accordingly, the absorption bands of P870 and BPh disappear and new bands that have been attributed to the radicals are formed. This is followed by the reduction of one quinone to produce a semiquinone radical, QA. The reactions occur extremely rapidly, essentially with a quantum efficiency of 1 and independent of temperature. This supports the idea that these components are held together tightly with little motion (Dutton, 1986). The electron of QA- is transferred to a second quinone, Qb This electron displacement is a much slower process and is temperature dependent. These reactions and the redox potentials are outlined in Fig. 10. The electron displaced from P870 is replaced from the c-type cytochrome, so the RC can respond again to light. After a second electron is removed from P870, the Qb has become fully reduced and Q2-b picks up two H+ from the cytoplasmic side of the membrane. The cytochrome bc1 complex oxidizes QBH2. These reactions are thought to proceed with a net proton efflux of as many as four H+ per two electrons (Dutton, 1986). The passage of these protons through a channel in the F0 portion of the ATP synthase is thought to be responsible for ATP synthesis (see Chapter 18). These details are summarized in Fig. 11. The series of reactions just discussed is entirely cyclic. The electron displaced from the photooxidation of P870 is replaced by the cytochrome bc1 complex, which in turn recovers it from the oxidation of quinone. Thus, the energy from the light absorption is converted into ATP with no net change in electrons. Most photosynthetic eubacteria carry out cyclic electron transport (Pierson and Olson, 1987).
Fig. 10 Kinetics and standard free energy changes of electron transfer steps in reaction centers isolated from Rb. sphaeroides. The rates of the transfers are indicated next to the arrows.
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C. Production of Reducing Equivalents in Bacteria What accounts for the production of reducing equivalents? In the RC-2 bacteria, NAD+ is probably reduced by reverse (energy-requiring) electron transport from the quinone pool. In contrast, green sulfur bacteria reduce ferredoxin directly from the secondary acceptor. Both cases require an external reductant such as succinate or H2S.
Fig. 11 Schematic representation of cyclic photophosphorylation in purple bacteria. Reducing equivalents are transferred from the P870 complex to the cytochrome bc1 complex by quinone (dissolved in the membrane), which is then returned to the P870 complex by cytochrome c (water soluble). The protons generated by the electron transport return through the ATP synthase, providing the energy to synthesize ATP from ADP and Pi.
Experiments with chromatophores isolated from Rhodospirillum rubrum and other nonsulfur purple bacteria show that electrons can be transferred from succinate to NAD+ in the dark, provided that ATP or pyrophosphate hydrolysis (presumably through the action of ATP synthase, proceeding in reverse) is available to supply energy (Jones and Vernon, 1969). In the absence of ATP, the reduction requires cyclic electron flow to pump electrons uphill from organic reductants, such as succinate. A block of the cyclic electron flow blocked NAD+ photoreduction but not the ATP-driven reduction. In addition,
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uncouplers of oxidative phosphorylation or photophosphorylation blocked the photoreduction and the ATP-dependent reduction (Hauska et al., 1983; Malkin, 1987). Uncouplers are thought to collapse the energy-dependent H+ electrochemical gradient, i.e., the proton motive force. Therefore, these experiments suggest that the energy is provided indirectly through the proton motive gradient. The reduction also requires the presence of a functional NADH dehydrogenase. The mechanism suggested by these experiments is summarized in Fig. 12a (Knaff and Kampf, 1987). In green sulfur bacteria, however, the energy is thought to be directly available, since there is no inhibition by uncouplers (Knaff and Kampf, 1987). The scheme consistent with the information available for these bacteria is summarized in Fig. 12b.
Fig. 12 Mechanism of NAD+ photoreduction in (a) purple bacteria and (b) green sulfur bacteria. UQ-Fe represents the Fe2+-quinone complex present at the primary quinone site of purple bacteria, although in some species menaquinone replaces ubiquinone. Fe/S represents the iron-sulfur center that functions as an early acceptor in green sulfur bacteria. The earliest electron acceptors have been omitted for both green and purple bacteria. The involvement of cyt b in S2- oxidation in green bacteria is speculative but is based on inhibition by antimycin A. Reproduced by permission from B. Knaff and C. Kampf, Photosynthesis, pp.199-212. Copyright ©1987 Elsevier Science Publishers, Amsterdam.
D. The Special Case of Bacteriorhodopsin Halobacterium halobium is halophilic (salt loving), red colored bacterium of tidal salt flats. Under conditions of oxygen deprivation, purple patches, referred to as purple membranes, appear in the carotenoid containing plasma membrane of Halobacterium. The purple membranes contain mostly protein (75% by weight) which corresponds almost entirely of bacteriorhodopsin (Brh) (see Blaurock and Stoeckenius, 1971; Oesterhelt and Stoeckenius, 1971). Brh acts as a transducer by coupling the
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absorption of light to the translocation of protons from the cytoplasm to the medium. The return of the protons to the cytoplasm is used to synthesize ATP via the ATP-synthase or the protons are exchanged for Na+ to transport it against a steep gradient, in the face of external NaCl concentrations as high as 4 M. The structure of Brh (see Khorana, 1988) has been deduced by electron microscopy and crystallographic techniques since this protein forms a highly organized lattice in the purple membrane. The molecule contains seven transmembrane α-helices (A-G) linked by loops. The purple color is the result of its binding to one molecule of all-trans retinal which absorbs with a maximum at a wavelength of 570 nm (see Khorana, 1988). Retinal changes from the all-trans form to the 13-cis-form, when it absorbs light and the bacteriorhodopsin pushes the proton through components of the seven helices to the outside (see below). The passage of the proton is through several intermediate states of the Brh (K-O), some accompanied by conformational changes in the transmembrane helices. Some of these conformations have been elucidated (see Kühlbrandt, 2000; selected articles are available through http://www.nature.com/nature/fow). One molecule of Brh contains one retinal molecule with one end of the retinal covalently bound to a lysine of helix G and the other end deep in the transmembrane segments of the Brh. For each photon absorbed by retinal, one H+ is transferred across the cell membrane against an electrochemical gradient (e.g., see Gennis and Ebrey, 1999). Very small movements (about 1 Å) of components of the Brh accompny the pumping. These movements are induced by the bending and unbending of retinal accompanying its light dependent isomerization (in the 13-cis-form one of the retinal terminals is twisted around a double bond and during the transition the pigment moves in relation to the protein). The movements of the Brh change the affinity to protons of neighboring side chains by changing their local chemical environment. The passage of the proton is through several intermediate states in the transmembrane helices in conduits involving several amino acid residues (e.g., aspartate, tyrosine, arginine and water molecules). In essence, the retinal acts as a valve in a conduit permitting the exchange in a single direction (see Kühlbrandt, 2000; selected articles are available through http://www.nature.com/nature/fow).
Go to Part 2 REFERENCES Search the textbook
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IV. THE TWO PHOTOCHEMICAL SYSTEMS OF CHLOROPLASTS As already discussed, chloroplasts have two distinct chlorophyll complexes in the RCs: P700 and P680. With their respective electron acceptors and donors, these complexes constitute two distinct assemblies, which are called photosystem I (PSI) and photosystem II (PSII), respectively. A summary of our present understanding of their organization is shown in Fig. 13 (Blakenship and Prince, 1986). The scale on the left represents the redox potentials of the electron carriers. The large arrows represent the excitation of PSI or PSII by light. As in the figures in Chapter 16, the small arrows represent electron transport steps. As shown in Fig. 13, PSI and PSII are connected in series, i.e., the electron transport chain that receives electrons from PSII replaces the electrons displaced by the light reaction in PSI. This arrangement also imposes two distinct features. One corresponds to a mechanism that replaces the electron lost by the photooxidation of PSII, carried out by the oxygen-evolving complex (OEC) (Dunahay et al., 1984), and the other corresponds to a series of reactions that accept the electrons from PSI to eventually reduce NADP+.
Fig. 13 The Z scheme for oxygenic photosynthesis constructed using excited-state redox potentials. The carriers are placed at the accepted midpoint redox potentials (at pH 7) where they have been measured directly; other potentials are estimated. OEC, oxygen-evolving complex; Z, donor to photosystem II
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(PSII); P680, reaction center chlorophyll of PSII; Ph, pheophytin acceptor of PSII; Q, quinone; cyt, cytochrome; FeSR, Rieske iron-sulfur protein; Pc, plastocyanin; P700, reaction center chlorophyll of PSI; A0 and A1, early acceptors of PSI (possibly chlorophyll and quinone species); FeSX FeSB, and FeSA, bound iron-sulfur protein acceptors of PSI; Fd, soluble ferredoxin; Fp, flavoprotein (ferredoxin-NADP reductase). FeSB and FeSA may operate in parallel. The dashed line indicates cyclic electron flow around PSI. The pathway of electron flow through the cyctochrome b6fcomplex is outlined by a box. Reproduced from Trends in Biochemical Sciences, vol. 10, R. Blakenship and R. C. Prince, pp. 382-383, copyright ©1985 Elsevier Science.
Biochemical fractionation of the chloroplast photosynthetic system has shown that the complete system consists of three separate transmembrane complexes: PSI, PSII, and cytochrome b6f complex. The electron transport between PSII and the cytochrome b6f complex is mediated by plastoquinone (PQ). Plastocyanin (PC) mediates the electron transport between the cytochrome b6f complex and PSI. The three complexes are represented in Fig. 14 in diagrammatic form (Cramer et al., 1985). Plastoquinone closely resembles ubiquinone (CoQ), a component of the electron transport chain of mitochondria. Plastocyanin is a water-soluble 1l-kDa protein containing copper ion coordinated to the side chains of cystein, methionine and two histidines. Reduced PC and oxidized PC contain Cu+ and Cu2+, respectively. The arrangement of Fig. 14 is reminiscent of the four complexes of mitochondria in which electron transport is carried out between complexes by either CoQ or cytochrome c (Chapter 16). The stoichiometry of the photosystems and the Chl distribution in higher plant chloroplasts are summarized in Table 4 (Glazer and Melis, 1987; Whitmarsh and Ort, 1984).
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Fig. 14 Arrangement of the three major transmembrane protein electron transport complexes in the thylakoid membrane. The photosystem II complex is involved in water splitting, in electron transfer to noncyclic electron transport chain and proton deposition to the lumen. It includes the 51-and 44-k Da chlorophyll-binding polypeptides that also bind the QA acceptor and (perhaps) the QD donor plastoquinone; two 32-kDa putative plastoquinone binding proteins to which herbicides and probably the QD plastoquinone also bind; and cytochrome b559 containing two hemes per P680 reaction center that are hypothesized to span the membrane as shown. Electrons from the PSII complex are transferred by a plastoquinone pool to the cytochrome b6f complex through a plastoquinone binding site, Qz. The b6 f complex consists of four subunits: 34-kDa cytochrome f, 23-kDa cytochrome b6 (with two heme groups), a 20-kDa polypeptide FeS redox center, and a fourth 17-kba polypeptide (not shown). The cytochrome bf complex is connected to photosystem I by plastocyanin (PC). The transmembrane PSI P700 reaction center complex possibly contains two large (Mr 70,000) P700 reaction center chlorophyll polypeptides and at least three smaller peptides that may contain the FeS electron receptor complex used to reduce ferredoxin (Fd) and NADP. The involvement of Fd in PSI cyclic phosphorylation, possibly through a plastoquinone binding site, Qc, is shown. The fourth membrane-spanning ATP synthase complex is not shown. Reproduced from Trends in Biochemical Sciences, vol.10, W.A. Cramer et al., pp.125. Copyright ©1985 Elsevier Science.
Table 4 Photosystem Stoichiometry and Chlorophyll Distribution in Wild-Type Higher Plant Chloroplasts
PSIIa
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Cyt b/fb
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Stoichiometry
1.7
1.0
Antenna size
330
200
Chl a
235
180
Chl b
95
20
Total Chl,%
63
37
1.10
a
Data from Glazer and Melis (1987). Reproduced, with permission, from the Annual Review of Plant Physiology, vol. 38 ©1987 Annual Reviews, Inc. b
Data from Whitmarsh and Ort (1984). Reproduced by permission.
The complexes are not randomly distributed in the membrane; rather, PSII is primarily located in the appressed grana and PSI primarily in the stroma lamellae (Anderson and Anderson, 1982). The interaction between the three complexes is thought to occur by the diffusion of plastoquinone in the membrane from PSII to the cytochrome b6f complex and by the location of the cytochrome complex close to PSI. The low molecular weight and high lipid solubility of plastoquinone would permit fast diffusion in the plane of the membrane, sufficient to account for the electron transport rate (Haehnel, 1984). This arrangement is shown in Fig. 15 (Haehnel, 1984).
Fig. 15 Schematic representation of photosynthetic electron transport distributions of the integral complexes in chloroplasts with grana stacks. PQH2, plastoquinol; b6f cytochrome b6f complex; Pc, plastocyanin; Fd, ferredoxin; and FNR, ferredoxin-NADP+ reductase. Not shown is the H+ uptake from outside. The long dashed arrows indicate assumed long-range diffusion of mobile electron carriers. From Haehnel (1984). Reproduced, with permission, from the Annual Review of Plant Physiology, volume 35, 1984 by Annual Reviews Inc.
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the effectiveness of various wavelengths. The effectiveness of light in photosynthesis can be expressed as the yield of O2 evolved per einstein of light. The einstein, the energy equivalent of photons, corresponds to Nhv, where N is Avogadro's number (6 x 1023), v is the frequency of the light (vibrations per second), and h is Planck's constant (1.6 X 10-34). Results of an experiment measuring the yield as a function of wavelength for the grana alga, Chlorella, are shown in Fig. 16 (Emerson et al., 1957). Curve 1 represents the yield with a single beam of light. Curve 2 represents the yield with two beams of light, the second beam of constant but of lower intensity and shorter wavelength. The yield is greater for curve 2, particularly at longer wavelengths. The effect must involve separate light-activated units, since it occurs even when the two beams are alternated (Fig. 17) (Myers and French, 1960). In these experiments, the oxygen release is measured with a Pt electrode and the actual current in µA recorded (ordinate). The excitatory beam is alternated between wavelenghts of 700 and 650 nm (as indicated in the upper tier of the figure). The O2 released is increased whenever the shorter wavelength is turned on (upper trace). However, when the alternating is done between 690 and 700 nm, there is no significant effect (lower curve and numbers in lower tier). The excitation of PSI and PSII is necessary for optimal functioning of chloroplast photosynthesis, but the two also have separate functional roles. The photochemical O2-generating system can be dissociated from other events by providing the system with an electron acceptor (e.g., indophenol blue). Such a reaction could be visualized as follows: 2OH- → 2e- = 2H+ + ½O2 The electrons would be picked up by the electron acceptor (not shown in the equation). On the other hand, the production of reducing equivalents in the form of NADPH would require an electron donor (e.g., ascorbate): NADP+ + H+ + 2e- → NADPH
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Fig. 16 Effect of supplementary light on quantum yield. Curve 1, control; curve 2, same conditions as used for curve 1 but with supplementary light. Reproduced with permission from R. Emerson, Science, 127:1059-1060. Copyright ©1958 by the American Association for the Advancemnet of Science.
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Fig. 17 Chromatic transients observed by alternating two light beams of intensities adjusted to sustain equal steady rates of photosynthesis. The current recorded from a platinum electrode is proportional to the oxygen concentration. Record A, alternating beams of 650 and 700 nm. Record B, alternating beams of 690 and 700 nm. From Myers and French (1960). Reproduced from The Journal of General Physiology, 1960, by copyright permission of the Rockefeller University Press.
Fig. 18 (Arnon, 1961) shows the wavelengths most effective for oxygen production or NADP reduction over the narrow wavelength range where the absorption spectra of the two chlorophylls do not overlap. Clearly, the evolution of O2 is most effectively produced by a lower wavelength than the reduction of NADP+, testifying to the involvement of PSII. On the other hand, NADP+ reduction has action peaks at much longer wavelengths, revealing an association primarily with PSI. Interestingly, ATP production seems to be associated with PSII.
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Fig. 18 (a) Effectiveness of monochromatic light, in the red region of the spectrum, in oxygen evolution and NADP reduction by isolated chloroplasts. Ascorbate was used as an electron donor for the photoreduction of NADP. (b) Effectiveness of monochromatic light, in the red region of the spectrum, in oxygen evolution and cyclic photophosphorylation by isolated chloroplasts. The 100 on the ordinate scale is equivalent to 0.16 atom oxygen evolved per micromole of light quanta absorbed; 60 on the ordinate scale is equivalent to 0.10 µmoles ATP formed per micromole of light quanta absorbed. Reproduced from D. Arnon, Bulletin of Torrey Botany Club, 88:215-259, with permission of Johns Hopkins Press, 1961.
As represented in Fig. 13 or 14, PSI and PSII are connected in series by the cytochrome b6f complex. The evidence for such an arrangement is the effect of illumination of either PSI or PSII on the redox state of the cytochrome complex. Stimulation of PSII reduces the cytochrome complex, whereas illumination of PSI oxidizes it. The experiment represented in Fig. 19 (Duysens et al., 1961), carried out with the red alga Porphyridium, shows the absorption of cytochromes. In this record, the upward deflections reflect oxidation and the downward arrows the turning off of light. Illumination at 680 nm corresponds to excitation of PSI and that at 562 nm corresponds to excitation of PSII. As predicted by the model in which the two systems are in series, light excitation of PSI oxidizes the cytochromes and excitation of PSII reduces them.
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Fig. 19 Time course of cytochrome oxidation in Porphyridium in light of 680 and 562 nm of intensities 4.4 x 10-10 and 5.3 x 10-10 einstein cm-2 s-1. Upward and downward arrows indicate that the light is switched on and off, respectively. Tracing 1 shows that cytochrome is oxidized by light of 680 nm but reduced by light of 562 nm. Curve 2 shows no effect by light of 562 nm. From Duysens et al. (1961). Reproduced with permission from Nature, 190:510-514, copyright ©1961 Macmillan Magazines Limited.
V. COMPONENTS OF THE CHLOROPLAST PHOTOSYNTHETIC SYSTEMS Knowledge of the topology and function of the various components of the thylakoid membrane has advanced rapidly thanks to the application of spectroscopic techniques, fractionation procedures, conventional reagents or antibodies, and the application of X-ray diffraction and cryoelectron microscopy (Chapter 1) accompanied by image reconstuction techniques. Sequencing of the appropriate cDNA (Chapter 1) and computer predictions of structure based on the hydrophobicity of the amino acid side chains of the polypeptides have also provided insights. This section presents some of the information that has been gained by the use of several of these techniques. A. PSII The LHC complex of PSII, referred to as LHC II, contains Chl a and b in approximately equal amounts. Part of the complex is tightly bound to PSII. A mobile or peripheral portion, not closely associated with PSII, accounts for 120 Chl molecules. The total corresponds to approximately 250 Chl molecules (Staehelin, 1986). The three dimensional structure of the reaction center of PSII has been analyzed using cryoelectron microscopy and image reconstruction techniques (Rhee et al, 1998) and more recently that of the entire PS II has been elucidated by X-ray diffraction (at a resolution of 3.8 Å) from crystals fully active in water oxidation (Zouni et al., 2001). . The arrangement is very similar to that of PSI (Krauss et al., 1996; http://www.albany.edu/~abio304/text/17part2.html (9 of 23) [5/5/2002 6:53:27 PM]
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Schubert et al., 1997) or that of purple bacteria (Deisenhofer et al., 1984, 1995). As discussed, the bacterial photochemical activity depends on two polypeptides, L and M, which span the membrane. The PSII reaction center contains a 32 kDa polypeptide, D1, and a 34 kDa polypeptide, D2. These two polypeptides have important homologies to L and M (Trebst, 1986) and are therefore thought to correspond in function to the bacterial polypeptides. In addition to D1 and D2, the isolated RC of PSII (Namba and Satoh, 1987) contains one molecule of cytochrome b559, five Chl, pheophytin and two βcarotenes. P680 is photooxidized on illumination (Doring et al., 1967). The primary electron acceptor is pheophytin (Klimov et al., 1980), a porphyrin identical to Chl a, but lacking Mg. A specialized plastoquinone, QA, is a secondary acceptor. These transfers produce an endergonic charge separation between P680 and pheophytin, followed by electron transfer to QA. An electron from Z replaces the electron removed by the photooxidation of P680 b. A tyrosine residue of D1 is thought to be the electron donor Z (Debus et al., 1988). Both Z+ and P680+ can be detected with EPR and the kinetics of the ESR changes implicate Z as the immediate donor (Boska et al., 1983). The electron lost by Z is replaced by the oxidation of water and the evolution of O2 carried out by the oxygen-evolving complex (OEC) (see Murata and Miyao, 1985), which is tightly bound to PSII. Each individual OEC undergoes a succession of increasing oxidation states from S0 to S4. Oxygen is liberated at S4 (Kok et al., 1970). The reactions of PSII are represented by Eq. (4) and the S-scheme is summarized by Fig. 20.
(4)
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Fig. 20 Scheme in which one electron is removed in each transition from S0 to S3. State S4 decays spontaneously, releasing O2.
B. PSI PSI of the thermophilic cyanobacterium Synechococcus elongatus contains 12 protein subunits and 127 cofactors comprising 96 Chls, 2 phylloquinones, 3 Fe4S4 clusters, 22 carotenoids and 4 lipids. 90 of the 96 Chls function as antennae; only 6 are involved in electron transfer. The structure of PSI has been studied in chloroplasts (see Fromme et al., 1996) and in cyanobacteria (Krauss et al., 1996; Schubert et al., 1997; Jordan, et al. 2001). These have many common features with that of purple bacteria (see Deisenhofer et al., 1995) and the RC of PSII (Rhee et al., 1998). As already discussed, P700 is the electron donor. The primary electron acceptor is A0 and the intermediate acceptor is A1. Three iron-sulfur centers, FX, FB and FA, have been identified. The sequence and kinetic constants are shown in Eq. (5).
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(5)
Fig. 21 A contemporary Z scheme depicting the sequence and oxidation-reduction midpoint potential (Em,7) of the electron carriers in oxygenic photosynthesis. This differs from the familiar Z scheme format in order to convey the concept that the photosynthetic electron transfer from H2O to ferredoxin (Fd) is accomplished by three electron transport complexes operating in series, interconnected by two pools of mobile carriers. Reproduced from D. R. Ort (1986), Encyclopedia of Plant Physiology, with permission. Copyright ©1986 Springer-Verlag, Germany.
P700 is a Chl molecule, although its nature and structure (e.g., whether it is a dimer or a monomer) are still unknown. A0 is also a Chl or a chlorophyll-like molecule and A1 is phylloquinone (vitamin K1) present at a stoichiometry of two per P700. P700 is associated with a core antenna (see Section VI) consisting of a 70-kDa tetramer (Vierling and Alberte, 1983), which also binds 130 Chl a and 16 carotenoid molecules. The purified PSI also contains other polypeptides (Bengis and Nelson, 1977).
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The structure of PSI has been studied in chloroplasts (see Fromme et al., 1996) and in cyanobacteria (Krauss et al., 1996; Schubert et al., 1997; Jordan, et al. 2001). It has many common features with that of purple bacteria (see Deisenhofer et al., 1995) and the RC of PSII (Rhee et al., 1998). The accessory light-harvesting system of PSI, LHC I (see Section VI) contains an additional 60 to 80 Chl a and b molecules, with approximately three times more Chl a. The LHC I polypeptides are in the range of 20 to 25 kDa and are immunologically distinct from those of PSII (Lam et al., 1984). Since LHC I can be separated into two fractions, each could have a distinct function as in the case of LHC II, where one is considered laterally mobile and the other is closely associated with PSII. C. The Cytochrome b6f Complex The cytochrome b6f complex (Cramer et al., 1987) containing plastoquinone and plastocyanin oxidoreductase, has been isolated (Hauska et al., 1983). In addition, the complex was found to include the 34-kDa polypeptide of cytochrome f, a 23-kDa polypeptide with two cytochrome b6 hemes, a 20-kDa FeS-Rieske protein, two other smaller polypeptides and bound plastoquinol. The aspects of the organization of the components of the thylakoid membranes discussed in Sections IV and V are summarized in Fig. 21 (Ort et al., 1986) and Fig. 22 (Anderson, 1981, 1987). Fig. 22a shows the organization of the complexes; Fig. 22b shows their organization in relation to the thylakoid structural arrangement.
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Fig. 22 (a) Arrangement of the supramolecular protein complexes and mobile electron transport carrier in thylakoid membranes. From J.M. Anderson, Photosynthesis, p.275, with permission of Elsevier Science Publishers, Amsterdam, ©1987. (b) Possible static representation of the lateral herterogeneity in the distribution of the supramolecular thylakoid complexes between appressed and nonappressed thylakoids. http://www.albany.edu/~abio304/text/17part2.html (14 of 23) [5/5/2002 6:53:27 PM]
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Reproduced from J.M. Anderson, FEBS Letters, 124(1), 1981, with permisssion.
VI. THE LIGHT-HARVESTING SYSTEM All photosynthetic processes depend on the efficient capture of energy to drive the biosynthetic processes. The problem is dealt with by the capture of light by light-harvesting complexes (LHCs) and the proximity between LHC and the RC which allows for the rapid transfer of energy. The number of Chl molecules per RC is very large. It has been estimated to be from 25 to several hundred, depending on the bacteria, to about 300 in the chloroplast. The function of these molecules is to act as an antenna, i.e., absorb light and transfer the energy to neighboring molecules by resonance transfer until it is trapped in the reactions of the RC. Part of the evidence for this mechanism stems from the fact that in oxygen-producing systems, the amount of oxygen evolved increases with intensity of the flash until a maximal saturation rate is obtained. The amount of O2 produced is very small in relation to the Chl content because the RCs are finite in number and must be reduced again before participating in another photooxidative event. Approximate calculations yield about 2500 Chl molecules per O2 evolved under saturating conditions or 300 per RC (8 photons are required per O2 see Fig. 20). The light-harvesting complexes (LHCs) contain Chl or BChl and a variety of other pigments, such as carotenoids (Zuber et al., 1987). Thus they can absorb light from a very broad region of the spectrum. The pigment molecules are noncovalently attached to integral proteins. A few pigment molecules are associated with each protein. The energy is transferred from short wavelength-absorbing to longer wavelength-absorbing pigments by inductive resonance and the formation of exciton states. In bacteria, the dominant LHC is LHCII which donates the energy to a second complex LHCI. How this takes place with high efficiency and high speed has been shown in studies of the atomic structure of the bacterial LHCII system with electron crystallography (McDermott et al., 1995). 18 bacteriochlorophyll a molecules absorbing at 850 nm (B850) are present in an overlapping ring closely associated with their neighbor and vertical to the plane of the membrane. Nine other chlorophyll a molecules (B800) are close to the cytoplasmic surface and are parallel to the plane of the membrane. The phytol chains of the bacteriochlorophyll intertwine. Nine carotenoids molecules span the membrane and make contact with both B850 and B800. The closeness between the B800 and B850 and the carotenoid molecules allows for the rapid and efficient transfer of energy. LHCI, which has a similar composition, is thought to have a similar arrangement. A model is shown in Fig. 23 (McDermott et al., 1995). The structure of LHCII from green plants which contains both Chl a and b has also been studied by electron crystallography. Each polypeptide has 232 amino acids and contains a minimum of 12 Chl molecules and 2 carotenoids. A representation is shown in Fig 24 (Kühlbrandt et al., 1994). Again all the components are close to each other allowing rapid transfer of energy. In this case, the role of carotenoid is mostly to protect the Chl from photodamage (Kühlbrandt et al., 1994). Apparently there are 6 trimers per RC and two additional polypeptides (CP43 and CP47) with 20 to 30 Chl, accounting for the total http://www.albany.edu/~abio304/text/17part2.html (15 of 23) [5/5/2002 6:53:27 PM]
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amount of pigment of the LHCII.
Fig. 23 a. The nanomeric complex viewed from the cytoplasmix side of the membrane. Protein units are shown in white, B800 BChl a in green, B850 BChl a in red and carotenoid in yellow. b. The complex viewed perpendicular to the symmetry axix. Broad ribbons are in the presumed hydrophobic region of the membrane. Reproduced with permission from Nature, McDermott, G., Prince, S.M., Freer, A.A., Hawthornwaite-Lawless, A.M, Papiz, M.Z., Cogdell, R.J. and Isaacs, N.W. (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria, 374:517-521, copyright ©1995 MacMillan Magazines Ltd.
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Fig. 24 Overall views of LCII of chloroplasts. a. Side view. Blue bands indicate the approximate location of the bilayer. Helices are labelled A to D. b. Stereodiagram showing the top view of the complex from the stromal side of the membrane, c. Stereodiagram of Chl tetrapyrroles and luteins. Residues 26 to 224 of the polypeptide are shown. The fit indicated by the dashed line for residues 100 to 116 is tentative. Dark green corresponds to Chl a, light green to Chl b, Mg atoms are pink, luteins are yellow and the membrane spanning helices are violet. Reproduced with permission from Nature, Kühlbrandt, W., Wang, D.N. and Fujiyoshi, Y. (1994) Atomic model of plant light-harvetsing complex by electron crystallography, 367:614-621, copyright ©1994 MacMillan Magazines Ltd.
VII. COORDINATION BETWEEN PSI AND PSII The efficient operation of the two systems, PSI and PSII, requires smooth coordination. Although PSI and PSII are likely to be excited equally by white light, shade favors PSI. Excess energy absorbed by either system would be dissipated as heat and lost to the system. The presence of components of the LHCs can be adjusted to decrease light absorption and the http://www.albany.edu/~abio304/text/17part2.html (18 of 23) [5/5/2002 6:53:27 PM]
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photosynthetic capacity can be adjusted to increase the utilization of the light energy. These mechanisms require synthesis and degradation of the photosyntiesis machinery. For more rapid adjustments, state transitions may take place. Unbalanced excitation of PSI is known as state I and that of PSII as state II. Apparently, these imbalances are corrected by what have been called state transitions. These are regulatory effects by which the flow of energy between the two systems is altered. The mechanism or mechanisms are still not entirely understood. Conceivably, the amount of energy normally transferred from PSII to PSI could be changed. Some role of Mg2+ or Na+ in the amount of energy transferred from PSII to PSI is suspected (Wang and Govinjee, 1979). Alternatively, the proportion of the light absorbed by the two photosystems could be altered, an effect referred to as a change in optical cross section. There is some evidence that the optical cross section changes. Barley mutants lacking LHC II fail to show state transitions (Canaani and Malkin, 1984), which suggests that LHC II is responsible for these transitions. Since PSI and PSII are in separate locations in the membrane, a change in the position of the movable portion of LHC II could favor the transfer of energy to either PSI or PSII. The phosphorylation and dephosphorylation of LHC II affect its distribution; phosphorylation favors a position closer to PSI (Bennett, 1983; Kyle et al., 1983). The phosphorylation is catalyzed by a membrane-bound Mg2+dependent kinase, whereas dephosphorylation is catalyzed by a phosphatase. Presumably, the phosphorylated LHC II moves laterally to approach PSI in the unstacked region, thereby favoring the transfer of energy from LHC II to PSI (Larsson et al., 1983). This could be the result of electrostatic repulsion. In contrast, a phosphatase dephosphorylates LHC II, favoring its displacement toward PSII. The phosphorylation cycle is thought to be regulated by the redox state of plastoquinone acting as a sensor of the energy distribution (Allen et al., 1981). Reduction of plastoquinone, resulting from the electron transport following the photoactivation of PSII, would favor the kinase, whereas the phosphatase would be stimulated by oxidation of plastoquinone. The redox state of plastoquinone also regulates the transcription of the reaction-center apoproteins of PSI and PSII, both coded by chloroplast genes (Pfannschmidt et al., 1999). The rate of transcription is much more rapid than that of nuclear genes. Reduction of plastoquinone induces apoproteins of PSI and oxidation represses them. PSII transcription is controlled in the opposite manner. VIII. PROTECTION FROM PHOTO-OXIDATION Oxygenic photosynthesis produces highly reactive oxidizing intermediates and byproducts that can damage the photosynthetic apparatus (see Niyogi, 1999). Photo-oxidative damage decreases the efficiency of photosynthesis and has been called photoinhibition (Kok, 1956). The reactions of photooxidation take place all the time during photosynthesis. However, they become particularly damaging when the absorption exceeds the capacity of the energy utilization system. Oxygenic photosynthetic organisms have developed a variety of mechanisms to prevent photo-oxidative damage. These mechanisms range from moving the leaves or moving chloroplasts within cells (to avoid exposure to light) to mechanisms involving molecular interactions. As we saw in Section VII, overexcitation of PSII relative to PSI, is thought to reduce the plastoquinone pool and activate a kinase
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that phosphorylates the peripheral LHC associated with PSII. Detachment of the phosphorylated LHC from PSII transfers it to PSI. This mechanism is not likely to play a role at high light intensity since the kinase is inactivated by intense light (e.g., Rintamäki et al., 1997). However, alternative electron transport pathways and thermal dissipation help to remove the energy form excess absorbed light. In addition, many antioxidant molecules and scavenging enzymes are present to deal with reactive oxygen species. The danger of photo-oxidation is greatest for the LHC associated with PSII, the PSII reaction center and the PSI acceptor side. Absorption of light causes Chl to enter the singlet excited state (1Chl). As already discussed, the energy is transferred to neighboring Chls in the LHC by resonance transfer. Before transferring to the RC, the triplet state (3Chl) can be formed from 1Chl. 3Chl is relatively long-lived and can produce singlet oxygen (1O2) from O2. The generation of 1O2 is much greater at PSII-LHC, because the average life time of 1Chl is longest in PSII-LHC (Foote, 1976). In addition to these reactions, P680+ and Z+ (see above) of PSII are capable of oxidizing nearby proteins and pigments. The acceptor side of PS1 can reduce O2 to the superoxide anion (O-2) radical and eventually hydroxyl radical. In excessive light, an increase in the thylakoid ∆pH regulates PSII by dissipating as much as 75% of the excess energy as heat (Demmig-Adams et al., 1996a). The pH dependent thermal dissipation occurs in the PSII- LHC and involves the de-epoxidation of xanthophyll pigments (e.g., Demmig-Adams, 1996b). Presumably, the interaction of the pigments with excited states of oxygen or Chl lead to a de-excitation state. The thermal energy dissipation can be estimated from the chlorophyll fluorescence emission. Although this emission involves a small portion of the chlorophyll, the fluorescent emission decreases with increases in thermal dissipation. This has been referred to as non-photochemical quenching (NPQ). Carotenoids facilitate thermal energy dissipation, as demonstrated in mutants that lack lutein an isomer of zeaxanthin, where dissipation is largely absent (see Niyogi, 1999) but where oxidative damage in the form of lipid peroxidation is evident (Havaux and Niyogi, 1999). A mutant of Arabidopsis thaliana with normal level of zeaxanthin, but deficient in energy dissipation, was isolated (Li et al, 2000). The mutant lacks a protein, chlorophyll binding protein of 22 kDa (CP22) [the product of the PSII gene ( psbS), Psb)]. CP22 is part of the LHC, one of the 30 components of LHC that have been identified (see Jansson, 1999). CP22 was found to be required for the thermal energy dissipation which also requires zeaxanthin and a pH gradient across the thylakoid membrane. In addition to the machinery described, chloroplasts contain many antioxidants that act as scavengers of reactive oxygen species. The scavengers include carotenoids, tocopherols, ascorbate and glutathione (see Niyogi, 1999). In addition, some enzymes are involved in scavenging, including superoxide dismutase (SOD) and ascorbate peroxidase (APX). SOD catalyzes the conversion of O2- to H2O2. APX reduces the http://www.albany.edu/~abio304/text/17part2.html (20 of 23) [5/5/2002 6:53:27 PM]
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H2O2 and produces monodehydroascorbate radicals which are reduced by PSI. IX. PHOTOSIGNALING SYSTEMS Light is essential for the survival of plants, and plants have evolved a remarkable ability to respond to light using sensory receptors (see Quail, 2002). Phytochrome (phy) is one family of these receptor molecules. Each phytochrome has specific physiological functions. The subunits form two main domains. Phys are a soluble homodimeric chromoproteins composed of two polypeptides approximately 125 kDa in size. Phys are encoded by multigene families (see Mathews and Sharrock, 1995). At the amino terminal of phy, a photosensitive domain is covalently attached to tetrapyrrole chromatophore (phytochromobilin). A domain at the carboxy terminal is responsible for dimerization. Light produces a switch from the inactive form (red-light-absorbing, Pr) to the biologically active form (far-red-light absorbing, Pfr). Phy molecules present in the cytoplasm in the Pr form are transferred to the nucleus following photoactivation (e.g., Kircher et al., 1999). Two hybrid screens (see Chapter 1) have identified some of the proteins interacting with phy. Among these the Phytochrome-interacting factor 3 (PIF3) a basic helix-loop-helix (bHLH) transcriptional regulator (Ni et al., 1998). PIF3 is present in the nucleus (Ni et al., 1998) bound to its DNA binding site, the G-box DNA sequence (CACGTG) which is also present in various other light activated promoters (Martinez-García et al., 2000). Analysis of the RNA shows the PIF3-phyB are responsible for activating two genes: circadian clock-associated protein 1 (CCA1) and late elongated hypocytyl (LHY) genes. These encode transcription-factor-related proteins which regulate the expression of chloroplast components and the circadian clock. Experiments using DNA-microarray technology showed that a large portion of the genes activated by far red light code for transcriptional regulators (Tepperman et al., 2001). These findings suggests direct targeting of light signals to the promoters of genes encoding key transcriptional regulators. These regulators orchestrate the expression of multiple genes. Another slower pathway also regulated by light involves the depletion in the nucleus of an E3 ubiquitin ligase initiated by ligt. The ubiquitin ligase is involved in the degradation pathway of the proteasome. This results in the accumulation of nuclear HYP5 protein, a transcription factor (see Osterlund et al., 2000; Schwechheimer and Deng, 2001). SUGGESTED READING General Reading Cramer, W.A., and Knaff, D.B. (1990) Energy Transduction in Biological Membranes, Chapters 5 and 6. Springer-Verlag, New York. Hall, D.O. and Rao, K.K., (1994) Photosynthesis, Cabridge University Press. Lawlor, D.W. (1987) Photosynthesis: Metabolism, Control and Physiology. Longman Scientific, http://www.albany.edu/~abio304/text/17part2.html (21 of 23) [5/5/2002 6:53:27 PM]
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London, and Wiley, New York. Other Allen, J.P. and Williams, J.C. (1998) Photosynthetic reaction centers, FEBS Lett. 438(1-2):5-9. (MedLine) Andersson, B. and Franzén, L.G. (1992) The two photosystems of oxygenic photosynthesis in Molecular Mechanisms in Bioenergetics, Ernster, L. ed. (New Comprehensive Biochemistry, vol.23) pp.121-143, Elsevier, Amsterdam, New York. Demmig-Adams, B., Gilmore, A.M. and Adams, W.W. III (1996) In vivo function of carotenoids in higher plants, FASEB J. 10:403-412. (Medline) Hunter, C.N. (1995) Rings of light, Curr. Biol. 5:826-828. (Medline) Knaff, D.B., and Kämpf, C. (1987) Substrate oxidation and NAD+ reduction by phototrophic bacteria. In Photosynthesis (Amesz, J., ed.), pp. 199-211. Elsevier, New York. Ort, D.R. (1985) Energy transduction in oxygenic photosynthesis: an overview of structure and mechanisms. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 165-196. Springer-Verlag. Berlin. Rutherford, A.W. (1989) Photosystym II, the water splitting enzyme. Trends Biochem. Sci. 14:227-232. (Medline) Sauer. R (1986) Photosynthetic light reactions-physical aspects. In Encyclopedia of Plant Physiology, New Series, Vol.19, pp. 85-97. Sprinder-Verlag, Berlin. Staehelin, L.A. (1986) Chloroplast structure and supramolecular organization of photosynthetic membranes. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 1-84. Springer-Verlag, Berlin. WEB RESOURCES Selected articles on the pump mechanism of bacteriorhodopsin are available through http://www.nature.com/nature/fow). REFERENCES Search the textbook http://www.albany.edu/~abio304/text/17part2.html (22 of 23) [5/5/2002 6:53:27 PM]
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Chapter 17: References
Back to Chapter 17
REFERENCES Allen, J.P. and Williams, J.C. (1998) Photosynthetic reaction centers, FEBS Lett. 438(1-2):5-9. (MedLine) Allen, J. F., Steinbach, K. E., and Arntzen, K. E. (1981)Chloroplast protein phosphorylation couples plastoquinone redoxstate to distribution of excitation energy between photosystems, Nature 291:25-29. Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. and Rees DC. (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors, Proc. Natl. Acad. Sci. USA 84:5730-5734. (MedLine) Anderson, J. M. (1981) Consequences of spatial separation of photosystem 1 and 2 in thylakoid membranes, FEBS Lett. 124:1-10. Anderson, J. M. (1987) Molecular organization of thylakoid membranes. In Photosynthesis (Amesz, J., ed.), pp. 273-297, Elsevier. New York. Anderson, J. M., and Anderson, B. (1982) The architecture of the photosynthetic membrane: lateral and transverse organization, Trends Biochem. Sci. 7:288-292. Arnon, D. I. (1961) Changing concepts of photosynthesis, BullTorrey Bot. Club 88:215-259. Balabin, I.A. and Onuchic, J.N. (2000) Dynamically controlled protein tunneling paths in photosynthetic reaction centers, Science 290:114-117. (MedLine) Bengis, C., and Nelson, N. (1977) Subunit structure of the chloroplast PSI reaction center, J. Biol. Chem. 252:4564-4569.(Medline) Bennett, J. (1983) Regulation of photosynthesis by reversible phosphorylation of the light-harvesting chlorophyll a/b protein,Biochem. J. 212:1-13.(Medline) Betts, J.N., Beratan, D.N. and Onuchic, J.N. (1992) Mapping electron tunneling pathways: an algorithm that finds the "minimum length"/maximum coupling pathway between electron donors and acceptor in proteins J. Am. Chem. Soc. 114:4043-4046.
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Blakenship, R. E., and Prince, R. C. (1986) State redox potentials andZ scheme of photosynthesis, Trends Biochem. Sci. 10:382-383. Blaurock, A.E. and Stoeckenius, W. (1971) Structure of the purple membrane, Nature New Biol. 233:152155.(Medline) Boska, M., Sauer. K., Buttner, W., and Babcock, G. T. (1983) Similarity of EPR signal II rise and P680+ decay kinetics in Tris-washed chloroplast II preparations as a function of pH, Biochim.Biophys. Acta 722:327-330. Canaani, O., and Malkin, S. (1984) Distribution of light excitationin an intact leaf between the two photosystems of photosynthesis.Changes in absorption cross-section following state 1-state 2transitions, Biochim. Biophys. Acta 766:513-524. Clayton, R. K. (1980) Photosynthesis: Physical Mechanisms and Chemical Patterns. Cambridge Univ. Press, London. Cramer, W. A., Widger, W. R., Herrmann, R. G., and Trebst. A.(1985) Topography and function of thylakoid membrane proteins,Trends Biochem. Sci. 10:125-129. Cramer, W. A.. Black, M. T.. Widger, W. R.. and Girivin. M. E.(1987) Structure and function of photosynthetic cytochrome b-c1and b6-f complexes. In The Light Reaction (Barber, J., ed.). pp.447-494. Elsevier, New York. Debus, R.J., Barry, B.A., Sithole, I., Babcock, G.T. and McIntosh, L. (1988) Directed mutagenesis indicates that the donor to P+680 in photosystem II is tyrosine-161 of the D1 polypeptide, Biochemistry 27:9071-9074. (Medline) Deisenhofer, J., Epp, O., Miki. K., Huber, R., and Michel, H.(1984) X-ray structure analysis of a membrane-protein complex, J.Mol. Biol. 180:385-398.(Medline) Deisenhofer, J., Michel, H., and Huber, R. (1985) Structural basis of photosynthetic light reactions in bacteria, Trends Biochem.Sci. 10:243-248. Deisenhofer, J., Epp, O., Sinning, I. and Michel, H. (1995) Crystallographic refinement at 2.3 Å resolution and refined model of the photosynthetic reaction centre from Rhodopseudomonas viridis, J. Mol. Biol. 246:429-457. (Medline) Demmig-Adams, B., Adams, W.W. III, Barker, D.H., Logan, B.A., Bowling D.R. and Verhoeven, A.S. (1996a) Using chrlorophyll, fluorescence to assess the fraction of absorbed light allocated to thermal dissipation of escess excitation, Physiol. Plant 98:253-264. http://www.albany.edu/~abio304/ref/ref17.html (2 of 8) [5/5/2002 6:53:33 PM]
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Demmig-Adams, B., Gilmore, A.M. and Adams, W.W. III (1996b) In vivo function of carotenoids in higher plants, FASEB J. 10:403-412. (Medline) Doring, G., Renger, G., Vater, J., and Witt, H. T. (1967) Properties of the photoactive chlorophyll-aII in photosynthesis, Z.Natyrforsch Teil B 1139-1143. Dunahay, T. G., Staehelin, L. A., Siebert, M., Ogilvie. P. D., and Berg, S. P. (1984) Structural, biochemical and biophysical characterization of four oxygen-evolving photosystem II preparations from spinach, Biochim. Biophys. Acta 764:179-193. Dutton, P. L. (1986) Energy transduction in an oxygenic photosynthesis. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 197-237.Springer-Verlag, Berlin. Duysens, L. N. M., Amesz, J., and Kamp, B. M. (1961) Two photochemicalsystems in photosynthesis, Nature 190:510-514. Emerson, R., Chambers, R., and Cederstrand, C. (1957) Some factors influencing the long-wave limit of photosynthesis, Proc. Natl.Acad. Sci. U.S.A. 43: 133-143. Foote, C.S. (1976) Photosensitized oxidation of singlet oxygen: consequences in biological systems, in Free Radicals in Biology, ed. Pryor, W.A., Academic Press, New York, 2:85-133. Fromme, P., Witt, H.T., Schubert, W.D., Klukas, O. Saenger, W. and Krauss, N. (1996) Structure of photosystem I at 4.5 Å resolution. A short review including evolutionary aspects Biochim. Biophys. Acta 1275:76-83. Fyfe, P.K. and Jones, M.R. (2000) Re-emerging structures: continuing crystallography of the bacterial reaction centre, Biochim. Biophys. Acta 1459:413-421. (MedLine) Gennis, R.B. and Ebrey, T.G. (1999) Proton pump caught in the act, Science 286:252-253.(Medline) Glazer. A. N. and Melis, A. (1987) Photochemical reaction centers, structure, organization, and function, Annu. Rev. Plant Physiol.38:11-45. Goodenough, U. W. and Staehelin, L. A. (1971) Structural differentiation of stacked and unstacked chloroplast membranes. Freeze etch electron microscopy of wild-type and mutant strains of Chlamydomonas, J. Cell Biol. 48:594-6l9.(Medline) Goodenough, U. W., Armstrong, J.J., and Levine, R. P. (1969) Photosynthetic properties of ac-31, a mutant strain of Chlamydomanas reinhardi devoid of chloroplast membrane stacking, Plant Physiol.
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44:1001-1012. Haehnel, W. (1984) Photosynthetic electron transport in higherplants, Annu. Rev. Plant Physiol. 35:659693. Hauska, G. A., Hurt. E., Gabellini, N.. and Lockau, W. (1983) Comparative aspects of quinol-cytochrome c/plastocyaninoxidoreductases, Biochim. Biophys. Acta. 726:97-133.(Medline) Hauska, G., Schoedl, T., Remigy, H. and Tsiotis, G. (2001) The reaction center of green sulfur bacteria Biochim. Biophys Acta1507:260-277. (MedLine) Havaux, M. and Niyogi, K.K. (1999) The violaxanthin cycle protects plants from photooxidative damage by more than one mechanism, Proc. Natl. Acad. Sci. USA 96:8762-8767.(Medline) Heathcote, P., Fyfe, P.K. and Jones, M.R. (2002) Reaction centres: the structure and evolution of biological solar power, Trends Biochem. Sci. 27:79-87. (MedLine) Jansson, S. (1999) A guide to the Lhc genes and their relatives in Arabidopsis/IT>, Trends Plant Sci. 4:236-240. (MedLine Jones C. W. and Vernon, L. P. (1969) Nicotinamide photoreductionin Rhodospirillum rubrum chromatophores, Biochim. Biophys. Acta.180:144-164.(Medline) Jordan, P., Fromme, P., Witt, H.T., Klukas, O., Saenger, W. and Krauss, N. (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 Å resolution, Nature. 2001 411:909-917. (MedLine) Khorana, H.G. (1988) Bacteriorhodopsin, a membrane protein that uses light to translocate protons, J. Biol. Chem. 263:7439-7442.(Medline) Kircher, S., Kozma-Bognar, L., Kim, L., Adam, E., Harter, K., Schafer, E. and Nagy, F. (1999) Light quality-dependent nuclear import of the plant photoreceptors phytochrome A and B, Plant Cell11:14451456. (MedLine),/a> Klimov, V. V., Dolan, E., Shaw, E. R., and Ke, B. (1980) Interactionbetween the intermediary electron acceptor (pheophytin) and a possible plastoquinone-iron complex in photosystem II reaction centers, Proc.Natl. Acad. Sci. U.S.A. 77:7227-7231. Knaff, D. B., and Kampf, C. (1987) Substrate oxidation and NAD+reduction by prototrophic bacteria. In Photosynthesis (Amesz, J.,ed.), pp. 199-212. Elsevier, New York.
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Kok, B. (1956) On the inhibition of photosynthesis by intense light, 21:234-244. Kok, B. Forbush, B., and McGloin, M. (1970). Cooperation of charges in photosynthetic 02 evolution. I. A linear four step mechanism, Photochem. Photobiol. 11:457-475.(Medline) Krauss, N., Schubert, W.D., Klukas, O., Fromme, P., Witt, H.T. and Saenger, W, (1996) Photosystem I at 4 Å resolution represents the first structural model of a joint photosynthetic reaction centre and core antenna system, Nature Struct. Biol. 3:965-973. (Medline) Kühlbrandt, W., Wang, D.N. and Fujiyoshi, Y. (1994) Atomic model of plant light-harvesting complex by electron crystallography, Nature 367:614-621.(Medline) Kühlbrandt, W. (2000) Bacteriorhodopsin--the movie, Nature 406:569-570. (MedLine) Kyle, D., Staehelin, L. A., and Amtzen, C. J. (1983) Lateral mobility of the light harvesting complex in chloroplast membranes controls excitation energy distribution in plants, Arch. Biochem.Biophys. 222:527541.(Medline) Lam, E., Ortiz, W., Mayfield, S., and Malkin, R. (1984) Isolation and characterization of a light harvesting chlorophyll a/b proteincomplex associated with PSI, Plant Physiol. 74:650-655. Lanyi, J.K. (1998) Understanding structure and function in the light-driven proton pump bacteriorhodopsin, J. Struct. Biol. 124:164-178.(Medline) Larsson, U. K., Jergil, B., and Anderson, B. (1983) Changes in lateral distribution of the light-harvesting chlorophyll a/b-protein complex induced by its phosphorylation, Eur. J. Biochem.136:25-29.(Medline) Li, X.-P., Björkman, O., Shih, C, Gross, A.R., Rosenquist, M., Jansoon, S. and Niyogi, K. (2000) A pigment-binding protein essential for regulation of photosynthetic light harvesting, Nature 403:391-395. Losada, M., Trebst, A. V., Ogata, S., and Amon, D. I. (1960) Equivalence of light and adenosine triphosphate in bacterial photosynthesis, Nature 186:753-760. Luecke, H., Schobert, B., Richter, H.T., Cartailler, J.P. and Lanyi, J.K. (1999) Structural changes in bacteriorhodopsin during ion transport at 2 Angstrom resolution, Science 286:255-261.(Medline) Maeda, H., Watanabe, T., Kobayashi, M. and Ikegami, I. (1992) Presence of two chlorophyll-a' molecules at the core of photosystem-I, Biochim. Biophys. Acta 1099:74-80. Malkin, R. (1987) Photosystem I. In The Light Reaction (Barber, J., ed.). pp. 495-560. Elsevier, New York. http://www.albany.edu/~abio304/ref/ref17.html (5 of 8) [5/5/2002 6:53:33 PM]
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Malkin, R., Chain, R. K., Kraichoke, S., and Knaff, D. B. (1981) Studies of the function of the membrane bound iron-sulfur center of the photosynthetic bacterium Cheomatium vinosum, Biochim.Biophys. Acta. 637:88-91. Martinez-García, J.F., Huq, E. and Quail, P.H. (2000) Direct targeting of light signals to a promoter element-bound transcription factor, Science 288:859-863. (MedLine) Mathews, S. and Sharrock, R.A. (1997) Phytochrome gene diversity, Plant Cell Environ. 20:666-671. McDermott, G., Prince, S.M., Freer, A.A., Hawthornwaite-Lawless,A.M, Papiz, M.Z., Cogdell, R.J. and Isaacs, N.W. (1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria, Nature 374:517-521. Murata, N., and Miyao, M. (1985) Extrinsic membrane proteins in photosynthetic oxygen-evolving complex, Trends Biochem.Sci.10:122-124. Myers, J., and French, C. S. (1960) Evidence from action spectrafor a specific participation of chlorophyll b in photosynthesis,J. Gen. Physiol. 43:723-736. Namba, O., and Satoh, K. (1987) Isolation of photosystem II reaction center consisting of D1 and D2 polypeptides an cytochromeb-559, Proc. Natl. Acad. Sci. U.S.A. 84:109-112. Ni, M., Tepperman, J.M. and Quail, P.H. (1998) PIF3, a phytochrome-interacting factor necessary for normal photoinduced signal transduction, is a novel basic helix-loop-helix protein, Cell 95:657-667. (MedLine) Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular approaches, Annu. Rev. Plant Physiol. Mol. Biol. 50:333-359. Nugent, J. (2001) Photosynthetic water oxidation, Biochim. Biophys. Acta. 1503:1. (MedLine) Ort, D. R. (1986) Energy transduction in oxygenic photosynthesis: an overview of structure and mechanisms. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 165-196. SpringerVerlag,Berlin. Oesterhelt, D. and Stoeckenius, W. (1971) Rhodopsin-like protein from the purple membrane of Halobacterium halobium, Nature New Biol. 233:149-152.(Medline) Osterlund, M.T., Hardtke, C.S., Wei, N. and Deng, X.W. (2000) Targeted destabilization of HY5 during light-regulated development of Arabidopsis Nature 405:462-466. (MedLine) http://www.albany.edu/~abio304/ref/ref17.html (6 of 8) [5/5/2002 6:53:33 PM]
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Park, R. B. (1966) Thin section of KMn04 fixed Spinacea oleraceachloroplast, x19,500. In The Chlorophylls (Vernon, L. P., and Seely, G. R., eds.), pp. 283-311. Academic Press, New York. Parson, W.W. (1987) The bacterial reaction center. In Photosynthesis(Amesz, J., ed.), pp. 43-62. EIsevier, New York. Pfannschmidt, T., Anders, N. and Allen, J.F. (1999) Photosynthetic control of chloroplast gene expression, Nature 397:625-628. Pierson, B. K., and Olson, J. M. (1987) Photosynthetic bacteria.In Photosynthesis (Amesz, J., ed.), pp. 2142. Elsevier, New York. Quail, P.H. (2002) Phytochrome photosensory signalling networks, Nature Rev. Mol. Cell Biol. 3:85-93. (MedLine) Rhee, K.H., Morris, E.P., Barber, J. and Kühlbrandt, W. (1998) Three-dimensional structure of the plant photosystem II reaction centre at 8 Å resolution, Nature 396:283-286. (Medline) Rintamäki, E., Salonen, M., Suoranta, U.M., Carlberg, I., Andersson, B. and Aro, E.M. (1997) Phosphorylation of light-harvesting complex II and photosystem II core proteins shows different irradiance-dependent regulation in vivo. Application of phosphothreonine antibodies to analysis of thylakoid phosphoproteins, J. Biol. Chem. 272:30476-30482.(Medline) Rutherford, A.W. and Faller, P. (2001) The heart of photosynthesis in glorious 3D, Trends Biochem. Sci. 26:341-344. (MedLine) Sauer, K. (1986) Photosynthetic light reactions. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 603-631. Springer-Verlag, Berlin. Schubert, W.D., Klukas, O., Krauss, N., Saenger, W., Fromme, P. and Witt, H.T. (1997) Photosystem I of Synechococcus elongatus at 4 Å resolution: comprehensive structure analysis, J. Mol. Biol. 272:741-769. (Medline) Schwechheimer, C. and Deng, X.W. (2001) COP9 signalosome revisited: a novel mediator of protein degradation, Trends Cell Biol. 11:420-426. (MedLine) Sprague, S. G., and Varga, A. R. (1986) Topography, composition and assembly of photosynthetic membranes. In Encyclopedia of PlantPhysiology, New Series, Vol. 19, pp. 603-631. Springer-Verlag, Berlin.
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Staehelin, L. A. (1986) Chloroplast structure and supramolecular organization of photosynthetic membranes. In Encyclopedia of Plant Physiology, New Series, Vol. 19, pp. 11-83. Springer-Verlag, Berlin. Stanier, R. Y. (1961) Photosynthetic mechanisms in bacteria and plants: development of a unitary concept, Bacterial. Rev. 25:1-17. Stemler, A., and Radmer, R. (1975) Source of photosynthetic oxygenin bicarbonate-stimulated Hill reaction, Science 190:457-458. Stowell, M.H., McPhillips, T.M., Rees, D.C., Soltis, S.M., Abresch, E. and Feher, G. (1997) Lightinduced structural changes in photosynthetic reaction center: implications for mechanism of electronproton transfer, Science 276:812-816. (MedLine) Tepperman, J.M., Zhu, T., Chang, H.S., Wang, X. and Quail, P.H. (2001) Multiple transcription-factor genes are early targets of phytochrome A signaling, Proc. Natl. Acad. Sci. USA 98:9437-9442. (MedLine) Trebst, A. (1986) The topology of the plastoquinone and herbicide peptides in photosystem II in the thylakoid membrane, Z.Naturforsch. Teil C5 41:240-245. Trebst, A. V., Tsujimoto, H. Y., and Arnon, D. I. (1958) Separation of light and dark phases in photosynthesis of isolated chloroplasts,Nature 182:351-355. Vierling, E., and Alberte, R. S. (1983) P700 chlorophyll a-protein. Purification, characterization and antibody preparation, Plant Physiol. 72:625-633. Webber, A.N. and Lubitz, W. (2001) P700: the primary electron donor of photosystem I, Biochim. Biophys. Acta 1507:61-79. (Medline) Whitmarsh, J., and Ort, D. R. (1984) Stoichiometries of electron transport complexes in spinach chloroplasts, Arch. Biochem. Biophys. 23:378-389.(Medline) Wang, D. and Govinjee (1979) Antagonistic effects of mono- and divalent cations on polarization of chlorophyll fluorescence inthylakoids and changes in excitation energy transfer, FEBS Lett.97:373-377. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger, W. and Orth, P. (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 Å resolution Nature 409:739-743. (MedLine) Zuber, H., Brunischolz, R., and Sidler, W. (1987) Structure and function of light-harvesting pigmentprotein complexes. In Photosynthesis (Amesz, J., ed.), pp. 233-272. Elsevier, New York.
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18. Energy Transduction: Oxidative Phosphorylation and Photophosphorylation
18. Energy Transduction: Oxidative Phosphorylation and Photophosphorylation I. Energy Transduction in Mitochondria A. Oxidative Phosphorylation Phosphorylation sites Thermodynamic considerations Proton production B. Transport of Ions II. Photophosphorylation III. Mechanisms of Coupling Alternatives IV. The ATP Synthase A. Structure of F0F1 B. Catalytic Activity of F1
Suggested Reading Web Resources References Back to List of Chapters The major function of the electron transport chains is to provide energy for the synthesis of ATP from ADP and Pi. ATP can be synthesized by either the oxidative reactions in mitochondria or bacteria (i.e., oxidative-phosphorylation) or the reactions of photosynthetic systems (i.e., photophosphorylation). As we saw in Chapters 15 and 16, the electron transport chain functions in either oxidative or photosynthesizing systems as a series of redox reactions. There is every indication that the mechanism of the coupling between electron transport and phosphorylation is also very similar in both systems. Section I deals with the reactions in mitochondria and Section II with photosynthetic systems. Finally, the mechanism of coupling is discussed in more detail in Sections III and IV. I. ENERGY TRANSDUCTION IN MITOCHONDRIA
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18. Energy Transduction: Oxidative Phosphorylation and Photophosphorylation
The involvement of mitochondria in both phosphorylation and ion transport is illustrated in the experiment of Fig. 1 (Chance, 1965). In this experiment, the respiration of isolated guinea pig kidney mitochondria is monitored by an electrode sensitive to O2 in the medium. The trace in the figure corresponds to a plot of the oxygen concentration (ordinate) with time (abscissa). Some respiration occurs without the addition of substrate, as indicated by the slight downward trace at the left-hand part of the graph; the mitochondria are presumably oxidizing an endogenous substrate. Addition of succinate (arrow 1) accelerates the respiration. Subsequent addition of ADP (arrow 2) to the suspension that already contains inorganic phosphate (Pi), produces a burst of respiration that coincides with oxidative phosphorylation. When all the ADP is used up, the respiration returns to a lower rate. Similar bursts of respiration occur after additions of Ca2+ (arrows 3 and 4), which is translocated into the mitochondrial lumen and precipitated as calcium phosphate. The rate of respiration before the addition of ADP, divided by the rate of respiration during phosphorylation, the respiratory control ratio, has been adopted as an index of the degree of coupling in
mitochondrial preparations. Fig. 1 Consumption of oxygen as a function of time, showing the increment in oxidation in the presence of ADP or Ca2+. Guinea pig kidney mitochondria and an assay medium containing 4 mM phosphate were used. Reproduced from B. Chance, Journal of Biological Chemistry, 240:2729-2748, 1965, with permission of the American Society of Biological Chemists, Inc.
Part A of this section concerns oxidative phosphorylation primarily; part B is concerned with the coupling to active transport of ions in mitochondria. A. Oxidative Phosphorylation The maximal phosphorylative yields of electron transport can be predicted from thermodynamic considerations (see below). However, these arguments cannot establish the actual yield, which can be determined directly in well-coupled mitochondria. The amount of ATP formed from ADP and Pi is carefully measured and the oxygen taken up by the system is estimated. Each oxygen g-atom represents an electron pair traversing the cytochrome chain. It has become customary to express the phosphorylative yield as a P/O ratio: the moles of inorganic phosphate esterified per oxygen atom taken up. Oxygen uptake can be conveniently assessed with an oxygen electrode (as done in the experiment depicted in Fig. 1). Phosphorylation can be evaluated by measuring the disappearance of Pi or ADP or the appearance of ATP. The incorporation into ATP of the radioactive 32Pi gives a reliable and convenient estimate of ATP http://www.albany.edu/~abio304/text/chapter_18.html (2 of 29) [5/5/2002 6:54:12 PM]
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synthesized. ATP can also be assayed by using biochemical reactions that are coupled to the hydrolysis of ATP. One of the most sensitive is the luciferin-luciferase reaction using enzymes purified from the firefly tail. The amount of light generated from the system is a function of the ATP concentration. The amount of ATP synthesized can also be estimated indirectly. When small amounts of ADP are added, phosphorylation proceeds until virtually all the ADP has been used up to synthesize ATP. Therefore, the ATP formed corresponds to the ADP added. This is the method used in the interpretation of Fig. 1. Phosphorylation sites The actual yield of ATP per electron pair transported will depend on the substrate used because some of the substrates enter at different sites in the electron transport chain. Substrates that are oxidized by NAD+ dehydrogenases, such as isocitrate, α-ketoglutarate and malate, make use of the entire electron transport chain and involve all the redox couples of respiration. The oxidation of α-ketoglutarate has one additional substrate-level phosphorylation not involving the cytochrome chain. In practice, the fatty acid βhydroxybutyric acid is commonly used. In liver mitochondria, this substrate is converted quantitatively into acetoacetate in a reaction that reduces one NAD+. The P/O ratio of β-hydroxybutyrate oxidation is listed in Table 1 (Copenhaver and Lardy, 1952); the value is between 2 and 3. The mitochondria are thought to be imperfectly coupled. Therefore, the higher figures are generally assumed to be correct and the synthesis of three ATP is thought to occur per electron pair traversing the entire chain (see Lee et al., 1996; for a discussion, Hinkle et al., 1991 and for alternative views Nicholls and Ferguson, 1992). Table 1 P/O Ratios in Isolated Mitochondria
Substrate β-Hydroxybutyrate Pyruvate+ malate Succinate +(rotenone)
ascorbate cytochrome c
P/O
Reference
2.4-2.5
a
2.9
b
1.7
a
1.7-1.9 0.88 0.61-0.68
b c c
aCopenhaver
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The contribution of the various portions of the chain can be examined in more detail using other substrates (e.g., Copenhaver and Lardy, 1952; Lee et al., 1996). Succinic dehydrogenase is a flavoprotein, and the oxidation of succinate does not involve NAD+; it skips the NAD+-ubiquinone segment of the electron transport chain (see Fig. 6, Chapter 16). Interestingly, with succinate as the substrate, electrons enter the chain below those for an NAD-linked substrate and the P/O ratio is approximately 2 (Table 1). The results in Fig. 1 show that the phosphorylation of 200 µM ADP results in the uptake of 108 µg-atoms of oxygen. In this case, the P/O ratio is 1.8. These results suggest that one phosphorylative step occurs in the span of the chain preceding CoQ (i.e., NAD-FP-CoQ) and the other two in the span below CoQ (i.e., CoQ-cyt bcyt c1-cyt c-cyt a-cyt a3-O2). Although no natural substrate interacts with the chain span below CoQ, it has been found that ascorbate reduces cytochrome c nonenzymatically. Frequently, the experiments hare carried out in the presence of catalytic amounts of the dye N,N,N',N'-tetramethyl-p-phenylene diamine (TMPD), which mediates the electron transfers between ascorbate and cytochrome c. The resulting P/O ratios approach 1 (Table 1) (Lehninger, 1955). Added reduced cytochrome c can also be oxidized by the system, also yielding a P/O of about 1. Therefore, the second phosphorylation must occur in the span between CoQ and cytochrome c, and the third between cytochrome c and oxygen, as shown in Fig. 2. For convenience, Chance and Williams (1956) numbered the various possible conditions of mitochondria. In the presence of substrate and O2, the mitochondria are said to be in state 4. With the addition of ADP, the phosphorylating mitochondria are said to be in state 3. When all the ADP is used up, the mitochondria undergo a transition from state 3 to state 4. Three other states, not discussed in this chapter, have been described: oxygenated mitochondria without added ADP or substrate (state 1), state 1 conditions with the addition of ADP (state 2), and the complete system in the absence of oxygen (state 5). In very tightly coupled mitochondria, little oxidation takes place in the absence of ADP. ADP is involved only at the phosphorylative sites; its absence should block electron flow at these sites. Accordingly, the components preceding the block should be more reduced and those following the block more oxidized. The electron transport chain should exhibit crossover points similar to those caused by a block in the chain by a specific inhibitor. In addition, since the oxidative phosphorylation reactions are reversible, the hydrolysis of ATP can reverse the flow of electrons. In this latter case, the components that precede the phosphorylative site in the course of normal electron transport should become more reduced and those that follow should become more oxidized. Therefore, the addition of ADP or ATP under the appropriate conditions aids in pinpointing the location of the phosphorylative sites.
Fig. 2 Diagram summarizing (a) the sequence of electron transport components and (b) the location of the
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phosphorylative sites.
The results of an experiment providing data to determine cross-over points is shown in Fig. 3. In this experiment, the hydrolysis of ATP reduces components at the phosphorylation site in the direction opposite to normal electron transport. The addition of ATP induces the reduction of pyridine nucleotide (Fig. 3a) at the expense of the oxidation of the cytochromes and flavoproteins. Na3S is used to block the cytochrome chain at the cytochrome a3 site. The two curves correspond to the changes in optical density at wavelengths that allow monitoring of the redox state of NAD (curve 1) and cytochrome c (curve 2) with time. The optical density changes have been calibrated in terms of NADH formed (upper ordinate) or expressed in terms of cytochrome oxidation (lower ordinate). In this kind of experiment, several respiratory carriers, including flavoprotein, are oxidized as shown in Fig. 3b. Similarly, other crossover points can be studied after isolating the relevant part of the chain by means of inhibitors. A block between cytochromes b and c with antimycin permits the demonstration of an ATPinduced oxidation of cytochrome a+a3 and reduction of cytochrome c. A slow reduction of cytochrome b, while components closer to the oxygen are oxidized, suggests a possible third point of interaction. The conclusions reached in experiments in which the reactions are driven backward agree with those obtained for the transition from state 3 to state 4. These two approaches, i.e., deductions from crossover points, can give insight into the sites responsible for the phosphorylation of ADP. However, it is obvious that phosphorylation can be coupled to the redox couples of the electron transport chain only when enough energy (i.e., ∆G) is available. Therefore the energetics of the system are pertinent as well. Thermodynamic considerations The energy available from the redox reactions (i.e., the ∆G) can be estimated directly by measuring the redox potential for the appropriate redox reactions. The redox potentials depend on the physical environment, therefore, redox potentials have a meaning only in a native system. The redox state of the system can be varied chemically by adding precise amounts of oxidants or reductants, and the redox potential of the system as a whole can be measured. As long as the system is anaerobic (i.e., reducing equivalents cannot be oxidized by oxygen), the system is closed. Equilibration of the electron carriers should result in the entire system being poised at the same potential (Eh). As discussed in Chapter 12, the ∆G and ∆E of a half-cell can be represented as shown in Eqs. (1) and (2), respectively.
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Fig. 3 Reversal of electron transport on addition of ATP. The cytochrome c is oxidized and the pyridine nucleotide is reduced in sulfide-inhibited pigeon heart mitochondria, in the absence and presence of ATP. The mitochondria were in a mannitol-sucrose medium, buffered with Tris at pH 7.4 and a temperature of 26C. Reproduced from B. Chance and C. Hollunger, Journal of Biological Chemistry, 236:1577-1584, 1961, with permission of the American Society of Biological Chemists, Inc.
In practice, the redox potential can be measured in the presence of low molecular weight mediators (see below) with two electrodes: a reference electrode (e.g., calomel electrode) and an indicator electrode (e.g., platinum electrode). The indicator electrode responds to the redox components of the system. Basically, each electrode acts as a half-cell. For the calomel electrode the reaction of the half-cell is Hg2Cl2 (solid) + 2e-↔ 2Hg (solid) + 2Cl- (3) For the platinum electrode the reaction corresponds to Pt2+ + 2e-↔ Pt (4) The electron transport chain in the mitochondrial inner membrane cannot interact directly with the platinum electrode. Catalytic amounts of low molecular weight synthetic carriers (electron donorsacceptors) are added in order to shuttle electrons from the mitochondria to the indicator electrode. For each level of oxidation-reduction of each component there will be a corresponding ratio [ox]/[red]. As
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shown by Eq. (2), when this ratio is unity, ∆E becomes ∆Eo, the half reduction potential (∆Em). The concentrations of oxidized or reduced components are determined either from their light absorption or, in the case of the iron-sulfur proteins, from the electron spin resonance of the oxidized form. By convention (see Chapter 10) the redox potentials are those that would have been obtained using a H2electrode half-cell. Those obtained with the more practical calomel half-cell can be readily converted to the appropriate values for the H2-electrode (usually indicated as Eh). The reaction at a H2-electrode would be: ox + H2 ↔ red + 2H+ (5) In Fig. 4 (Wilson et al., 1983), components of the electron transport chain are displayed as a function of their redox potential (∆Eh and ∆Eoh). The various redox potentials seem to fall into three groups of approximately the same potentials. However, between the equipotential groups there is a change of about 250 mV corresponding to a ∆G of -5.7 kcal/mol. Considering that two electrons are needed to go through the cytochrome chain per phosphorylation site, with -7 to -10 kcal needed for the synthesis of ATP, the calculated -11.4 kcal would be sufficient. The redox potentials of three components (cyt a3, cyt b1, and a flavoprotein) appear to depend on whether the mitochondria are uncoupled (lower value) or coupled and in the presence of ATP (higher value). In the original studies, this finding was attributed to a direct involvement of these reactions in the synthesis of ATP. However, other interpretations are possible. For example, the ox/red ratio could have been altered by reversed passage of electrons at the expense of energy from ATP hydrolysis, as discussed above. The order of the electron transport carriers shown previously and the data summarized in Fig. 4 (Wilson et al., 1971) support the location of the three phosphorylation sites shown by the jumps in potentials in the figure, schematically expressed in Fig. 2.
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Fig. 4 Thermodynamic profile of the oxidation-reduction components of the respiratory chain of pigeon heart mitochondria. Each component is represented by a rectangle, which is centered on its half-reduction potential at pH 7.2 and extends from the potential at which the component is 9% reduced to the potential at which it is 91% reduced. The positions of the rectangles are not intended to indicate the sequence of electron transfer. On the left side are indicated the Eh values of the isopotential groups in pigeon heart mitochondria under state 4 conditions, with succinate and glutamate as substrate. Two values are indicated for cytochromes bt and a3, one measured for uncoupled mitochondria and the other measured for coupled mitochondia in the presence of excess ATP. Reproduced from D. P. Wilson, et al., Current Topics in http://www.albany.edu/~abio304/text/chapter_18.html (8 of 29) [5/5/2002 6:54:12 PM]
18. Energy Transduction: Oxidative Phosphorylation and Photophosphorylation
Bioenergetics, 5:234-265, 1973, with permission of Academic Press.
Proton production The chemiosmotic model proposes that a flux of H+ generated by electron transport produces a proton electrochemical gradient, the protonmotive force. A flow of protons in the direction of this gradient is coupled to the phosphorylation of ADP. Some of the details of the proposal can be questioned (see section III). However, a link between H+-flux and the synthesis of ATP is, undeniable. Therefore, sites of proton production must be equivalent to the phosphorylative sites. The reactions translocating protons have been shown in Chapter 16 (Section IIIC). They correspond to the electron transport sites deduced from the experiment just discussed. 4H+ are generated per site involving the NADH-ubiquinone oxidoreductase, the ubiquinol-cyt c oxidoreductase and the cytochrome c oxidase. Currently, 3H+ are thought to be needed per ATP synthesized (see summary of Cramer and Knaff, 1990, p.118). B. Transport of Ions As shown in Fig. 1, the addition of certain cations to mitochondrial suspensions results in a burst of respiration. This is the result of the active transport of the ion from the medium to the mitochondrial matrix. Energy-dependent uptakes have been demonstrated for a number of divalent cations (e.g., Mg2+, Mn2+, and Sr2+) and monovalent cations (e.g., K+, Na+, or Li+). The translocations can be supported by oxidative reactions or by the hydrolysis of ATP. As might be expected, the active transport is blocked by uncouplers. However, there are indications that the transport of ions differs significantly from the processes leading to phosphorylation, as indicated by the effect of the antibiotic oligomycin. Oligomycin binds to the ATP-synthase complex (F0F1) and blocks both the phosphorylation of ADP and the ATPase activity of the enzyme. Oligomycin also blocks the transport of cations powered by ATP hydrolysis. However, oligomycin does not block the transport of cations powered by respiration. The results of the experiment of Table 2 (Brierley et al., 1962) show the uptake of Mg2+ by beef heart mitochondria. The uptake in the complete system is shown in line 2. The uptake of Mg2+ requires energy, as indicated by the need for a substrate (line 5) and by the inability to transport in the presence of an uncoupler (line 8) or the inhibitor of electron transport, antimycin (line 9). Transport and oxidative phosphorylation compete as the uptake is reduced in the presence of ADP (line 6) or ADP plus hexokinase and glucose (line 7) (the latter to regenerate ADP by phosphorylating glucose). However, oligomycin, which blocks phosphorylation, fails to affect the transport (line 10), as discussed in more detail later. In the experiments of Table 2, Mg2+ is accompanied by Pi. In fact, accumulation of a cation is generally accompanied by the passage of an anion, maintaining electric neutrality; alternatively, an internal cation must leave. The uptake of a divalent cation in the presence of Pi results in precipitation of the salt. The anion is, therefore, functioning in a dual capacity, accompanying the cation and acting as a trapping agent. Under these conditions, massive amounts of the salt can be accumulated. However, the anion need not be phosphate; arsenate, acetate, and many others will do. Without removal of the accumulated ions by precipitation (as in the case of acetate), the uptake is not as marked.
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The uptake of cations is sometimes accompanied by a countermovement of H+. The precise magnitude of the exchange depends on the conditions. For example, in the absence of an excess of penetrating anion, two H+ appear in the medium per Ca2+. This ratio can be lowered considerably by the presence of acetate or some other anion to which the membranes are permeable, and in some cases no protons are ejected. Do the same steps in the electron transport chain provide the energy for ion transport and for the phosphorylation of ADP? Several experiments suggest that this the case. The effectiveness of the substrates linked to the various coupling sites (e.g., Brierley and Murer, 1964, Penniston et al., 1966) and the cross-overs in the presence and absence of Ca2+ (e.g., Chance and Hollunger, 1961) suggest that the transport of ions involves the same sites in the electron transport chain as oxidative phosphorylation. Table 2 Pi and Mg2+ Accumulation by Isolated Heart Mitochondria
Conditions 1. Mitochondria alone 2. Complete system 3. Mg2+ omitted 4. Pi omitted 5. Substrate omitted 6. ADP added (10 µmoles) 7. ADP + hexokinase system 8. Complete + dinitrophenol
Pia
Mg2+a
30 1010 10
50 1800 35
10
95
10 130 30 8
75 206 85 75
40 980
75 1800
(0.3 µmoles) 9. Complete + antimycin (6 µg) 10. Complete + oligomycin (12 µg)
Reproduced with permission from G.P. Brierley, et al., Proc. Natl. Acad. Sci. USA 48:1928-1935, 1962.
II. PHOTOPHOSPHORYLATION As we saw in Chapter 17, the electron transport chain and the phosphorylative reactions in photosynthetic systems are very similar to those of the mitochondrial system. Analogous diagrammatic schemes can be
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designed, as shown in Fig. 5 (Hall, 1976). Figure 5 shows not only the various electron transport components and their position in the photosynthetic electron transport chain, but also inhibitors, electron acceptors and donors. Electron acceptors and donors can functionally isolate part of the chain, as previously shown for the study of PSI or PSII (Chapter 17). For example, with methylviologen (MV) or ferricyanide (FeCy), the electron flow that normally reduces NADP can be bypassed. Similarly, benzidine and ascorbate can bypass the reactions that normally release oxygen. The relationship between electron transport and phosphorylative reactions can be studied in the chloroplasts by methodology analogous to that used in the study of mitochondria. Figure 6 (Hall, 1976) shows a record of oxygen release in an experiment in which ferricyanide is used as an electron acceptor. It is seen that light in the presence of ferricyanide increases the amount of O2 released. When ADP is added (state 3), the amount of O2 released increases. When the ADP is used up, the O2 released returns to the state 4 value (no ADP). The P/O ratios calculated from these results approach 2. However, such high ratios can be calculated only after correcting for the basal electron transport (see Horton and Hall, 1968; Winget et al., 1965). In addition to determining phosphorylative yield, it is possible to calculate photosynthetic control (PC) ratios analogous to respiratory control (RC) ratios in mitochondria, by dividing the oxygen released in state 3 by that released in state 4. If the components associated with PSI or PSII are isolated, the portions of the electron transport chain appropriately marked in Fig. 5 can be shown to be involved in the synthesis of ATP. These results suggest one such site per span.
Fig. 5 Scheme for noncyclic electron transport and phosphorylation. From Hall (1976), with permission. Abbreviations are as follows. Electron donors: DPC, diphenyl carbazide; FD, ferrodoxin; K3, vitamin K3 (menadione); NQ, napthoquinone; DAD, diaminodurene; TMI, tetramethylindamine; DAB, diaminobenzidine; PMS, phenazine methosulphate. Electron acceptors: ST, silicotungstate; BBMD, benzl-abromomalodinitrile; DADox, diaminodurene; DMQ, dimethyl-p-benzoquinone; MV, methyl viologen; FeCy, ferricyanide; BQ, benzoquinine. Inhibitors: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Tris, tris(hydroxy- methlaminomethane); DBMIB, 2,5-dibromo-3-methyl-6-isopopyl-p-benzoquinone;EDAC,1ethyl-3-(3-dimethylaminopropyl)cardodiimide; DSPD, disalicyclidene propane diamine; PADR, phosphoadenosine diphosphate ribose. Reproduced with permission from D.O. Hall, The Intact Chloroplast. Copyright ©1976 Elsvier Sceince Publishers, Amsterdam.
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Fig. 6 Record of oxygen release over time. The electron acceptor is ferricyanide (FeCy). Reproduced with permission from D.O. Hall, The Intact Chloroplast. Copyright ©1976 Elsevier Science Publishers, Amsterdam.
The results for photophosphorylation appear analogous to those for oxidative phosphorylation. For this reason the two are thought to involve the same mechanisms, as discussed in Section III. III. MECHANISMS OF COUPLING The chemiosmotic hypothesis of Mitchell (1966, 1967) proposes the formation of a proton electrochemical gradient by the coupling of electron transport to the flux of H+, an efflux in the case of mitochondria and an influx in the case of the thylakoid vesicles. The return of H+ in the direction of its electrochemical gradient involving the ATP-synthase is coupled to the phosphorylation of ADP. The transducing membranes are assumed to be extremely impermeable to ions so that the electrochemical gradient is not passively dissipated. Translocations of ions are assumed to occur mostly by electrically neutral exchanges in which external ions are exchanged for internal ions of the same charge (antiport), or oppositely charged ions are simultaneously transferred in the same direction (symport). http://www.albany.edu/~abio304/text/chapter_18.html (12 of 29) [5/5/2002 6:54:13 PM]
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Fig. 7 Diagram summarizing the possible interpretation of the data. A chemiosmotic model.
The system can be operated as an ATPase or as a phosphorylative mechanism, as shown for mitochondria in Fig. 8. The operation of the cytochrome chain to produce an H+ ion gradient or a membrane potential is represented in Fig. 8a. The operation of the ATP synthase either in phosphorylation (heavy lines) or as an ATPase (dashed lines) is shown in Fig. 8b. The pH differential or an internal negative potential resulting from the expulsion of H+ during oxidation would drive the H+ inward for phosphorylation. The functioning of the system in the transport of cations, in this example Ca2+, is shown in Fig. 8c. Fig. 8 represents the chemiosmotic hypothesis as proposed in mitochondria. In the case of the thylakoid vesicles, all the events would have the opposite polarity, since the H+ transport occurs inwardly.
Fig. 8 Diagram representing the chemiosmotic hypothesis. (a) Formation of a membrane potential or a pH gradient by passage of the electrons through the cytochrome chain. (b) ATPase functioning either to hydrolyze ATP (dashed line) or to phosphorylate ADP (solid line). (c) Exchange of cations for H+.
The possible role of the chemiosmotic model can be evaluated through two basic questions: (1) Can the flux of H+ in the direction of its electrochemical gradient produce ATP from ADP and Pi? (2) During normal electron transport, is there an H+ electrochemical gradient sufficient to play a role in the various transducing systems (mitochondria, thylakoid vesicles and bacteria)?
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The answer to the first question is yes for thylakoid vesicles, as shown by the classic experiment of Jagendorf and Uribe (1966), summarized in Table 3. In this experiment, leaky chloroplasts, probably equivalent to thylakoid vesicles, were maintained in the dark or in the presence of the appropriate inhibitor. The chloroplasts were first equilibrated with organic acid and then shifted to an alkaline medium. Presumably, the inside would initially be acidic, as it would be with illumination; the proton flow is in the direction opposite to that in mitochondria. The external alkalinization would then provide the appropriate gradient for an H+ efflux. The first item (a) corresponds to the experimental determination with the complete system. The amount of ATP formed was measured either with the luciferin-luciferase reaction (column 4) or by measuring the amount of phosphate incorporated into ATP (column 6); the two methods show that ATP was formed by this procedure. On the other hand, ATP was not synthesized when the initial incubation was at pH 7.0 (item b), or when any of the necessary components were left out (such as PO4, item c; ADP, item d; Mg2+, item e; or chloroplasts, item f). Analogous demonstrations are available for submitochondrial particles or reconstituted vesicular preparations containing only ATP synthase, FF, (e.g., Sone et al., 1977). The latter experiments also establish that F0F1 (and no other protein) is necessary for the synthesis of ATP. Table 3 Formation of ATP by Acid-Base Transitionsa
ATP (estimated by luciferase assay)
ATP (estimated by phosphomolybdate extraction)
Reaction mixture
pH of acid
Total
Net synthesis
Total
Net Synthesis
(1)
(2)
(3)
(4)
(5)
(6)
a. Complete
3.8
141
129
166
163
b. Complete
7.0
12
--
3
--
c. Pi omitted
3.8
12
--
--
--
d. ADP omitted
3.8
4
--
3
--
e.. Mg2+ omitted
3.8
60
48
48
45
f.. Chloroplasts omitted
3.8
7
--
3
--
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Reproduced with permission from A.T. Jagendorf and E. Uribe, Proc. Natl. Acad. Sci. USA 55:170-177, 1966. aThe
chloroplast fragments were first exposed to acid then to an alkaline medium (pH 8). The results are given in micromoles/mg protein. Even though the experiments just described support the chemiosmotic hypothesis, experiments with intact mitochondria do not. In mitochondria, because the H+ pumping is outward, the matrix will become alkaline. In isolated mitochondria, a small alkaline shift in the medium induces ATP synthesis (Malenkova et al., 1982). Experiments with base followed by acid shifts do not produce ATP. Since in mitochondria a H+ influx should be coupled to ATP synthesis, these experiments are at variance with the present chemiosmotic model. The answer to the second question is also not clear. The electrochemical potential gradient (the protonmotive force of Mitchell) contains two terms: one referring to the H+ concentration gradient and the other to the electrical potential across the transducing membrane, Eq. (6):
In these equations, the subscripts i and o refer to location inside or outside the lumen, F is the Faraday and corresponds to the membrane potential. The stoichiometry was thought to correspond to n=2. However, more recently, n=3 has been favored. The two terms have been evaluated in a number of studies. The mitochondrion will be used for the discussion of the technique used to calculate the pH. Analogous reasoning can be applied to the thylakoid vesicles. In metabolizing mitochondria in which the interior is alkaline in relation to the outside, the pH has generally been evaluated by measuring the distribution of a weak acid (HA). The acid is assumed to permeate the inner mitochondrial space rapidly and primarily in its undissociated form. With these assumptions, at equilibrium, [HA]i = [HA]o (7) The dissociation constant would be the same for the two phases:
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[H+]i[A-]i = [H+]o[A-]o (9) so that
Therefore, in mitochondria, the pH can be readily calculated from the distribution of the weak acid. In practice, only HA + A- can be determined. Equation (10) can be used readily for these cases (which requires the appropriate pKA). A more complex equation permits the calculation when the pKA is too low for Equation (15) to be applicable. For thylakoid vesicles, the inside is acid in relation to the medium, and the analogous technique uses weak bases. For illuminated chloroplasts at steady state, the values calculated by a similar technique suggest a pH as high as 3 to 3.5 (Pick et al., 1974). In fact, little phosphorylation occurs below a pH of 2.5. In effect, this would allow for 8.4 kcal (at pH=3), which would barely suffice to synthesize one ATP if two H+ were transferred. However, it is generally thought that 3 H+ are transferred per ATP synthesized. In contrast, with mitochondria, the pH under physiological conditions depends on the tissue. In rat liver, it is generally approximately 0.5 (Addanki et al., 1968), not enough to play a significant role (although sometimes values as high as 1 have been reported for mitochondria from other tissues). Calculations of membrane potentials follow a different rationale. In a number of cells, it is possible to estimate the electric potential across the plasma membrane by indirect techniques, using the distribution (i.e., the ratio of concentrations) of a cation completely dissociated at biological pH values. Commonly, these techniques have been applied to mitochondria or chloroplasts. Equation (11) shows the free energy available from the distribution of a monovalent cation per mole transferred.
At equilibrium ∆G = 0, so Eq. (12), the Nernst equation, is applicable.
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Where it is possible to calculate the potential from the distribution of a cation, the procedure assumes that (1) the system is in equilibrium in relation to the cation used as a probe, (2) the ion distribution has not disturbed the system, and (3) the cation has distributed solely as the result of the potential across the membrane. The third item tacitly makes the a priori assumption that no other mechanism is responsible for the distribution. Presumably, the same reasoning should apply for the thylakoid vesicles. However, in this case the potential across the membrane would be positive inside and an anion would have to be used as a probe. In the case of the thylakoid vesicles, the distribution shows that the potential is negligible in magnitude, at least at steady state. The predominantly accepted view at this time is that there is a significant membrane potential across the inner mitochondrial membrane. This ∆Ψ is the major portion of the driving force for the phosphorylation of ADP or the transport of ions. In support of this view, the rate of influx of Ca2+ in isolated mitochondria conforms to that predicted from the calculated ∆Ψ (Gunter and Pfeiffer, 1990). However, this may result from a common transport mechanism for the various cations, sharing the driving force and the energy requirements. In the case of mitochondria, there are preparations that phosphorylate in the absence of a significant potential (as calculated by the same method). In other preparations, however, it is possible to calculate a substantial membrane potential, as high as -200 mV. However, there is evidence that a process other than the potential itself is responsible for the distribution, that is, a transport system involving an H+/C+ stoichiometric exchange (for a review, see Tedeschi, 1981). Microelectrode impalement of giant mitochondria also shows that phosphorylation takes place in these mitochondria in the absence of a significant membrane potential (e.g., Campo et al., 1984). In addition, the protons that have to be pumped out in the absence of ion transport to produce a potential across the membrane of -200 mV can be easily calculated (see Eq. (6) of Chapter 21) to be approximately 1 x l0-6 moles per g protein, as done by Mitchell (1966). When checked experimentally, a significant H+ efflux has never been observed in the absence of ion transport (e.g., Archbold et al., 1979). Other findings also suggest that the calculated from cation-probe distributions does not have a role in energy coupling. For example, the presence or absence of respiration has no effect on the rate constant of the efflux of cationic probe molecules; it affects only the rate constant of the influx (Skulskii et al., 1983). Both should be affected (in opposite ways) if a membrane potential were responsible for distribution. Furthermore, in at least some experiments, the membrane potential and the protonmotive force measured by conventional means remain essentially the same when both respiration and phosphorylation are decreased in parallel by the use of inhibitors (e.g., Mandolino et al., 1983). http://www.albany.edu/~abio304/text/chapter_18.html (17 of 29) [5/5/2002 6:54:13 PM]
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If we discarded the chemiosmotic model, we would be left with a major dilemma: how could we explain the synthesis of ATP in the experiments in which an H+ flux has been imposed? Several models have now been proposed that depend on localized effects (e.g., see Dilley and Schreiber, 1984; Hong and Junge, 1983; Kagawa, 1984; Westerhoff et al., 1984). In additon, current evidence indicates that protons generated at the surface of a bilayer membrane diffuse laterally (Heberle et al., 1994; Scherrer et al., 1994; Alexiev et al., 1995), suggesting that these localized protons can be directly coupled to the oxidative- or photo-phosphorylation of ADP (see Ferguson, 1995). Ferguson (1995) suggests that despite a localization of protons, at steady state there would be "an equilibrium with the bulk aqueous phase." This conclusion is highly unlikely under phosphorylative conditions because the proton flux generated by electron transport in one direction is likely to be be matched by the flux of the protons responsible for the phosphorylation in the opposite direction. In contrast, we would expect an equilibration in the absence of oxidative phosphorylation. This dichotomy would provide a switch between oxidative-phosphorylation (localized mechanism) and cation transport (supported by a membrane potential). Although this latter possibility would seem extremely attractive, it should be noted that we have also demonstrated in giant mitochondria, the accumulation of Ca2+ in the absence of a membrane potential (Maloff et al. 1978); there is also evidence that the transport of cations occurs electroneutrally by a stoichiometric exchange with protons (see Tedeschi, 1981). Fortunately, the same molecular mechanisms can be invoked as long as a role of H+ is postulated. The electron transport chain could deliver H+ to the ATP synthetase either in the presence or absence of a conventional protomotive force. Fig. 9 (Senior, 1979) illustrates a model that can operate either with or without a proton gradient. The proton directly delivered to the ATP synthase (step I) produces the release of ATP (by changing the conformation and hence the dissociation constant) in step II. This apparently corresponds to the energy-requiring step. The simultaneous attachment of ADP and Pi (step II) results in the release of H+ (step III), presumably by decreasing the binding constant for H+. The synthesis of bound ATP is shown in step IV. There is considerable evidence that the formation of bound ATP does not require a significant amount of energy (Boyer et al., 1973; Feldman and Sigman, 1982, 1983). Bound ATP, (ATPb), can be present in the presence of an uncoupler (Boyer et al., 1973; Feldman and Sigman, 1983) and furthermore, in preparations of F1 alone, at very low concentrations, the equilibrium constant for [(ATP)b]/[ADP][Pi] is approximately 0.6, corresponding to a ∆G of 0.3 kcal. Similarly, there is evidence for conformational changes of ATP synthase dependent on energy input (e.g., Gogol et al., 1989a, 1989b, 1990) or the presence of ATP (Dilley and Schreiber, 1984), and these changes result in different binding constants of nucleotides. The synthesis of ATP by isolated ATP synthase induced by a shift to an alkaline medium, supports a conformational model (Blumenfeld et al., 1987), although not the details of Fig. 9.
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Fig. 9 The conformational model: Cyt, cytoplasm; mat, mitochondrial matrix. Reproduced from Senior (1979), by courtesy of Marcel Dekker, Inc.
IV. THE ATP SYNTHASE The ATP synthase or proton-ATPase, F0F1, is a complex of many polypeptides and can function either forward in oxidative phosphorylation or photophosphorylation or backward, i.e., to hydrolyze ATP. A. Structure of F0F1 Present understanding of the structure of ATP synthase of Escherichia coli is shown in the model depicted in Fig. 10 (Junge et al., 1997). The figure incorporates data from many biophysical approaches. In order to show the arrangement in some detail, the front α and β subunits are not shown in the figure (the αβ complex is actually a hexamer). It includes the water-soluble portion (F1) and the components associated with the inner mitochondrial membrane (Fo). The ATP synthase subunits are highly conserved in organisms ranging from E. coli and thermophilic bacteria to the so-called higher eukaryotes including the http://www.albany.edu/~abio304/text/chapter_18.html (19 of 29) [5/5/2002 6:54:13 PM]
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synthase of chloroplasts. In addition, there is a high degree of homology between subunits α and β that contain the catalytic site. The mechanism of action of the ATP synthases of the various organisms is probably identical, since recombination of the various components from different sources produces a
hybrid complex that is functional. Fig. 10 Model of the general structure of ATP synthase from E. Coli. The Fo portion represents integral and proton-conducting proteins. The components of F1 contain the nucleotide binding sites. The stator portion (see text) includes a , b, δ and (α β)3. The rotor portion includes cn, ε and γ. From Junge et al., 1997 by permission.
The representation differs from previous versions by the presence of a double stalk, one including γ and ε, components of F1, and the other including b2, a component of Fo, in close contact with δ (a subunit of F1). The γ portion has been shown to rotate in relation to the (αβ)3 complex. As discussed later, the assembly of a, b, δ and the complex of (αβ)3 acts as a stator, that is the stationary part of a machine. In contrast, c, γ and ε correspond to the rotor of the motor (e.g. see Junge et al., 1997; Elston et al., 1998). The polypeptide composition of F1 is listed in Table 4 for E. coli and beef heart mitochondria (Senior, 1988). With the exception of ε from beef heart mitochondria, the subunits from the two different systems appear to be very similar. However, the nomenclature, i.e., the Greek letter denoting each polypeptide, differs. The components are present with the stoichiometry (αβ)3 γδ ε. The composition of the Fo portion is shown in Table 5 (Senior, 1988). The E. coli Fo proteins a, b and c are present in the stoichiometry a1b2cn, where n has been estimated to be between 10 and 12. Many other components have been shown in mitochondria: factor B, 11-15 kDa; F6, 89 kDa; subunit 8,6-8 kDa; an uncoupler binding protein, 30 kDa; and the inhibitor polypeptide, 9.6 kDa. The inhibitor peptide probably functions in blocking the ATPase activity in favor of the synthase activity. Subunit 6 of mitochondrial Fo of 25 to 29 kDa is homologous to subunit a of E. coli, whereas subunit 9, an http://www.albany.edu/~abio304/text/chapter_18.html (20 of 29) [5/5/2002 6:54:13 PM]
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8-kDa proteolipid, is homologous to subunit c. Table 4 Subunit Composition of E coli and Beef Heart Mitochondrial F1
Subunit composition Total molecular size
E. coli
Beef Heart mitochondria
α3β3γδε 381 kDa
α3β3γδε 371 kDa
513 55.2 kDa
509 55.2 kDa
459 50.15 kDa
480 51.6 kDa
286 31.43 kDa
272 30.14 kDa
177 19.33 kDa
190 21 kDa
138 14.92 kDa
146 15.1 kDa
α subunit No. of residues molecular size β subunit No. of residues molecular size γ subunit No. of residues molecular size δ subunit No. of residues molecular size ε subunit/ δ subunita No. of residues molecular size ε subunit No. of residues molecular size
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50 5.65 kDa
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Reproduced by permission from A.E. Senior, Physiological Reviews 68:177-231. Copyright ©1988, American Physiological Society. aNote
that in E.. coli is analogous to oligomycin sensitive conferral protein, OSCP, E. coli is analogous to mitochondrial and mitochondrial has not counterpart in E. coli Table 5 Subunits of Fo
E. coli
Beef heart mitochondria
271 30.3 kDa
226 24.8 kDa
Subunit a No. of residues Molecular size Subunit b No. of residues Molecular size
156 17.2 kDa
Subunit c No. of residues Molecular size
79 8.3 kDa
75 7.4 kDa
Not present
66 8.0kDa
Not present a1b2cn
Likely Unknown (cn)
A6L/aapl No. of residues Molecular size Other subunits Stoichiometry
Reproduced by permission from A.E. Senior, Physiological Reviews, 68:177-231. Copyright ©1988 the American Physiological Society.
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B. Catalytic Activity of F1 Each F1 complex contains six nucleotide binding sites. Three of these exchange rapidly when ATP is added to the medium, and are presumed to be catalytic sites. The other three exchange only slowly, and might be regulatory sites. In fact, occupancy of the presumed noncatalytic sites by ATP is not required for ATPase-catalysis (Weber et al., 1994). The three catalytic sites of the complex function in a highly cooperative fashion. Functioning as an ATPase, the rate of hydrolysis at any one of the three sites is slow, but it is accelerated by several orders of magnitude by binding to a second site (see Senior, 1988; Boyer, 1993). Binding studies with isolated F1 show that each α or β subunit binds ATP or ADP. The nucleotides of each subunit exchange rapidly with those in the medium. Affinity labeling of F1 with nucleotide analogs labels mostly β, although there is a suggestion that α may also be involved, perhaps at the interface between the two. Binding affinity for substrates and products at one catalytic site was found to be profoundly affected by binding at a second site by ADP aid Pi, which appeared to be needed for net ATP synthesis or release at the first site. The model presently favored proposes that ADP and Pi tightly bound at one catalytic site can form ATP. However, binding of ADP and Pi to a second catalytic site is obligatory for release of ATP. As discussed previously, the energy for the release would come from the proton gradient. The model involves alternating catalytic sites; each β subunit acts in the binding of ADP and Pi, in ATP synthesis and in its release, alternately. Fig. 11 (Senior, 1988) incorporates these features in a model in which three alternating sites are present (see Cross, 1981). In this respect, it is of considerable interest that hybrid F1 containing one inactive β subunit along with two normal β subunits is inactive.
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Fig. 11 ATP synthesis: proposed cyclical mechanism using all three catalytic sites of F1. (A) catalytic site 1 is a high-affinity site, undergoing reversible synthesis of ATP from tightly bound ADP + Pi Site 2 is a catalytic site of intermediate affinity, which must be occupied by ADP + Pi, and site 3 is a loose catalytic site. Energy from the proton gradient causes synchronous binding affinity changes at each site to give state B. (B) Site 1 has become lowest-affinity site, number 3, and ATP has been released and replaced by ADP and Pi from the medium. Site 2 has become high-affinity site, number 1, and reversible ATP synthesis has commenced. Site 3 has become an intermediate-affinity site, now number 2. Another proton gradientinduced binding-affinity change will release ATP and switch sites again to give the situation in state C. Reproduced from Senior (1988), with permission.
NMR, crosslinking data (see Engelbrecht and Junge, 1997) and EM studies (Wilkens et al., 1997; Wilkens and Capaldi, 1998) together with the crystallographic information discussed below, suggest the structure for FoF1 shown in Fig.10. The structure of F1 has been revealed by X-ray crystallography in three distinct configurations (Abrahams http://www.albany.edu/~abio304/text/chapter_18.html (24 of 29) [5/5/2002 6:54:13 PM]
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et al., 1994) at a 2.8 resolution. One corresponded to the empty site, the other was filled by AMP-PNP, a non-hydrolyzable ATP analog, and one with ADP. These three configurations represent snapshots of the rotary mechanism of catalysis and suggest that the γ-subunit moves like a crankshaft, coupled to the opening and closing of the catalytic-binding sites of F1. The rotational hypothesis was supported by several experiments. Reversible disulfide cross-links between engineered β and γ subunits showed that ATP hydrolysis switched the β-γ partners (see Duncan et al., 1995; Zhou et al., 1996; Zhou et al., 1997). The rotation was also demonstrated with eosin-linked to γ in CF1 immobilized in the (αβ) 3 portion. The polarization anisotropy relaxed with ATP hydrolysis, indicating a rotation (Sabbert et al., 1996; 1997). The occurrence of a rotation in individual molecular assemblies was demonstrated dramatically in recombinant F1 of thermophilic bacteria (Noji et al., 1997). Noji et al., observed the rotation induced by Mg-ATP after attaching fluorescently labelled actin fibers to the γ-subunit. The (αβ)2 of F1 was immobilized. The rotation was counter-clockwise (viewed from the membrane side). The three lines of evidence are summarized in Fig. 12. More details on the rotational movement of the ATP-synthase were provided by experiments following the same experimental design as that of Noji et al. (1997). In this case (Sambongi et al., 1999), the fluorescently labelled actin filament was attached to a subunit of c bound to the components of F1. The c oligomer was found to rotate using ATP-hydrolysis as the source of energy and generating approximately the same amount of force as shown for the γ subunit in the earlier experiments. Considering the structural data that show a close association of the c oligomer to the γ and δ subunits of F1 (e.g., Stock et al., 1999), the results indicate that the c-oligomer and the γ subunit of F1 are attached and rotate together. Studies of the rotation of the γ subunit of ATP-synthase at lower ATP concentrations, provided some significant information (Yasuda et al., 1998). This study found that the actin tag attached to the γ subunit moves in discrete 120o steps. Yasuda et al., calculate that the work carried out in each step is very close to the energy available from the hydrolysis of 1 ATP molecule (implying close to 100% efficiency). The energy expended by the work was calculated from the equation of Hunt et al. (1994) to estimate frictional drag. The energy available from the hydrolysis of 1 ATP was assumed to be approximately 80 pN.nm (corresponding to approximately -11.5 kcal; 1 N = 105 dynes) and approximately the same as the energy required for one step of the rotation. The actual numbers can only be considered approximate. However there is no doubt that the efficiency of the ATP-synthase operating as a motor is extremely high. This is not too surprising for a reversible pump. In fact, rough calculations for another ion transport pump, the Na+, K+-ATPase, while operating in transport, suggest an efficiency as high as 80% (see Chapter 12 and Tedeschi and Kinnally, 1992). When a small gold bead is attached to the γ as a marker (rather than actin, to reduce frictional drag) and high speed photography is used, the 120o steps can be subdivided into substeps of approximately 90o and 30 o ( Yasuda et al., 2001). The beads were observed either with transmission light microscopy or with laser dark-field microscopy (i.e., from the light scattered by the http://www.albany.edu/~abio304/text/chapter_18.html (25 of 29) [5/5/2002 6:54:13 PM]
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beads) . The first phase is likely to correspond to the binding of ATP, and the second to the hydrolysis step. It is interesting to note that the movement is induced by ATP binding (see discussion in Schnitzer, 2001). Other movements that use ATP binding are the stepping motion of kinesin (see Vale and Milligan, 2000) and the chaperone GroEl . For the latter, binding to one ring structure induces the opening of the other ring structure releasing the folded protein (see Sigler et al., 1998).
Fig. 12. Illustration of the techniques used to demonstrate rotation of γ in relation to (αβ) 3 during the hydrolysis of ATP by F1. (a) Schematic diagram showing cleavage and reformation of disulfide bridges between engineered γ and β during ATP hydrolysis, producing new γ-β pairing that could occur only with rotation. (b) Polarized absorption recovery of eosin after photobleaching on immobilized (αβ) 3 showing large scale rotation. (c)Fluorescence microscopy showing movement of a fluorescent actin filament attached γ recorded by video. From Junge et al., 1997. Reproduced by permission.
The question remains of how to couple proton movement and rotation. In Escherichia coli, the proteolipid subunit c is folded like a hairpin where two transmembrane α-helices, separated by a short loop, face the cytoplasmic side of the membrane (Girvin and Fillingame, 1994; Girvin et al., 1998). These subunits are present in multiple copies probably forming a ring as suggested by images on the EM (Birkenhäger et al., 1995) and atomic force microscope (Singh et al., 1996). Two monomers of c are thought to associate to form a dimer where the flattened front surface of one unit interacts with the flattened back surface of the other. Part of the evidence was derived from cross-linking with doubly Cys-substituted variant of the c subunit (Jones et al., 1998). Data from extensive cross-linking indicate 12 subunits per c oligomer(Jones and Fillingame, 1998). However, X-ray crystallography suggests 10 (Stock et al., 1999). In the oligomer, the α helices form two concentric rings with the amino- and carboxy-terminals located in the inner or outer ring, respectively (Dmitriev et al., 1999). In a model of 12 subunits, the oligomer forms a hollow cylinder with an outer diameter of 55-60 Å and an internal diameter of 11 Å. The Asp-61 residue, corresponding to the H+-transport residue, is in center of four transmembrane helices of two interacting subunits. Subunits a http://www.albany.edu/~abio304/text/chapter_18.html (26 of 29) [5/5/2002 6:54:13 PM]
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and b are outside the c oligomer. Subunit a consists of as many as five transmembrane helices and subunit b has a single transmembrane stretch and a long hydrophilic head (Fillingame, 1996). A model for the a
and c of Fo is shown in Fig. 13. Fig. 13. Model for the generation of torque in Fo driven by protonmotive force. Subunit c proteolipid subunits form a ring. Subunit a has two proteon access channels for protons on either side of the membrane. From Junge et al., 1997. Reproduced by permission.
A possible mechanism would require that: (a) the carboxyl groups in the c-ring (corresponding to Asp 61 of subunit c) must be protonated and electroneutral when they face the lipid core, (b) when facing subunit a, they can be deprotonated and charged and (c) random Brownian motions become directed when constrained by requirements (a) and (b) (Junge et al., 1997; see also Junge, 1999; Dimroth et al., 1999). As shown in Fig. 13, the subunits of the ring can move only if a proton is bound. When a charged group of c facing a hits the boundary with the lipid, it is reflected back. When the lower channel of a is more acidic than the upper channel, the ring will turn clockwise (from the bottom). It will turn counter-clockwise when the gradient is reversed. The model proposed by Elston et al. (1998) is very similar. It proposes an important role for Arg 210 in the a subunit by contributing to the the deprotonation of the carboxyl group when the movement proceeds in the unfavorable direction. Both Asp 61 (Dmitriev et al., 1995) in each c subunit and Arg 210 in the single a subunit (Vik and Antonio, 1994; Valiyaveetil and Fillingame, 1997) are essential for proton translocation as revealed by mutagenesis. However, there are indications that the deprotonation of Arg 61 induces a conformational change in the c subunit, a 140o rotation of the carboxy-terminal helix with respect to the amino-terminal helix (Rastogi and Girvin, 1999). This rotation drives the movement of the γ-portion. Two possible mechanisms are suggested by Rastogi and Girvin (1999). The a subunit may move with the carboxy terminal helix of the c subunit. This would produce a 30o rotation of the ring with respect to the a subunit. An alternative model proposes the movement of a negatively charged "proton hole" which traverses the ring until it arrives at a ε subunit, which moves to an adjacent c subunit, providing a 30o rotation of the ε-γ stalk. SUGGESTED READING Overall view
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Saraste, M.(1999) Oxidative phosphorylation at the fin de siècle, Science 283:1488-1493. (Medline) Boyer, P.D. (1999) What makes ATP synthase spin? Nature 402:247-249. (Medline) Capaldi, R.A. and Aggeler, R. (2002) Mechanism of the F1F0-type ATP synthase, a biological rotary motor, Trends Biochem. Sci. 27:154-160. (MedLine) Others Boyer, P. D. (1989) A perspective of the binding change mechanism for ATP synthesis, FASEB J. 3:2l642178. (Medline) Cross, R.L. (1994) Our primary source of ATP, Nature 370:594. (Medline) Cramer, W. A., and Knaff, D. B. (1990) Energy Transduction in Biological Membranes, Chapter 8. Springer-Verlag, New York. Nakamoto, R.K. (1999) Molecular features of energy coupling in the FoF1 ATP synthase, News Physiol. Sci. 14:40-46. Nicholls, D.G. and Ferguson, S.J. (1992) Bioenergetics 2, Academic Press, New York. Penefsky, H. S., and Cross, R. L. (1991) Structure and mechanisms of F0F1 type ATP synthases and ATPases. Adv. Enzymology, 64:173-214. (Medline) Senior, A. E. (1988) ATP synthesis by oxidative phosphorylation, Physiol. Rev. 68:177-231. (Medline) Senior, A. E. (1990) The Proton-translocating ATPase of Escherichia coli, Annu. Rev. Biophys. Biophys. Chem. 19:7-41. (Medline) Tzagaloff, A. (1982) Mitochondria, Chapters 6-9. Plenum Press, New York. Special Aspects Ferguson, S.J. (1995) Protons fast and slow, Current Biol. 5:25-27. (Medline) Kagawa, Y. (1984) Proton motive ATP synthesis. In Bioenergetics (Ernster. L., ed.), pp. 149-186. Elsevier. New York. Tedeschi. H. (1981) The transport of cations in mitochondria, Biochim. Biophys. Acta 639:157-196; see http://www.albany.edu/~abio304/text/chapter_18.html (28 of 29) [5/5/2002 6:54:13 PM]
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pp. 162-170. (Medline) Weber, G. (1972) Addition of chemical and osmotic energies by ligand protein interactions, Proc. Natl. Acad. Sci. U.S.A. 69:3000-3003. (Medline) WEB RESOURCES Noji, H., Yasuda, R., Yoshida, M. and Kinosita Jr, K. Direct observation of the rotation of F1-ATPase Movie 1 Movie 2 Diwan, J.J. Oxidative phosphorylation: chemiosmotic coupling, http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/oxphos.htm Oxidative phosphorylation: F1Fo ATPase, http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/f1fo.htm REFERENCES Search the textbook Search the textbook
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Chapter 18: References
Back to Chapter 18
REFERENCES Abrahams, J.P., Leslie, A.G.W., Lutter, R. and Walker, J.E. (1994) Structure at 2.8 resolution of F1ATPase from bovine heart mitochondria, Nature 370:621-628.(Medline) Addanki, S., Dallas, F., Cahill, F. S., and Sotos, J. F. (1968) Determinations of intramitochondrial pH and intramitochondrial extramitochondrial pH gradient of isolated heart mitochondria by the use of 5,5dimethyl 1-2,4 oxazolinedione, J. Biol. Chem. 243:2337-2348. Alexiev, U., Mollaaghaba, R., Scherrer, P., Khorana, H.G. and Heyn, M.P. (1995) Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk, Proc. Natl. Acad, Sci. USA 92:372-376.(Medline) Archbold, G. P. R., Farrington, C. L. Lapping S. A., McKay, A. M., and Malpress, F. H. (1979) Oxygen pulse curves in rat liver mitochondrial suspensions: Some observations and deductions, Biochem. J. 180:161-174.(Medline) Birkenhäger, R., Hoopert, M. Deckers-Hebestreit, G., Mayer, I., and Altendorf, K.(1995) The Fo complex of Escherichia coli ATP synthase. Investigation by electron spectrocopic imaging and immunoelectron microscopy, Eur. J. Biochem. 230:58-67.(Medline) Blumenfeld, M., Goldfeld, G., Mikoyan, V. D., and Soloyev, I. S. (1987) ATP synthesis by isolated coupling factors from chloroplasts during acidic and alkaline pH shifts. [translated in Mol. Biol. 21:268274 (1987)] Molekulyarnaya Biologiya 21:323-329. Boyer, P.D. (1993) The binding change mechanism for ATP-synthase - some probabilities and possibilities, Biochim. Biophys. Acta 11440:215-250.(Medline) Boyer, P.D., Cross, R.L., and Momsen, W. (1973) A new concept in energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions, Proc. Natl. Acad. Sci. USA 70:2837-2839.(Medline) Brierley, G. P., and Murer, E. (1964) Ion accumulation in heart mitochondria supported by reduced cytochrome c., Biochem. Biophs. Res. Commun. 14:437-442.(Medline)
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Chapter 18: References
Brierley, G. P., Bachman, E., and Green, D. E. (1962) Active transport of inorganic phosphate and magnesium ions by beef heart mitochondria, Proc. Natl. Acad. Sci. U.S.A. 48:1928-1935. Campo, M. L., Bowman, C. L., and Tedeschi, H. (1984) Assays of ATP synthesis using single giant mitochondria, Eur. J. Biochem. 141:1-4.(Medline) Capaldi, R.A., Ageler, R., Turina, P. and Wilkens, S. (1994) Coupling between catalytic sites and the proton channel in the F1Fo-ATPases, Trends in Biochem Scie. 19:284-289. Chance, B. (1965) The energy-linked reaction of calcium with mitochondria, J. Biol. Chem. 240:27292748. Chance, B., and Hollunger, G. (1961) The interaction of energy and electron transfer reactions in mitochondria. VI. The efficiency of the reaction, J. Biol. Chem. 236:1577-1584. Chance, B., and Williams, G. R. (1956) The respiratory chain and oxidative phosphorylation, Adv. Enzymol. 17:65-134. Copenhaver, J. H., Jr., and Lardy, H. A. (1952) Oxidative phosphorylation: pathways and yield in mitochondrial preparations, J. Biol. Chem. 195:225-238. Cramer, W. A., and Knaff, D. B. (1990), Energy Transduction in Biological Membranes, SpringerVerlag, New York. Cross, R. L. (1981) The mechanism and regulation of ATP synthesis by F1F0-ATPases, Annu. Rev. Biochem. 50:681-714.(Medline) Dilley, R. A., and Schreiber, U. (1984) Correlation between membrane-localized protons and flashdriven ATP formation in chloroplast thylakoids, J. Bioenerg. Biomembr. 16:173-193.(Medline) Dimroth, P., Wang, H., Grabe, M. and Oster, G. (1999) Energy transduction in the sodium F-ATPase of Propionigenium modestum, Proc. Natl. Acad. Sci. USA 96:4924-4929.(Medline) Dmitriev, O.Y., Altendorf, K. and Fillingame, R.H. (1995) Reconstitution of the Fo complex of Escherichia coli ATP synthase from isolated subunits: varying the number of essential carboxylates by co-incorporation of wild-type and mutant subunit c after purificationon organic solvents, Eur. J. Biochem. 233:478-483.(Medline) Dmitriev, O.Y., Jones, P.C. and Fillingame, R.H. (1999) Structure of the subunit c oligomer in the F1Fo ATP synthase: model derived from solution structure of the monomer and cross-linking in the native http://www.albany.edu/~abio304/ref/ref18.html (2 of 8) [5/5/2002 6:54:16 PM]
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enzyme, Proc. Natl. Acad. Sci. USA 96:7785-7790.(Medline) Duncan, T.M., Bulygin, V.V., Zhou, Y., Hutcheon, M.L. and Cross, R.L. (1995) Rotation of subunits during catalysis by Escherichia coli Fo1-ATPase, Proc. Natl. Acad. Sci. USA 92:10964-10968.(Medline) Engelbrecht, S., and Junge, W. (1997) ATP synthase: a tentative structural model, FEBS Lett. 414:485491.(Medline) Elston, T., Wang, H. and Oster, G. (1998) Energy transduction in ATP synthase, Nature 391:510513.(Medline) Feldman, R.I., and Sigman, D.S. (1982) The synthesis of enzyme bound ATP by soluble chloroplast factor 1, J. Biol. Chem. 257:1676-1683.(Medline) Feldman, R.I., and Sigman, D.S. (1983) The synthesis of ATP by membrane bound ATP synthase complex from medium P under completely uncoupled conditions, J. Biol. Chem. 258:1217812183.(Medline) Ferguson, S.J. (1995) Protons fast and slow, Current Biol. 5:25-27.(Medline) Fillingame, R.H. (1996) Membrane sectors of F- and V-type H+-transporting ATPases, Curr. Opin. Struct. Biol. 6:491-498. Girvin, M.E. and Fillingame, R.H. (1994) Hairpin folding of subunit c of F1Fo ATP synthase: 1H distance measurements to nitroxide-derivatize aspartyl-61, Biochemistry 33:665-674.(Medline) Girvin, M.E., Rastogi, V.K., Abildgaard, F., Markley, J.L. and Fillingame, R.H. (1998) Solution structure of the transmembrane H+-transporting subunit c of the F1F0 ATP synthase, Biochemistry 37:88178824.(Medline) Gogol, E. P., Aggeler, R., Sagermann M., and Capaldi, R. A. (1989a) Cryoelectron microscopy of Escherichi coli F1 Adenosinetriphosphatase decorated with monoclonal antibodies to individual subunits of the complex, Biochem. 28:4717-4724.(Medline) Gogol, E. P., Lücken, U., Bork, T., and Capaldi, R. A. (1989b) Molecular architecture of Escherichia coli F1 Adenosinetriphosphatase, Biochem. 28:4709-4716.(Medline) Gogol, E. P., Johnston, E., Aggeler, R., and Capaldi, R. A. (1990) Ligand-dependent structural variation in Eschierichia coli F1 ATPase revealed by cryoelectron microscopy, Proc. Natl. Acad. Sci. 87:9585http://www.albany.edu/~abio304/ref/ref18.html (3 of 8) [5/5/2002 6:54:16 PM]
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9589.(Medline) Gunter, T.E. and Pfeiffer, D.R.(1990) Mechanisms by which mitochondria transport calcium, Am. J. Physiol. 258 (Cell Physiol. 27): C755-786.(Medline) Hall, D. O. (1976) The coupling of photophosphorylation to electron transport in isolated chloroplasts. In The Intact Chloroplast (Barber, J., ed.), pp. 135-170. Elsevier, New York. Heberle, J. Riesle, J. Thiedemann, G., Oesterhelt, D. and Dencher, N.A., (1994) Proton migration along the membrane surface and retarded surface to bulk transfer, Nature 370, 379-382.(Medline) Hinkle, P.C., Kumar, M.A., Resetar, A. and Harris, D.L. (1991) Mechanistic stoichiometry of mitochondrial oxidative phosphorylation, Biochemistry 30:3576-3582.(Medline) Hong, Y. Q., and Junge, W. (1983) Localized or delocalized protons in photophosphorylation. On the accessibility of the thylakoid lumen for ions and buffers, Biochim. Biophys. Acta 722:197-208. Horton, A. A., and Hall, D. O. (1968) Determining stoichiometry of photosynthetic phosphorylation, Nature 218:386-388. Hunt, A.J., Gittes, F., Howard, J. (1994) The force exerted by a single kinesin molecule against a viscous load, Biophys. J. 67:766-781.(Medline) Jagendorf, A. T., and Uribe, E. (1966) ATP formation caused by acid-base transition of spinach chloroplasts, Proc. Natl. Acad. Sci. U.S.A. 55:170-177.(Medline) Jones, P.C. and Fillingame, R.H. (1998) Genetic fusions of subunit c in the F0 sector of H+ transporting ATP synthase. Functional dimers and trimers and determination of stoichiometry by cross-linking analysis, J. Biol. Chem. 273:29701-29705.(Medline) Jones, P.C., Jiang, W. and Fillingame, R.H. (1998) Arrangement of the multicopy H+ -translocating subunit c in the membrane sector of the Escherichia coli F1F0 ATP synthase, J. Biol. Chem. 273:1717817185.(Medline) Junge, W. (1999) ATP synthase and other motor proteins, Proc. Natl. Acad. Sci. USA 96:47354737.(Medline) Junge, W., Lill, H. and Engelbrecht, S. (1997) ATP synthase: an electrochemical transducer with rotary mechanics, Trends in Biochem. Scie. 22:420-423.(Medline)
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Kagawa, Y. (1984) A new model of proton motive ATP synthesis: acid-base cluster hypothesis, J. Biochem.95:295-298.(Medline) Lee, C.P., Gu, Q., Xiong, Y., Mitchell, R.A. and Ernster, L. (1996) P/O ratio reassesed: mitochondrial P/O ratios consistently exceed 1.5 with succinate and 2.5 with NAD-linked substrates, FASEB J.10:345350.(Medline) Lehninger, A. L. (1955) Oxidative phosphorylation, Harvey Lect. 49:176-215. Malenkova, I. V., Kuprin, S. P., Davydov, R. M. and Blumenfeld, L. A. (1982) pH-jump-induced ADP phosphorylation in mitochondria, Biochim. Biophys. Acta 682:179-183.(Medline) Maloff, B.L., Scordilis, S.P. and Tedeschi, H. (1978) Assays of the viability of single giant mitochondria: experiments with intact and impaled mitochondria, J. Cell Biol. 78:214-226.(Medline) Mandolino, G., De Santis, A., and Melandri, B. A. (1983) Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus), Biochim. Biophys. Acta 723:428-439. Mitchell, P. (1966) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation, Biol. Rev. 41:445-502.(Medline) Mitchell, P. (1967) Proton-translocation phophorylation in mitochondria, chloroplasts and bacterianatural fuel cells and solar cells, Fed. Proc. 26:1370-1379.(Medline) Nicholls, D.G. and Ferguson, S.J. (1992) Bioenergetics 2, Academic Press, New York. Noji, H., Yasuda, R., Yosida, M. and Kinosita, K., Jr. (1997) Direct observation of the rotation of F1ATPase, Nature 386:299-302.(Medline) Penniston, J. T., Zande, H. V., and Green, D. E. (1966) Mitochondrial particles resolved for ion translocation. I. Preparation and properties of a particle coupled only at the phosphorylation site III of the electron transfer chain, Arch. Biochem. Biophys. 113:507-511.(Medline) Pick, U., Rottenberg, H., and Avron, M. (1974) The dependence of photophosphorylation in chloroplast on pH and external pH, FEBS Lett. 48:32-36.(Medline) Rastogi, V.K. and Girvin, M.E. (1999) Structural changes linked to proton translocation by subunit c of the ATP synthase, Nature 402:263-268.(Medline)
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Sabbert, D., Engelbrecht, S. and Junge, W. (1996) Intersubunit rotation in active F-ATPase, Nature 381:623-625.(Medline) Sabbert, D., Englebrecht, S. and Junge, W. (1997) Functional and idling rotatory motion within F1 ATPase, Proc. Natl. Acad. Sci. (1997) 94:4401-4405.(Medline) Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y. and Futai, M. (1999) Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation, Science 286:1722-1724.(Medline) Scherrer, P, Alexiev, U., Marti, T., Khorana, H.G. and Heyn, M.P. (1994) Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin 1. Proton migration along the surface of Bacteriorhodopsin micelles and delayed transfer from surface to bulk, Biochem. 33:1368413692.(Medline) Schnitzer, M.J. (2001) Molecular motors: Doing a rotary two-step, Nature 410:878-881. (MedLine) Senior, A. E. (1979) The mitochondrial ATPase. In Membrane Proteins in Energy Transduction (Capaldi, R. A., ed.), pp. 233-278. Marcel Dekker, New York. Senior, A. E. (1988) ATP synthesis by oxidative phosphorylation, Physiol. Rev. 68:177-231.(Medline) Sigler, P.B., Xu, Z., Rye, H.S., Burston, S.G., Fenton, W.A. and Horwich, A.L. (1998) Structure and function in GroEL-mediated protein folding, Annu. Rev. Biochem. 67:581-608. (MedLine) Singh, S., Turina, P., Bustamante, C.J., Keller, D.J. and Capaldi R. (1996) Topographical structure of membrane-bound Escherichia coli F1F0 ATP synthase in aqueous buffer, FEBS Lett. 397:3034.(Medline) Skulskii, I. A., Saris, N. E. L., and Glusunov, V. V. (1983) The effect of the energy state of mitochondria on the kinetics of unidirectional cation fluxes, Arch. Biochem. Biophys. 226:337-346.(Medline) Sone, N., Yoshida, M., Hirata, H., and Kagawa, Y. (1977) Adenosine triphosphate synthesis by electrochemical proton gradient in vesicles reconstituted from purified adenosine triphosphatase and phospholipids of thermophilic bacterium, J. Biol. Chem. 252:2956-2960.(Medline) Stock, D., Leslie, A.G. and Walker, J.E. (1999) Molecular architecture of the rotary motor in ATP synthase, Science 286:1700-1705.(Medline) Tedeschi, H. (1981) The transport of cations in mitochondria, Biochem. Biophys. Acta 639:157-196; see http://www.albany.edu/~abio304/ref/ref18.html (6 of 8) [5/5/2002 6:54:16 PM]
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pp. 162-170.(Medline) Tedeschi, H. and Kinnally, K.W. (1992) Bionergetics, in Fundamentals of Medical Cell Biology, JAI Press Inc., Greenwich, CT, vol 3B, pp. 609-642. Vale, R.D. and Milligan, R.A. (2000) The way things move: looking under the hood of molecular motor proteins, Science 288:88-95. (MedLine) Valiyaveetil, F.I. and Fillingame, R.H. (1997) On the Role of Arg-210 and Glu-219 of Subunit a in Proton Translocation by the Escherichia coli FoF1-ATP Synthase, J. Biol. Chem. 272:3263532641.(Medline) Vik, S.B. and Antonio, B.J. (1994) A mechanims of proton translocation by FoF1 ATP synthases suggested by double mutants of the a subunit, J. Biol. Chem. 269:30364-30369.(Medline) Weber, J., Wilke-Mounts, S., Grell, E. and Senior, A.E. (1994) Tryptophan fluorescence preovides a direct probe on nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase, J. Biol. Chem. 269: 11261-11268.(Medline) Westerhoff, H. V., Melandri, B. A., Venturoli, G., Azzone, G. F., and Kell, D. B. (1984) Mosaic protonic coupling hypothesis for free energy transduction, FEBS Lett. 165:1-5.(Medline) Wilkens, S., Rodgers, A., Ogelvie, I. and Capaldi, R.A. (1997) Structure and arrangement of the δ subunit in the E. coli ATP synthase, Biophys. Chem. 68:95-102.(Medline) Wilkens, S. and Capaldi, R.A. (1998) ATP synthase's second stalk comes into focus, Science 393:29.(Medline) Wilson, D. F., Dutton, P. L., and Wagner, M. (1973) Energy transducing components in mitochondrial respiration, Curr. Top. Bioenerg. 5:234-265. Winget, G. D., Izawa, S., and Good, N. E. (1965) The stoichiometry of photophosphorylation, Biochem. Biophys. Res. Commun. 21:438-443.(Medline) Yasuda, R., Noji, H., Kinosita, K. Jr. and Yoshida, M. (1998) F1-ATPase is a highly efficient molecular motor that rotates with discrete 120o, Cell 93:1117-1124.(Medline) Yasuda, R., Noji, H., Yoshida, M., Kinosita, K. Jr. and Itoh, H. (2001) Resolution of distinct rotational substeps by submillisecond kinetic analysis of F1-ATPase, Nature 410:898-904. (MedLine)
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Zhou, Y., Duncan, T.M. and Cross, R.L. (1997) Subunit rotation in Escherichia coli FoF1-ATP synthase during oxidative phosphorylation, Proc. Natl. Acad. Sci. USA 94:10583-10587.(Medline) Zhou, Y., Duncan, T.M., Bulygin, V.V., Hutcheon, M.L. and Cross, R.L. (1996) ATP hydrolysis by membrane-bound Escherichia coli FoF1 causes rotation of the γ subunit relative to the β subunits, Biochim. Biophys. Acta 1275:96-100.(Medline)
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19. The Cell Membrane: Transp ort and Permeability
19. The Cell Membrane: Transport and Permeability I. Characterization of Transport A. Diffusion and Channels; the Aquaporins B. Transporter Mediated Translocations II. Molecular Mechanisms of Transport; The Glucose Transporters III. Vectorial Enzymes IV. Strategies for the Isolation of Transport Proteins Suggested Reading Web Resources References Back to List of Chapters The integrity of the cell depends on the presence of the plasma membrane. The membrane prevents the loss of internal components and metabolites. Its ability to exclude some solutes and to permit the passage of others allows the maintenance of an internal environment that differs from the medium external to the cell. The low concentration of Ca2+ in the cytoplasm permits its use as a sensitive signal for a variety of physiological regulatory effects. Similarly, the creation of an Na+ electrochemical gradient provides a reservoir of energy that can be used for the transport of other solutes, such as amino acids and sugars. Furthermore, the low internal Na+ and high K+ and the channels that allow their passage, are indirectly responsible for the electrical potential of cells, excitation and conduction (Chapter 22). The membrane controls the entrance of substrates used in either energy metabolism (e.g., glucose) or the synthesis of cell components (e.g., amino acids). The present chapter is directed primarily to general aspects of transport through the plasma membrane. Chapters 20 and 21 discuss the transport of ions. As we shall see the translocation through the plasma membrane involves in many cases the mediation of channels or transporters. Interestingly, although translocations through channels and transporters represent distinct mechanisms, transporter molecules can act as channels when reconstituted into bilayers ( see Chapter 21). This indicates that part of the translocation mediated by transporters is through channel like structures (as also suggested by present models of transport). In addition, at least the glutamate transporter of the mammalian nervous system also can act as a channel in vivo (see Slotboom et al., 2001). Enzymes act to increase the rates of reactions (Chapter 13) and are subject to a variety of regulatory mechanisms affecting either their activity (Chapters 13 and 14) or their turnover (Chapters 15). No less important is the accessibility of the cell's interior to substrates and cofactors which could also control the rate of metabolism. I. CHARACTERIZATION OF TRANSPORT Passage of a solute from one biological compartment to another may involve passage of the molecule through a biological membrane. This process need not differ from ordinary diffusion. The passage from, say, compartment 1 to compartment 2, is proportional to the concentration of solute in the first http://www.albany.edu/~abio304/text/chapter_19.html (1 of 18) [5/5/2002 6:54:39 PM]
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compartment [S1], as shown in Eq. (1). J12 = k[S1] (1) In this equation, k is a constant related to the diffusion coefficient (diffusion coefficient/thickness of the membrane). Equation (1) is a common sense equation, expressing Fick's law. The probability of the molecules going through a barrier must obviously increase with the number of molecules present. The flux (J) can be measured experimentally, simply by measuring the passage from 1 to 2 (J12) of a radioactive isotope before the concentration in the second compartment [S2] is sufficiently high to produce a significant flux in the opposite direction. The passage in the opposite direction (J21) is given by an identical relationship. The net passage is given by the differences between the two fluxes. J21 = k[S2] (2) J12 - J21 = k([S2] - [S1]) = dS1/dt (3) Equation (3) predicts an equilibrium (dS1/dt = 0) when [S1] = [S2]. It also predicts that the net passage, due to diffusion alone, will be in the direction of the concentration gradient. The chemical potentials (see Chapter 12) for the solute in compartments 1 and 2 are, respectively, µ1 = µo + RT ln[S1] (4) µ2 = µo + RT ln[S2] (5) µ1 - µ2= ∆ G and at equilibrium ∆ G = 0 and [S1] = [S2]. When µ1 is not equal to µ2 and the difference or ∆G is positive, energy would have to be expended to transfer the solute against the concentration gradient. Conversely, when negative, the chemical potential could be used to drive another system to perform work. Either event would require a special mechanism to couple the energy providing reaction to the energy expenditure. Some of these topics were addressed in Chapter 12. For certain cases, the mechanism of coupling is the topic of Chapters 20 and 21. The constant k of Eq. 3 will depend on the nature of the solute and the properties of the biological membrane in question. The rate constant will also depend directly on the surface area exposed to the exchanges. The constant k, corrected for surface area (A), is the permeability constant (P). k/A = P (6) Solute molecules could pass through the phospholipid bilayer of the plasma membrane. First they must break away from the water phase of one http://www.albany.edu/~abio304/text/chapter_19.html (2 of 18) [5/5/2002 6:54:39 PM]
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compartment, then they must dissolve into the bilayer to be released to another aqueous phase after diffusing through the hydrocarbon environment. However, evidence has accumulated for the involvement of protein channels in many of these translocations. Channels are responsible for the passage of ions during excitation and conduction of electrical impulses (see Chapter 22) or other cellular events. In addition, specialized channels or pores have been implicated in the passive passage of water and certain non-electrolytes. A. Diffusion and Channels: the Aquaporins The cell membrane has a much higher permeability to water than phospholipid bilayers. Furthermore, passage of water in response to an osmotic gradient is much more rapid than diffusion of water, as would be expected if channels are present. In addition, mercurial sulfhydryl reagents block water transport in the cell membrane so that it becomes as impermeable to water as phospholipid bilayers. A dependence on sulfhydryl groups suggests an involvement of proteins, most likely forming channels. Further studies showed that these expectations were correct. An integral membrane protein of approximately 28 kDa has been identified in red blood cells and renal tubules: the aquaporin channel forming integral protein (CHIP or aquaporin-1). Other water channel proteins associated with other tissues were found later. All these channels are generally referred to as aquaporins (AQPs). The AQPs (Deen and van Os, 1998) are members of a family of channel forming proteins called the major intrinsic proteins (MIP). Members of this family have been partially or fully sequenced. 150 MIPs are known including 10 mammalian AQPs. The MIP proteins constitute a family of proteins widely present in plants, animals and bacteria (Park and Saier, 1996; Deen and van Os, 1998). AQP1-mediated water channel activity was first demonstrated in Xenopus laevis oocytes. These oocytes have a low water permeability to survive in fresh water ponds. The mRNA corresponding to AQP1 was transcribed in vitro using the appropriate cDNA as a template. The newly synthesized mRNA was then microinjected into the cells (Preston et al., 1992; Preston et al., 1993). The increased water permeability closely followed the characteristics of the red blood cells that contain AQP1. The transport of several nonelectrolytes is also sensitive to reagents that are likely to react with proteins. Therefore, several studies proposed a transfer of these substances through protein channels. AQP0, AQP3, AQP7 and AQP9 are also permeable to glycerol, and AQP3, AQP7 and AQP8 to urea. AQP1 has been found to be a homotetramer of the 28 kDa subunit (Denker et al., 1988; Smith and Agre, 1991). AQP1 has been sequenced (Preston and Agre, 1991) and has been found to be an integral protein with 6 hydrophobic domains capable of spanning the membrane, connected by loops A to E. The monomer is thought to have a long polygalactoseaminoglycan. One molecule has two repeats of three α helices which are 180o mirror images. Each repeat consists of an asparagine-proline-alanine (i.e., NPA) sequence in loops B and E (the hourglass model). In this model, the highly conserved loops B and E are essential for the formation of the water pore. The three dimensional structure has been reported (see Fig. 1) from studies using cryo-electron microscopy and electron diffraction (Li and Jap, 1997; Walz et al., 1997; Cheng et al., 1997; Murata et al., 2000) and more recently X-ray crystallography (Sui et al., 2001). The unit cell contains two tetramers oriented in the bilayer in opposite direction (e.g., Walz et al., 1997). The helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their connections to the surrounding helices. A model representing an AQP1 monomer is shown in Fig. 1, top (from Walz et al., 1997). A side representation of the six-helix barrel is shown in Fig. 1, middle (Cheng et al., 1997), and Fig. 1, bottom, shows the arrangement of the helices in the tetramer as seen from above (Cheng et al., 1997). Despite its presence as a tetramer, a single subunit can act as a channel, as shown by radiation inactivation, which reveals a target size of about 30 kDa (Van Hoek et al., 1991, 1992). This method works on the principle that the larger the http://www.albany.edu/~abio304/text/chapter_19.html (3 of 18) [5/5/2002 6:54:39 PM]
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molecule, the easier it is to hit by radiation (e.g., X-rays) and thereby be inactivated. In mammals, the expression of AQP1 is regulated by several factors. In rat fetal lung cells ( King et al., 1996), corticosteroids induce AQP1. In addition, this protein increases with hypertonic stress ( Jenq et al., 1999) in several kinds of cells. Proteasome inhibitors increase AQP1 expression, indicating that the this protein is degraded via the ubiquitination- proteasome pathway (see Chapter 15). In addition, the half-life of the AQP1 protein of cells exposed to hypertonic medium is significantly longer than under isotonic conditions ( Leitch et al., 2001), indicating an osmotic regulation of the degradation. The specificity of AQP1 provides a challenging mystery. This channel protein allows the rapid flow of water but not H+ (e.g., Zeidel et al., 1992). Recent electron crystallographic data (Murata et al., 2000) with a resolution of 3.8 Å, obtained from AQP1, allows the construction of an atomic model that may have resolved this question. Hydrophobic residues line the water channel. They allow rapid water transport in single file (based on studies carried out with gramicidin A; gramicidin is an antibiotic which forms well defined channels in bilayers) (see Finkelstein and Andersen, 1981). The water molecules can serve as proton wires (e.g., see Pomès and Roux, 1996). Supposedly, the protons interact with the oxygen of the water and are passed along the water column in a succession of hops. In gramicidin A channels, protons move fifteen times faster than K+ (which have the same mobility in bulk water) and eight times faster than the water molecules themselves (see Pomès and Roux, 1996)! In the AQP1 molecule, selectivity is probably provided by a constriction of the channel with a diameter of about 3 Å over a span of one residue. At the point of constriction, water can form hydrogen bonds via its oxygen with the free amino residues and the consequent rearrangement of the water molecules in the column interrupts the continuous chain of hydrogen bonds needed for the proton transfer. Consequently, H+ is unable to get past this point. A similar mechanism has been proposed to explain the H+ impermeability of the E.coli glycerol channel and it has been suggested that the same mechanism may operate for all AQPs (Tajkhorshid et al., 2002)
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Fig. 1 Representation of aquaporin. Top: model showing the predicted position of the helices and loops. From Walz et al., 1997. Reproduced by permission. Middle: representation of the six-helix barrel parallel to the bilayer. From Cheng et al., 1997. Reproduced by permission. Bottom: top view of the arrangment of the helices in a tetramer. From Cheng et al., 1997. Reproduced by permission. Copyright ©1997 MacMillan Magazines Ltd.
. The structure of the glycerol conducting channel from Escherichia coli, containing 3 glycerol molecules also has been studied crystallographically (Fu et al., 2000). A region (termed the selectivity filter) is large enough to allow the passage of only 1 glycerol. In addition, the 3 glycerols are present in single file. The pathway is amphipatic and matches successive CH-OH groups. The polar interactions take place only on one side of the pathway. The alkyl portion of glycerol is held against a hydrophobic environment. Successive hydroxyl groups form hydrogen bonds with a pair of acceptor and donor http://www.albany.edu/~abio304/text/chapter_19.html (5 of 18) [5/5/2002 6:54:39 PM]
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atoms. The passage of solutes is relatively specific allowing the entry of polyhydroxy-alcohols provided that the carbon backbone is lined along the channel axis and the molecule is sufficiently small. The distribution and role of AQPs in mammalian organisms have been recently reviewed (King and Agre, 1996; Deen and van Os, 1998) and some of these are summarized in the table. AQP
Tissue
AQP0 lens fiber cells (1)
Function water/glycerol transport
Malfunction cataracts (1)
AQP1 Kidney, red blood cells, lungs, eye, choroid plexus, bile duct, Non- water/water reabsorption in kidney impaired water reabsorption in mice (3) fenestrated epithelia (2) AQP2 renal collecting duct cells (apical side) (e.g. 11)
nephrogenic diabetes insipidus (12)
AQP3 renal collecting duct cells (basolateral side) GI tract airway epithelia, eye conjuctive meningeal cells (4) renal collecting duct cells (basolateral side) AQP4 glial and ependymal cells of brain and others (4)
reabsorption of cerebrospinal fluid?
AQP5 apical plasma membrane of pneumocytes, secretory epithelium and in upper airway and salivary glands (5)
secretion of fluid
AQP6 Kidney (6) AQP7 Late spermatids and sperms Kidney and heart (7) AQP8 Hepatocytes, pancreas, heart and testes (8 and 9) AQP9 Adipocytes (also heart, kidney and small intestine) (10)
(1) Shiels and Bassnet, 1996 (2) King and Agre, 1996 (3) Ma et al., 1998 (4) Lee et al., 1997 (5) Nielsen et al., 1997 (6) Ma et al., 1996 (7) Ishibashi et al., 1997 (8) Koyama et al., 1997 (9) Ma et al., 1997 (10) Kuriyama et al., 1997 (11) Saito et al., 1997 (12) Deen and Knoers, 1998 http://www.albany.edu/~abio304/text/chapter_19.html (6 of 18) [5/5/2002 6:54:39 PM]
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AQPs in mammals have a central role in the reabsorption of water in the kidney. Most of the reabsorption occurs in the proximal tubules and the descending loop of Henley. The remaining 10% takes place in the collecting duct principal cells and is regulated by antidiuretic hormone (ADH) also know as vasopressin. AQP3 and AQP4 are present in the basolateral membrane and AQP2 in the apical membrane of these cells. The release of ADH from the pituitary (when the blood’s level of Na+ is too high or the blood volume is too low) favors the increase in channels. The vasopressin binds to its receptor of the basolateral membrane of the principal cells. This is followed by the formation of cAMP in the cytoplasm and activation of PKA. The PKA phosphorylates several proteins, including AQP2 in intracellular vesicles (Fushimi et al., 1997; Katsura et al., 1997). AQP3 and 4 are not affected in this manner. The vesicles containing the phosphorylated AQP2 fuse to the apical membranes and are responsible for the osmotically driven reabsorption. When the level of vasopressin decreases, the vesicles are removed from the plasma membrane by endocytosis. In addition, the AQP2 and AQP3 levels are increased as a result of increased transcription. The cAMP-responsive elements in the AQP2 promoter region are involved in ther case of AQ2 (Matsumura et al., 1997). Exocytotic delivery of AQPs is likely to be a general mechanism for increasing water transport. For example, the hormone secretin is known to stimulate ductal bile secretion by cholangiocytes. These cells transport water through AQP1. Secretin stimulates exocytosis in cholangiocytes and causes up to a 3fold increase in the amount of AQP1 in plasma membranes and a proportional decrease in the amount of the water channel in microsomes. These findings suggest that secretin induced redistribution of AQP1 from intracellular to plasma membranes. The redistribution was found to be dependent on vesicular translocation and microtubular function (Marinelli et al., 1997). Similar mechanisms involve the glucose transporter GLUT4. Stimulation of glucose transport by insulin, is linked to translocation of the GLUT4 glucose transporter from an intracellular pool to the cell surface (e.g., see Kanai et al., 1993). B. Transporter Mediated Translocations Equation (3) expresses the net flux of a solute transferred by a diffusional process and indicates that the net passage of solute should be directly proportional to the concentration gradient. As we saw, a steady state should be reached eventually in the form of a true equilibrium at which [S1] = [S2]. Although not explicitly stated by the equation, the passage should be largely independent of the presence of other substances. Furthermore, we would expect a similar rate transfer for isomers. For example, transfer of the D form of a compound should generally be about the same as transfer of the L form. It must have been very exciting for the early investigators to find glaring exceptions to these predictions. In many cases, the rate of entry was not found not to be a linear function of the concentration of the penetrant. In addition, some of the translocations were found to be surprisingly specific and against the concentration gradient. Fig. 2 (Christensen et al., 1963) shows some of the characteristics of the transport of leucine by Ehrlich ascites tumor cells (a line of mouse peritoneal cells). Fig. 2a shows the rate of transport of L-tert-leucine as a function of concentration of the amino acid in the medium. Fig. 2b is the corresponding Lineweaver-Burk plot and Fig. 2c shows the ratio of internal over external concentration of leucine with time of incubation showing that the uptake corresponds to an active transport. In addition, L-tert-leucine is taken up more rapidly than the D-isomer, as shown in Fig. 2c. Consequently, the steady-state level reached is greater for the L isomer. The ability to distinguish between isomers is known as stereospecificity. Other apparent anomalies have also been observed. Where many amino acids are used simultaneously, they interfere with each other as if they were http://www.albany.edu/~abio304/text/chapter_19.html (7 of 18) [5/5/2002 6:54:39 PM]
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competing for entry. Some of the findings may be explained without changing the diffusion model significantly. The saturation effect shown in Fig. 2(a) could be the result of passage of the amino acid through a restrictive channel that could admit only a certain number of molecules at a time. However, most of the other observations cannot be explained by such a simple concept. We would not expect, for example, that a simple channel would be able to distinguish between an L and a D isomer. Neither would we expect accumulation against a chemical or electrochemical gradient.
Fig. 2 (a) Kinetics of the transport of L-tert-leucine. (b) Lineweaver-Burk plot of L-tert-leucine concentrations against its rate of uptake. See text for details. Vm, 3.0 µmol ml-1 min-1; Km 1.0 x 10-3 M. (c) Time course of the uptake of the isomers of tert-leucine by Ehrlich ascites tumor cells. The cells were incubated as 4% suspensions in 1 mM L- or D-tert[1 - 14C]leucine. The distribution ratio represents the ratio of radioactivity per kilogram of cellular water to the radioactivity of the suspending solution. Reproduced from Biochimica et Biophysica Acta,vol. 78, H.N. Christensen, et al.,pp. 206-213, copyright ©1963 Elsevier Science. http://www.albany.edu/~abio304/text/chapter_19.html (8 of 18) [5/5/2002 6:54:39 PM]
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In order to explain these anomalies, scientists postulated that the transport is mediated by a transporter or carrier molecule. The present discussion will favor the term "transporter" because the term "carrier" has been used in the past to imply a movement of the whole transporter molecule from one membrane interface to the other. The translocation through a transporter mechanism requires reversible binding of the transported molecule to specific sites in the transporter molecule, followed by transfer of the sites to the opposite interface of the membrane and release of the ligand. These properties are very similar to that of the enzymes discussed in Chapter 13. The model reactions describing the general patterns of enzyme substrate interactions could also be used here, as shown in Eq. (7). In this equation, T = transporter which is restricted to the membrane phase (indicated by the brackets); S is the substance being transported; the subscripts o and i indicate the location in the inner and the outer phase. The brackets indicate that the reaction occurs in the membrane. So + [T↔ TSo] + Si (7) Where the accumulation is against a concentration gradient, a coupled reaction supplying the needed energy expenditure has to be added to this simple scheme. The transporter itself, or other molecular elements involved in the reaction between substrate and transporter, could have the requisite stereospecificity. As in the case of enzymes, the number of transporter molecules would necessarily be limited and the kinetics of the transfer should therefore exhibit saturation. This saturation could explain the constancy of the rate of transfer with large increases in substrate concentration (Fig. 2a). Except for the localization of the system in a membrane and the fact that the reaction corresponds to a vectorial displacement rather than catalysis, the model formally corresponds to that discussed for enzyme reactions (Chapter 13). In fact, the equations that are applicable are the same, such as Eq. (8).
As shown in Fig. 2a, when the substrate concentration increases, the rate (or velocity) of the reaction approaches a limiting value (Vm). As for the action of enzymes, and as shown in Eq. (8), a plot of 1/V as a function of 1/So (a Lineweaver-Burk plot), should yield the constants Km and Vm. In this case, the Vm is 3 mol ml-1 min -1 and the Km is 1 x 10-3 M. The Km of Eq. (8) depends on the various rate constants of the reactions represented in Eq. (7) (see Christensen 1975, pp. 115-119). Obviously, other features of the transport system will also be the same as those of enzyme reactions, such as competitive inhibition by analogs or a block by certain chemicals that have been shown to react with proteins. The representation of Eq. (7) does not include the coupling to a reaction supplying energy as required in active transport. The coupling may be through the phosphorylation of the transporter (see Chapters 20 and 21) or involve the cotransport of another ion in the direction of its electrochemical gradient. Eq. (8) does not always predict the rate of net transport accurately, because independent processes such as a diffusional component may also be involved. http://www.albany.edu/~abio304/text/chapter_19.html (9 of 18) [5/5/2002 6:54:39 PM]
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Many transport processes have the characteristics just discussed. When the transport is not against an electrochemical gradient, the process is referred to as facilitated diffusion. Transport against a gradient is referred to as active transport. II. MOLECULAR MECHANISMS OF TRANSPORT: THE GLUCOSE TRANSPORTERS What is the molecular mechanism for the transport of solutes? The information amassed over the years is beginning to provide answers (see Chapter 21). The transporters have been isolated and sequenced. These and other data have permitted eliminating many alternative models. However, examination of several models, for example those shown in Fig. 3, helps us define more precisely the characteristics of the transport systems and the mechanisms of transport. The transporter-substrate complex could be transferred from one interface of the membrane to the other by diffusion of the complex through the membrane (model 1). A larger molecule spanning the membrane could also transfer the solute, perhaps by rotating or moving in some other way (model 2). However, the movement need not involve a large portion of the molecule, but simply a small portion (not shown) or a conformational movement of the molecule or molecules constituting the pore structure (model 3). These three models involve movement of either the whole transporter or a portion of the transporter molecule (Fig. 3a). In contrast, fixed groups capable of binding the substrate arranged through a membrane pore (Fig. 3b) could also transfer the solute. The mechanisms represented by the two sets of general models - (a) or fixed (b) - can be distinguished experimentally (Fig. 4). Part a of the figure focuses for simplicity on model 1. However, the reasoning would be identical if we were to consider any of the mobile models. Let us examine the situation in which the substrate does not normally accumulate against a concentration gradient. On reaching a steady state (here, a true equilibrium), the inside and outside concentrations are the same. The inward flux is precisely the same as the outward flux. A competitor should decrease the flux by making the transporter less available to the substrate. As shown in Fig. 4a, if the transporter is mobile, the competitor, present on only one side, would naturally block the passage of substrate in a single direction (i.e., if the competitor is outside, it should affect only the inward flux). The flux in the opposite direction should remain largely unaffected. The inhibition would, therefore, be asymmetric. This situation could result in temporary accumulation against a gradient. The counterflow of a substrate against a concentration gradient involves the dissipation of chemical potential due to the flow of a second substance in the direction of its own concentration gradient. Counterflow is also known as trans facilitation (trans indicating that the competitor is on the other side of the membrane from the substrate). This phenomenon is apparently also aided by the fact that the rate of movement of the transporter is considerably higher when it is loaded than when it is empty (Gorga and Lienhard, 1981) (see below). For the case of a fixed carrier (Fig. 4b), both the inward and outward fluxes should be equally inhibited and no trans facilitation should take place. Many experiments clearly establish that the transporter is mobile (see also below). We already examined the case of the monosaccharide transport of red blood cells (Rosenberg and Wilbrandt, 1958) discussed in Chapter 12.
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Fig. 3 Diagrammatic representation of carrier models. Circle, carrier; S, substrate. (a)
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Fig. 4 Diagrammatic representation of transporter models and their behavior in relation to competitive inhibitors. T, Transporter; S, substrate; I, inhibitor; filled circles indicate binding sites. (a) Mobile transporter; (b) fixed transporter.
The movement of the transporter probably corresponds to a relatively small conformational change. As we saw in Chapter 4, integral membrane proteins clearly neither move from one surface to the other (model 1), nor rotate (model 2). In the case of the glucose transporter, the intrinsic fluorescence of the protein changes with binding to the substrate (Gorga and Lienhard, 1981), an indication that the components responsible for the fluorescence have been moved to a different molecular microenvironment. A conformational change is also demonstrated by changes in its sensitivity to chemicals. Similarly, the galactose-binding protein of E. coli shows an increase in intrinsic fluorescence and electrophoretic mobility when attached to its substrate (Boos et al., 1972). Model 3 is the most likely. It shows two different parts of the transporter forming a channel which exposes the binding groups alternatingly to the two phases separated by the membrane. A version of this model will be discussed in more detail in Chapter 21 for ion transport. The glucose transporters have been studied in detail. These transport systems are of obvious importance in the physiology of cells since the use of glucose as a major food is widespread. Furthermore, homeostatic mechanisms required in mammals for maintenance of glucose concentration are extensive and the diseases resulting from malfunction of this homeostatic control, such as in diabetes mellitus, are of obvious significance. Initially, the study of glucose transport was facilitated by the availability of red blood cells and the abundance of the glucose transporter in these cells, estimated to correspond to 5% of the total membrane proteins (e.g., Allard and Lienhard, 1985). The later DNA sequencing and cloning (see Chapter 1) provided considerable information on the widespread existence of this family of transporters, referred to as major facilitators (MFs). Until 1998, 133 have been sequenced, where sugar transporters form a subgroup of 18 members (see Walmsley et al., 1998). Six glucose transporters have been identified in mammals, these are referred to as GLUT1 to GLUT5 and GLUT7 (GLUT6 is non-functional as a transporter). Similar sugar transporters are present in http://www.albany.edu/~abio304/text/chapter_19.html (12 of 18) [5/5/2002 6:54:39 PM]
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bacterial systems. The transport in bacteria, however, is active via proton symporters (see Henderson, 1993), where glucose and H+entry are linked. The Na+-linked sugar transporters present, for example, in the kidney and small intestine, appear to be unrelated (see Hediger et al., 1987). In mammals, each GLUT has a characteristic tissue distribution (e.g., see tabulation of Gould and Holman, 1993). GLUT1 is one of the major proteins of the red blood cell. This transporter is present mostly in brain, adipose tissue and the blood brain barrier. GLUT2 is present in liver, pancreas β cells, kidney and small intestine, GLUT4 in muscle, heart and adipocytes and GLUT5 primarily in the small intestine. GLUT7 has a different function since it is located in the ER membrane. Some mammalian cells, such as brain have GLUT protein which are constitutive and mostly present in the plasma membranes. In others such as muscle and adipose tissue most of the transporters are recruited to the surface in response to biological signals (e.g., insulin or exercise) In part, the regulation of GLUT1 and GLUT4 uses an exocytotic mechanism similar to the one discussed for the aquaporins. Functional GLUT1 increases following stresses (such as viral infection, heat shock or alkaline pH) because the transporter is transferred from intracellular sites in the perinuclear region to the cell surface (Pasternak et al., 1991; Widnell et al., 1990). However, prolongued stress, particularly glucose deprivation, increases the total amounts of GLUT-1-mRNA and GLUT1 in the cell (Shetty et al., 1992; Yamada et al., 1983). White and brown fat, skeletal muscle and heart, transfer GLUT4 from the cell's interior to the cell surface in response to insulin (see Pessin et al., 1999). The transfer is rapid and produces as much as a 20-fold increase (Holman et al., 1990). In heart and brown adipocytes, immunological techniques have determined that, in the basal state, 99% of the GLUT4 is in the cell's interior in the trans-Golgi and tubular-vesicular structures (Slot et al., 1991a, b). The GLUT4 delivered to the plasma membrane in response to insulin appears to be present in part in small vesicles, 50 nm in diameter, which contain the proteins thought to be necessary for the exocytosis to the plasma membrane and these proteins are depleted with insulin treatment (e.g., Ramm et al., 2000). The activity of GLUT4, but not its distribution in the cell, is also regulated by other hormones such as adrenocoticotropic hormone (ACTH) and glucagon (see Simpson and Cushman, 1986). The regulation of GLU4 has been found to be central to the control. of glucose utilization and in diabetes in mammals (e.g., see addendum). GLUT1, a glycoprotein with an apparent molecular weight of 55 kDa (on SDS PAGE, this is band 4.5), has been extensively studied because of its presence in readily available red blood cells (Allard and Lienhard, 1985). The protein is heavily glycosylated and hydrophobicity plots (see Chapter 4) suggest 12 helices spanning the membrane (e.g., see Mueckler et al., 1985). Hydrogen exchange studies (by dialyzing reconstituted vesicles containing GLUT1 in a [2H]2O medium) indicate that a large fraction of the helices are accessible to water, evidence that they may be arranged to form a water filled channel (e.g., Alvarez et al., 1987). Supporting this view, the amino acid sequences of segments III, V, VII, VIII and XI were found to suggest amphipathic α-helices (Mueckler et al., 1985) since they contain several serine, threonine, glutamine and asparagine residues that would be localized on the same face of the α-helix. Both the carboxy and the amino terminals are in the highly charged cytoplasmic surface (Baldwin, 1993; Hresko et al. 1994). The carbohydrate moiety is external, attached to Asp45. Experiments with proteolytic digestion and antibody binding (see Chapter 4) confirm this general arrangement (e.g., Shanahan and D'Artel-Ellis, 1984; Cairns et al., 1984, 1987; Davies et al., 1987). The mechanism of transport currently favored is that shown in Model 3 of Fig. 3, where the transporter alternates between inward and outward-facing conformations (e.g., see Baldwin, 1993). In this model, the reorientation of the transporter is thought to be the rate-limiting step in the translocation of the glucose. The evidence supporting this model is as follows. Fluorescence spectroscopy shows that the tryptophan fluorescence changes during transport indicating that there is a change in the conformation of the transporter (see Appleman and Lienhard, 1985, 1989). In addition, essentially unidirectional http://www.albany.edu/~abio304/text/chapter_19.html (13 of 18) [5/5/2002 6:54:39 PM]
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conversion of one conformation to the other was accomplished by rapidly mixing purified transporter with either 4,6-ethylidene-D-glucose, which preferentially binds to the external phase of GLUT1 or phenyl beta-D-glucoside, which preferentially binds to the internal phase. Other evidence also supports the view that during transport there is a movement of binding sites from one surface to the other. One approach shows that cleavage of the transporter by the protease thermolysin is favored by the antibiotic cytochalasin B (CB) but retarded by the azidosalicoyl derivative of bis(D-mannose) (ASA-BMPA) (Holman and Rees, 1987). This finding suggests that cytochalasin B recruits the transporter site to the inner surface, whereas ASA-BMPA does the opposite. Cytochalasin B binds to the inward facing conformation since it is a competitive inhibitor of glucose efflux but a non-competitive inhibitor of influx. Similarly, ASA-MPA must bind to the outer surface because it is an impermeable competitive inhibitor of glucose transport. Thermolysin, a proteolytic enzyme isolated from bacteria, hydrolyzes internal peptide bonds adjacent to hydrophobic residues. Two other strategies have demonstrated movement of binding sites from one surface to the other by showing recruitment to the side that binds the competitor. In one case the binding of ligands (e.g., ethylidene glucose) at the external surface of the transporter was shown to interfere with cytochalasin B binding on the inner surface (Deves and Kupka, 1978; Gorga and Lienhard, 1981). In the other approach, the transport sites were shown to be recruited to the external surface by using impermeant, reversible inhibitors. The initial availability of transporter sites was assayed by measuring the initial rate of glucose influx. After treatment with the inhibitor, the subsequent addition of glucose and dilution of inhibitor produced an initial rate of transport considerably faster than the steady state transport in the absence of pretreatment (Lowe and Walmsey, 1987; Critchley and Lowe, 1991). However, the transport sites that are available to two sets of inhibitors - CB at the inner surface and sugar derivatives at the outer surface - are distinct. UV light cross-links CB to GLUT1 between helices X and XII, and D-glucose inhibits the binding. Sugar derivatives that bind to the external site attach between helices IX and X (see Walmsley, 1988). These sites may be on different transmembrane segments, however, they may be very close together in the tertiary structure. For example, a hydrophilic cleft is predicted between spans VII and VIII and XI and XII (see Baldwin, 1993). The two inhibitor binding sites are very close together since the two labels are separated by a small span of protein and can be separated by some chemical fragmentation procedures (see Holman and Rees, 1987). The two labels can be isolated together down to fragments as small as 7 kDa. A tryptophan cleavage at residue 388, divides the cytochalasin B site and the ASA-BMPA site. Mutagenesis studies have determined the amino acids residues that are important for the transport (Walmsley et al., 1994). In addition, the orientation of the glucose during transport by GLUT1 has been determined (Barnett et al., 1975). It enters the external face C-1 first and C-4 and C-6 enter last. Other GLUTs bind glucose with a similar polarity (Colville et al., 1993). Transmembrane chain V of GLUT1 is one of the segments of GLUT1 that has been postulated to form an amphipathic transmembrane helix (Mueckler et al., 1985) that might line the translocation pathway. This notion was put to test (Mueckler and Makepeace, 1999). Each residue supposed to be in segment V were mutated to cysteine and expressed in Xenopus oocytes (see e.g., Chapter 1). The mutants had transport activity. At least six of these cysteine residues were found accessible to p-chloromercuribenzoatesulfonate (pCMBS) which blocks transport. pCMBS is presumed to enter only a hydrated space because of its polarity. However, the six residues were clustered at the external surface of the plasma membrane and, therefore, represent only a small portion of segment V. Many ATP powered transports (e.g., see Chapter 21) and secondary active transports (i.e., powered by a gradient for another solute and not by ATP) have been shown to alternate binding groups between the inner and the outer face (e.g., see Hirayama et al., 1997). The case of the Na+-dependent http://www.albany.edu/~abio304/text/chapter_19.html (14 of 18) [5/5/2002 6:54:39 PM]
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glucose transport has been studied in detail (Panayotova-Heiermann et al., 1995; Hirayama et al., 1994). Binding of Na+, Li+ or H+ induce a change in conformation and increases the affinity for the sugar. The rate of transport is also determined by the cation. III. VECTORIAL ENZYMES In many ways, transport resembles an enzyme catalyzed process in which a protein molecule mediates translocation of the substrate rather than a chemical change. However, there is no reason why a protein could not pick up a substrate on one side of the membrane and simultaneously catalyze a chemical change with the discharge of the product on the other side of the membrane. In this case, the reaction would differ from one catalyzed by an enzyme in solution in that it has directionality. The reaction is said to be vectorial. A vectorial reaction is thought to be involved in the simultaneous translocation and phosphorylation of glucose and related monosaccharides in E. coli, Salmonella typhimurium and Bacillus subtilis. The sugars are translocated from the external medium into the cell, where they are released in a phosphorylated form. A similar process is thought to take place in yeast (e.g., Van Steveninck, 1972) and in Aerobacter aerogenes (Kelker and Anderson, 1972). Simultaneous translocation and enzyme action are by no means restricted to microorganisms. They are thought to take place, for example, in the intestinal transport of sugars, where sucrose is hydrolyzed and the products of the reaction, fructose and glucose, are released into cells. The most direct evidence for a role of vectorial reactions in translocation comes from studies of the phosphoenolpyruvate phosphotransferase reaction in vesicles obtained from E. coli membranes. The enzyme is responsible for the transfer of phosphate from phosphoenolpyruvate to sugar molecules. The experiment is represented in Fig. 5 (Kaback, 1968), where the ordinate represents the amount of sugar taken up as a function of time. The interior of the vesicles was preloaded with [14C]glucose, and [3H] glucose and phosphoenolpyruvate were added to the outside. The phosphorylation and the transfer of the phosphate showed a preference for the outside [3H]glucose. The phosphorylation did not precede the transfer, since the vesicles were unable to take up external glucose-6-phosphate.
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Fig. 5 Uptake and phosphorylation of[3H]glucose by E. coli membranes previously loaded with [14C]glucose. Reproduced with permission from H. R. Kaback, Journal of Biological Chemistry 243:3711-3724. Copyright ©1968 American Society of Biological Chemists, Inc.
In some bacteria, the phosphorylation translocation of sugars involves the phosphoenolpyruvate phosphotransferase reaction, as in the example of Fig. 5. In this reaction, sugars are phosphorylated by the transfer of phosphate from phosphoenolpyruvate (PEP). The reaction occurs in two steps: one in which the phosphate is transferred from PEP to a protein (HPr) and another in which it is transferred from the protein to the sugar. This can be represented as:
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HPr is small protein (9.4 kDa) that is phosphorylated at the histidine residue. The phosphotransferase system of bacteria may well account for the first reaction of many metabolized sugars for which no active kinase has been found. The apparent absence of an appropriate kinase has been one of the most intriguing questions in the physiology of some microorganisms (Wood, 1966). There are many indications that the phosphotransferase system plays a role in the translocation of sugars and that they are accumulated in the phosphorylated form. Mutants of S. typhimurium lacking any of the components of the system, fail to accumulate sugars (Simoni et al., 1967). However, it should be recognized that the mutants may be pleiotrophic (Kennedy and Scarborough, 1967) and have more than one deficiency. In some cases, the substrate is accumulated as a phosphorylated intermediate, as in the case of β-glucosides (Fox and Wilson, 1968; Wang and Morse, 1968). It has been proposed that the PEP system is involved in the transport of β-glycosides in E. coli. The protein HPr is deficient in treated E. coli cells that have impaired β-galactoside transport, and the addition of HPr reestablishes function. On the other hand, the finding that E. coli mutants deficient in enzyme I can transport β-galactosides makes this interpretation unlikely (Asensio et al., 1963). IV. STRATEGIES FOR THE ISOLATION OF TRANSPORT PROTEINS Our insights into a biological system cannot be considered sufficient until all molecular components and their characteristics are recognized. The ultimate test of our understanding consists of reestablishing biological activities in the test tube after putting the component molecules back together. A number of transport proteins have been isolated, and with many of them it has been possible to reconstitute transport by recombining them with artificial membranes. Isolation of a transport protein requires disruption of the membrane and some means of recognizing the presence of the molecules through the various fractionation procedures. Detergents must be used because the transporters are integral proteins. Recognition is not always a simple matter. Transport proteins may be recognized by their ability to bind either substrates or inhibitors. The task is simplest when a specific labelled inhibitor strongly binds to the transporter molecule. The anion transporter of the red blood cell has been recognized in this fashion. The usefulness of this approach can be much extended with the use of modified substrates that can be cross-linked at the sites of attachment, perhaps in a light-dependent fashion (photoaffinity labeling). Some active transports are coupled to a reaction which hydrolyzes ATP. These transporters are called P-ATPases. Their ATPase activity depends on the presence of the appropriate ions (Ca2+ for the Ca2+-ATPase, Na+ and K+ for the Na+, K+-ATPase). Therefore, the appropriate ATPase activity can serve to identify the transporter in the various protein fractions during the fractionation procedure. The ability of binding either substrates or inhibitors can also be directly exploited. These substances (e.g., glucose, ATP) can be covalently attached to a column and will bind the transporter, but allow other proteins through (this is known as affinity chromatography). An excess of the free substrate or inhibitor can then be used to set free the transporter by competing for binding with the substances attached to the column in a process known as elution. A group of related transport proteins with ATPase activity has been identified. The members of this super-family of traffic ATPases are also referred to as the ATP-binding casette proteins (ABC) (Hyde et al., 1990). They include the multidrug resistance P-glycoprotein (MDR), the cystic fibrosis gene http://www.albany.edu/~abio304/text/chapter_19.html (17 of 18) [5/5/2002 6:54:39 PM]
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product (CPTR), and a variety of prokaryotic permeases. CPTR probably functions as a Cl--conducting channel (Anderson et al., 1991; Kartner et al., 1991). Many years of work by many people have brought us close to unraveling one of the most intriguing problems of cell physiology: how a cell can control its integrity by controlling the flow of material in and out of its internal compartments. In this chapter, we have dealt primarily with the translocation of nonionic components. The next two chapters (Chapters 20 and 21) discuss the systems responsible for the transfer of ions in the cell. SUGGESTED READING Christensen, H.N. (1975) Biological Transport, Chapters 1, 4-8. Benjamin, Reading, Mass. Gould, G.W. and Holman, G.D. (1993) The glucose transporter family: structure, function and tissue-specific expression, Biochem. J. 295:329-341. (Medline) Macey, R. I. (1987) Mathematical models of membrane transport processes. In Membrane Physiology, 2d ed. (Andreoli, T. E., Hoffman, J. F., Fanestil, D. D., and Schulz, S. G., eds.), pp. 111-132, Plenum Medical Book Company, New York. Schafer, J. A. and Andreoli, T. E. (1987) Principles of water and nonelectrolyte transport across membranes. In Membrane Physiology, 2d ed. (Andreoli, T. E., Hoffman, J. F., Fanestil, D. D., and Schulz, S. G., eds.), pp. 177-190. Plenum Medical Book Company, New York. Schulz, S. G. (1987) Ion-coupled transport of organic solutes across biological membranes. In Membrane Physiology, 2d ed. (Andreoli, T. E., Hoffman, J. F., Fanestil, D. D., and Schulz, S. G., eds.), pp. 283-294. Plenum Medical Book Company New York. Stein, W. D. (1986) Transport and Diffusion Across Cell Membranes, Chapters 1, 2, 4, and 5. Academic Press, New York. Zeuthen T. (2001) How water molecules pass through aquaporins, Trends Biochem. Sci. 26:77-79. (MedLine) WEB RESOURCES Diwan, J.J., Membrane Transport, http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm">http://www.rpi.edu/dept/bcbp/molbiochem/MBWeb/mb1/part2/carriers.htm
REFERENCES Search the textbook
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Chapter 19: References
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REFERENCES Abel, E.D., Peroni, O., Kim, J.K., Kim, Y.B., Boss, O., Hadro, E., Minnemann, T., Shulman, G.I. and Kahn, B.B. (2001) Adipose-selective targeting of the GLUT4 gene impairs insulin action in muscle and liver, Nature 409:729-733. (MedLine) Allard, W.J. and Lienhard, G.E. (1985) Monoclonal antibodies to the glucose transporter from human erythrocytes, Identification of the transporter as a Mr = 55,000 protein, J. Biol. Chem. 260:86688675.(Medline) Alvarez, J., Lee, D.C., Baldwin, S.A. and Chapman, D. (1987) Fourier transform infrared spectroscopic study of the structure and conformational changes of the human erythrocyte glucose transporter, J. Biol. Chem. 262:3502-3509.(Medline) Anderson, M.P., Berger, H.A., Rich, D.P, Gregory, R.J., Smith, A.E. and Welsh, M.J. (1991) Nucleoside triphosphates are required to open the CFTR chloride channel, Cell 67:775-784. (MedLine) Appleman, J.R. and Lienhard, G.E. (1985) Rapid kinetics of the glucose transporter from human erythrocytes. Detection and measurement of a half-turnover of the purified transporter, J Biol. Chem. 260:4575-4578.(Medline) Appleman, J.R. and Lienhard, G.E. (1989) Kinetics of the purified glucose transporter. Direct measurement of the rates of interconversion of transporter conformers, Biochemistry 28:82218227.(Medline) Asensio, C., Avigad, G., and Harecker, B. L. (1963) Preferential galactose utilization in a mutant strain of E. coli. Arch. Biochim. Biophys. 103:299-309. Baldwin, S.A. (1993) Mammalian passive glucose transporters: members of an ubiquitous family of active and passive transport proteins, Biochim. Biophys. Acta. 1154:17-49.(Medline) Barnett, J.E., Holman, G.D., Chalkley, R.A. and Munday, K.A. (1975) Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system, Biochem. J. 145:417429.(Medline)
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Chapter 19: References
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20. The Cell Membrane: Transport of Ions
20. The Cell Membrane: Transport of Ions I. II. III. IV. V.
The P-ATPases Functional Significance of the Na+-K+ Transport System Coupling of ATP Hydrolysis to the Transport of Na+ The Na+-K+ Transport ATPase System Secondary Transports A. Na+-Glucose Transporters (SGLTs) B. H+-Peptide-Transporters Suggested Reading References Back to List of Chapters
The active transport of ions has many facets and variations. The translocation of a solute may be coupled to the translocation of another. These have been called secondary transports and are discussed in Section IV, below. When the two are translocated in the same direction, the transporter has been referred to as a symport; when in the opposite direction, as an antiport. The translocation of a solute can be powered by different mechanisms. In either a symport or an antiport, one of the solutes may be transferred in the direction of its electrochemical gradient and this transfer can pay for the active translocation of the accompanying solute. The active influx of sugars and amino acids in animal cells is powered by a symport in which Na+ is translocated in the direction of its electrochemical gradient. One of the mechanisms for pumping Ca2+ out of cells depends on an antiport which transfers Na+ in the direction of its gradient. Other transporters are powered by the coupled hydrolysis of ATP and they function as ion-dependent ATPases. I. THE P-ATPASES The P-type ATPases are self phosphorylating enymes which use ATP to phosphorylate a conserved aspartic residue. These include the Ca2+-ATPase of the plasma membrane or that of the sarcoplasmic reticulum (SR), the Na+,K+-ATPase of the plasma membrane, the gastric mucosa ATPase which transports H+ outwards in exchange for K+ and the H+-ATPase of the plasma membrane of plants and yeasts maintains the intracellular pH and membrane potential (see Møller et al., 1996). The translocation
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20. The Cell Membrane: Transport of Ions
reactions of these ATPases are outlined in Eq. (1) to (3). In these equations, the location of the component, inside or outside, is indicated by the subscript i or o, respectively. These four are probably the only active transport systems coupled to ATP hydrolysis in the plasma membrane of animal cells. 2Ko+ + 3Nai+ + MgATPi ↔ MgADPi + Pi + 3Nao+ + 2Ki+ (1) Ho+ + 2Cai2+ + MgATPi ↔ MgADPi + Pi + 2Cao2+ + Hi+ (2) nKo+ + nHi+ + MgADPi ↔ MgADPi + Pi + nHo+ + nKi+ (3) Each of the four proteins responsible for the ATP-powered active transport of these ions has been isolated (Kyte, 1971; McLennan, 1969; Sachs et al., 1976; see Nakamoto and Slayman, 1989). The Ca2+ and the Na+-K+ transport systems have been reconstituted in artificial lipid membranes from the purified ATPases and phospholipids. The various transport ATPases resemble each other strikingly. All are polypeptides of approximately 900 to 1200 amino acids and their activity involves phosphorylation in an aspartate residue. The Ca2+ and Na+,K+-ATPases have been studied in more detail. They appear to share many similarities, in their amino acid sequence and in exhibiting similar hydrophobic segments and adenine nucleotide binding sites. All four involve the transport of one cation in one direction, and another in the opposite direction. Because of the stoichiometry of the exchange, at least the Ca2+ pumps and the Na+, K+-ATPase are electrogenic. In its native form, the Na+,K+- ATPase has two polypeptides: a larger polypeptide (α of 94 to 106 kDa and a smaller sialoglycoprotein (β) of 41 to 52 kDa (Kyte, 1974), which may have a role in the transport and membrane assembly of the α-subunit (McDonough et al., 1990). The two polypeptides are present in equimolar amounts, but it is generally agreed that the α-component corresponds to the transporter; the βsubunit can be removed without changing the ATPase activity (Freytag, 1983). Furthermore, the very similar Ca2+-ATPase of the SR lacks the smaller polypeptide. The Na+-K+ transport system is associated with almost all cells that have been studied, including those of specialized tissues which carry out Na+ transport, such as the kidney and the rectal gland of elasmobranchs which function to excrete Na+. The Ca2+-ATPase functions to maintain a low cytoplasmic Ca2+ concentration. One Ca2+-ATPase is associated with the plasma membrane, while another distinct ATPase functions to sequesters Ca2+ in the sarcoplasmic reticulum of muscle cells (referred to as CERCA)(seeChapter 23). Similar Ca2+ sequestering vesicles are likely to be present in the cytoplasm of other cells. In contrast, the H+,K+-ATPase is the means by which acid is accumulated by the gastric mucosa (Sachs et al., 1976). A similar ATPase in the fungus, Neurospora, (Scarborough, 1980) transports H+ outward and is responsible for the high membrane potential of its cells; in this case, the transport is strongly electrogenic.
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This chapter primarily treats the functional significance and characteristics of the Na+, K+-ATPase transport system. The next chapter will discuss the characterization of the proteins responsible for the transport of cations, together with possible models of transport. The Na+, K+-ATPase and the Ca2+ATPases are so similar that the two are frequently discussed interchangeably in that chapter. II. FUNCTIONAL SIGNIFICANCE OF THE Na+-K+ TRANSPORT SYSTEM In all cells, there seems to be some mechanism for controlling the concentration of ions in the internal medium. A high internal concentration of K+ and a low concentration of Na+ is generally the rule. The range of internal K+ concentration in vertebrates is narrow: 100 to 200 mM. In freshwater organisms, the internal level of K+ is frequently much smaller, 15 to 30 mM, but this is remarkably high compared to the external environment, in which K+ may be present only in trace amounts. The capacity to control the internal concentration is such that certain organisms can grow at extreme conditions of salinity and still concentrate K+ selectively, despite the high Na+ concentration of the medium and the relative absence of K+. Halobacterium salinarium concentrates K+ in the face of an external NaCl concentration of 4 M and can attain the remarkable internal K+ concentration of 4 M (Christian and Waltho, 1962). Examples of the concentrations of Na+ and K+ in various organisms are listed in Table 1 (Steinbach, 1963). The reasons for the universality of these ion distributions are still a subject for speculation. As we shall see in Chapter 22, the plasma membrane's resting and action potentials depend on this ion concentration imbalance between the inside and the outside of the cell. These potentials play a role not only in the signal conduction of nerves and muscle in higher organisms, but in other functions. In protists, they play a role in their responses to environmental stimuli. Furthermore, the internal concentration of cations has to be controlled (in this case by pumping out Na+) to maintain cell volume within physiological limits. The Na+ tends to accumulate inside (Tosteson and Hoffman, 1960) to neutralize the negative charges of the macromolecules trapped in the cytoplasm. Inhibition of the Na+ pumping activity therefore leads to osmotic swelling. There is no doubt that the exclusion of Na+ plays a role in cell function and integrity in at least animal cells. However, it is difficult to argue that these needs are universal. Some organisms may not use electrical signals and the rigid walls of plant cells and microorganisms are effective in limiting swelling. Other reasons may therefore exist for the universality of the control of the cell's internal environment. Evidence accumulated over the years indicates that K+ is generally required for growth (Steinbach, 1963). Potassium ions are required for protein synthesis in a number of unrelated organisms or preparations (Table 2). This requirement probably explains the effect of K+ on growth. In addition, K+ is required for maximal activity of a number of enzymes concerned primarily with other functions, such as 6-phosphofructokinase and pyruvate kinase (Lubin, 1964 lists a total of 23 important enzymes). Table 1 Selected Values for Na+ and K+
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Erthyrocytes (mmol/kg wet wt.) Animal
Plasma or Serum (mmol/kg wet wt.)
Na
K
Na
K
Human
11
91
138
4.2
Sheep (average)
82
11
160
4.8
Dog
106
5
150
4.4
Rabbit
16
99
158
4.1
Elephant seal
95
7
142
4.5
Rat
12
100
151
5.9
Duck
7
112
141
6.0
Chicken
18
119
154
6.0
Dolphin (mammal)
13
99
153
4.3
Fish (mackerel)
-
-
183
10.0
Frog
-
-
105
4.8
Reptile (turtle)
-
-
140
4.6
Plant
Na
K
Asparagus
1
50
Beet, leaves
60
130
Beet, roots
30
75
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Lettuce
6
60
String beans
1
60
Broccoli
7
75
Celery
50
90
Yucca, leaves
30
120
Yucca, stem
19
70
Vetch
40
156
Salt grass (sea shore)
70
45
Salt bush
23
63
Rye, tops
4
120
Clover, tops
20
150
The K+ requirement for protein synthesis is elegantly shown in an experiment with a mutant strain of E. coli that is incapable of transporting K+. The cells can be easily depleted of K+ and the internal concentration of K+ mirrors that in the medium. The cells are incubated in a medium containing leucine labeled with [14C]. The ordinate in Fig. 1 (Lubin and Ennis, 1964) represents the labeled leucine incorporated by the bacteria, the abscissa, the intracellular K+ concentration. In this experiment, leucine incorporation serves as an indicator of protein synthesis. Clearly, the K+ inside the cells is needed for the incorporation of amino acid into protein. Similar results were obtained in experiments carried out with B. subtilis mutants and mammalian tumor cells in culture in which the K+ transport has been inhibited by the drug amphotericin B. From these experiments, it is clear that K+ accumulation has a fundamental role in protein synthesis. In addition, it activates a number of metabolic enzymes.
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Fig. 1 Incorporation of [14C]leucine into protein at various concentrations of intracellular K+ at 37C. Reproduced from Biochimica et Biophysica Acta, vol.80, M. Lubin and H. Ennis, pp.614-631, copyright ©1964 with permission from Elsevier Science.
III. COUPLING OF ATP HYDROLYSIS TO THE TRANSPORT OF Na+ The active transport of Na+, K+, Ca2+, or H+ in the plasma membrane is coupled to the hydrolysis of ATP. For practical reasons, the Na+, K+-ATPases studied most intensely have been those of giant nerve cell axons and red blood cells. As we shall see, they have obvious advantages. The high internal K+ and the low Na+ concentration of these cells, as in many others, are maintained in the presence of high Na+ and low K+ concentrations in the external medium. Here we will consider the evidence for an active transport mechanism that functions as an ATPase. Table 2 Dependence of Protein Synthesis on K+
System
Reference
Liver extract
Sachs, H. J. Biol. Chem. 228:23 (1957)
Pancreas extract
Gazzinelli, G., and Dickman, S.R. Biochim. Biophys. Acta 61:980 (1962)
Sea urchin egg
Hultin, T. Exp. Cell Res. 25:405 (1961)
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E. coli intact mutant or extract
Lubin, M. Nature 213:415 (1967)
Bacillus subtilis intact mutant
Lubin, M. Fed. Proc. Fed. Am. Soc. Exp. Biol. 23:994 (1964)
Sarcoma 180, intact, pharmacological manipulation
Lubin, M. Nature 213:451 (1967)
Giant axons of certain invertebrates are ideal experimental material for the study of ionic transfers and electrical phenomena. Their size, as much as 0.5 mm in diameter, greatly facilitated manipulation, for example, in the internal injection of compounds with micropipettes, even in the days when the fabrication of pipettes was fairly primitive. In addition, considerable data can be collected from a single experiment, since these axons are sturdy enough to allow prolonged observations. With the squid axon, a number of incisive experiments have explored a whole spectrum of problems related to ion transfer and excitability, including that of the coupling of Na+ transport to energy expenditure. When the radioactive isotope [22Na+] is injected into the nerve fiber, the exit of Na+ can be followed by placing the axon in unlabeled medium and simply measuring the level of [22Na+] in the external medium. Oxidative metabolism can be blocked with certain inhibitors (e.g., CN-). The role of high-energy phosphates in Na+ transport can be tested directly by injecting these compounds into the axon after blocking the metabolic reactions that would regenerate them (see Fig. 2a). Results of such an experiment are shown in Fig. 2b and c. The addition of 2 mM cyanide to the system considerably reduces the Na+ efflux from the axon (arrow 1, Fig. 2b or c). However, injections of ATP (arrow 4) increase the efflux of [22Na+] significantly, whereas the injection of breakdown products of ATP is ineffective (arrow 2). The results with arginine phosphate, another phosphate ester with a high phosphate group-transfer potential, are similar (Fig. 2c). Arginine phosphate is thought to transfer its terminal phosphate to ADP to regenerate ATP. In the experiments shown, the effects of the ATP or arginine phosphate are transient, but they can be prolonged for several hours if higher concentrations are used. Although injections of ATP or arginine phosphate into the axon are effective, their addition to the medium is not. Thus the system is asymmetric, as might be expected in a transport ATPase which pumps Na+ outward. In both experiments, after about 4 h, the cyanide is washed off and the high rate of Na+ efflux (arrow 4) returns. From these experiments, it is relatively simple to calculate the Na/P ratio in much the same way as done for the mitochondrial system. The ratio was found to be about 0.7 using either arginine phosphate or ATP. More recent results show that the value is more likely to be close to 3. The lower value is probably the result of the activity of other cytoplasmic ATPases or phosphatases. Regardless of stoichiometry, these results agree with the notion that an ATP-powered transport is taking place in these axons.
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Fig. 2 (a) System for measuring [22Na] efflux in giant axon. (b)[22Na] efflux; the effect of CN- and ATP and arginine phosphate. Reproduced with permission from P.C. Caldwell, et al. , Journal of Physiology, 152:561-590. Copyright ©1960 The Physiological Society, Oxford, England.
Similar experiments have been carried out with intact red blood cells or isolated membranes. Erythrocytes are generally sturdy and, of course, are available in large quantities. Analysis of the internal medium or the suspending solution, can be carried out readily after separating the cells or other vesicles from the medium by centrifugation or filtration. The erythrocytes can be lysed (hemolyzed) by hypotonic conditions or the use of detergents. The intact cells are shaped like slightly concave plates. When suspended in a hypoosmotic medium, they swell to a spherical shape so that the surface membrane is stretched. At this point, they become extremely permeable and lose their internal contents, including hemoglobin; what is left are the erythrocyte ghosts. Because of their high permeability, the ghosts tend to equilibrate with the external medium. Interestingly, the empty sacs can at least partially regain their low permeability under the appropriate conditions, as if their membranes were capable of "resealing." Because of this property, it is possible to vary the internal contents of the cells not by injection but by hemolysis in media containing the desired components and then resealing them. This procedure is illustrated in Fig. 3 (Whittam, 1962), which shows the good correlation between the Na+ composition inside the resealed ghosts (ordinate) and that of the suspension medium used for hemolysis (abscissa). Comparable results have been shown for other solutes contained in the hemolysis medium. Ghost fragments are more amenable to biochemical studies, since the absence of permeability barriers avoids unnecessary complications in experimental design (for example, changes in concentration of ions due to the presence of a permeability barrier). A number of other cell membrane preparations, such as leaky brain and kidney microsomes, are similar to these ghost fragments. Incorporation of ATP into the ghosts gives results very similar to those obtained with the giant axon. Such an experiment is represented in Fig. 4. Curve 1 shows the results obtained with ATP, and curve 2 http://www.albany.edu/~abio304/text/chapter_20.html (9 of 22) [1/9/2003 12:14:18 PM]
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shows the inhibition of the ATP effect by the use of an inhibitor of the Na+ transport system, ouabain (Gstrophanthin, a glycoside used as a cardiac drug). Table 3 (Hoffman, 1962) compares the Na+ exit in the presence of ATP (row 1) with that occurring in its absence (row 5). The effects of several other compounds of high phosphate group transfer potential (rows 2-4) are also shown. From the results, it can be concluded that ATP serves as the source of energy for the translocation of Na+ in the two systems discussed.
Fig. 3 Ionic composition of resealed ghosts as a function of the composition of the hemolysate. ( (
) Na+,
) K+. From Biochemistry Journal, 84:110-118, copyright ©1962 The Biochemical Society, London.
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Fig. 4 Release of [24Na] induced by ATP (curve 1) and its inhibition by ouabain (curve 2). Reproduced from J. Hoffman, Circulation 26:1201-1213, 1962, by permission of the American Heart Association, Inc.
Table 3 Influence of Different Incorporated Nucleotides on the Activation of Transport in Depleted Ghosts
24Na
Incorporated substrate
released in 80 min (%)
Alone
+Ouabain
1. ATP
50.6
22.8
2. ITP
27.6
22.2
3. GTP
25.9
25.5
4. UTP
33.4
33.2
5. Control
29.1
24.2
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Reproduced from J. Hoffman, Circulation 26:1201-1213, 1962, by permission of the American Heart Association, Inc.
The effect of ouabain is a useful one: this drug inhibits the active transport of Na+ in many different kinds of cells and organisms. With it, the active transport of Na+ can easily be distinguished from nonspecific events, such as those brought about by a change in permeability. A parallel change would be expected for an ATPase involved in transport; other unrelated ATPases are not likely to be affected. The ATPases of both nerve and erythrocyte ghosts appear to be sensitive to the inhibitor. At least in squid axon, which has been studied in this respect, cardiac glycosides are effective only when added to the outside; their injection into the axon does not inhibit Na+ transport. This is a polarity opposite to that of ATP, which is effective only on the inside. IV. THE Na+ K+-TRANSPORT ATPase SYSTEM The experiments just discussed show that the energy available from the hydrolysis of ATP can be used for the transport of Na+ (and K+). In order to function as an ion pump powered by ATP hydrolysis, the ATPase must reside in the plasma membrane. Erythrocyte ghosts are virtually plasma membranes. The results depicted in Fig. 4 were obtained using ghosts and they show considerable oubain-sensitive ATPase. Similar evidence has also been obtained in neurons, where the individual nerve cell sheaths can be mechanically isolated by microdissection, as shown in Fig. 5 (Cummins and Hyden, 1962). The activity of the material isolated in this way is shown in Table 4 (Cummins and Hyden, 1962). It is interesting to see that, in this case, ouabain is capable of inhibiting the membrane ATPase entirely. These results leave little doubt that the Na+, K+-transport ATPase is located in the plasma membrane. Many other membrane preparations from widely different tissues, including plants (Lai and Thomson, 1971), have been shown to contain Na+ and K+-dependent ATPases. These experiments opened the way for a more detailed examination of this transport. The maintenance of a high internal concentration of K+ and a low internal concentration of Na+ may be explained most simply by a model, such as that represented in Fig. 6 (Glynn, 1957). Here, a high phosphate group transfer potential form of a carrier, Y, in the inner phase (step 1) permits the translocation of Na+ to the external phase (steps 2-4) against an electrochemical gradient. In this process, Na+ complexes with the carrier Y (step 2) and, after movement of the complex NaY to the external surface (step 3), the NaY complex dissociates (step 4). The transition from a high-energy form of the carrier (Y) to a low-energy form (X) (step 5) fulfills the thermodynamic requirement of an energy expenditure. It also presents K+ with a carrier for its translocation to the internal phase (steps 6-8).
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Fig. 5 Microsurgical procedure to obtain nerve cell membrane. (a) Whole nerve cells; (b) initial incision; (c) the incision; (d) final membrane preparation. Reproduced from Biochimica et Biophysica Acta, vol. 60, J. Cummins and H. Hyden, pp.271-283. Copyright ©1962 with permission from Elsevier Science.
The cyclic operation of this model could account for the Na+ efflux and K+ influx coupled to ATP hydrolysis. NaY and KX could be moved inside the membrane from one membrane interface to the other. However, a mechanism in which the binding groups of the transporter are alternatively exposed to the two membrane interfaces through a conformational rearrangement is more likely (see Chapter 21). The form of the energy expended is not specified in Fig. 6; however, the evidence discussed for the Na+K+ transport shows that ATP hydrolysis is coupled to the transport, so that the model can be modified to include this detail, as represented in Fig. 7. Table 4 Effect of Ouabain on the ATPase of Nerve Cell Membranea
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Concentration of ouabain (M)
ATPase activity (pmoles ATP hydrolyzed per membrane/h)
0
0.4
2 x 10-6
0.2
2 x 10-5
0
Reproduced with permission from J. Cummins and H. Hyden, Biochim. Biophys. Acta 60: 271-283. Copyright ©1962 Elsevier Publishing, Amsterdam. a
Composition of saline was NaCl, 66 mM,; KCl, 33 mM; MgCl2, 5 mM, Tris, 25 mM (pH 8.0); final volume 6 µl. 15 pmol of radioactive ATP added (250 counts/min). Total of 10 experiments consisting of 8-10 membranes each.
Fig. 6 Shaw's hypothesis of a K+ carrier that is converted to an Na+ carrier by the expenditure of energy. Reproduced with permission from Progress in Biophysics and Molecular Biology, 8:241-307, I. M. Glynn, copyright ©1957 Pergamon Journals, Ltd.
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Fig. 7 Shaw's hypothesis modified to specify the high-energy intermediate as a phosphorylated form.
Figure 7 specifies a phosphorylated form of the carrier as the transporter of Na+ (step 1) and another phosphorylated form (formed in step 5) as the transporter of K+. Otherwise, it does not differ from the model of Fig. 6. As shown in Fig. 7, the transport system, and hence the energy-expending step 1, cannot operate without the presence of Na+ and K+. Hence, an ATPase involved in Na+/K+ transport must depend on the concentrations of these two ions. In addition, since cardiac glycosides inhibit the transport, any ATPase activity reflecting this transport must be inhibited. ATPases from many systems have been found to fulfill these requirements, as shown in Table 5 (Bonting and Caravaggio, 1963), and, today, it is generally assumed that they correspond to the Na+/K+ pump. The dependence of the membrane ATPase on Na+ and K+ is represented in Fig. 8a and b (Post et al., 1960). The ATPase activity of erythrocyte ghosts is expressed as Pi released per hour in the ordinate. The total osmotic pressure of the solution was maintained constant by varying the proportion of the two cations, as shown in the abscissa. Figure 8a represents the experiment over a wide range of Na+ concentrations (0-100 mM) with K+ varied accordingly from 20 to 140 mM. The experiment essentially provides an approximate constant (apparent Km, 24 mM) for Na+, as indicated by the half-maximal concentration of Na+. The variation in the K+ concentration in part a does not interfere with the results, because saturation levels of K+ are used. Figure 8b shows the variation of activity with K+ concentration, which is varied over a much narrower range (0-25 mM). Accordingly, Na+ is at high concentration (120145 mM) again at saturation levels. This experiment provides an apparent Km, of 3 mM for K+. The dependence of the Na+ transport on Na+ concentration is shown in Fig. 8c. The constant for this process (Km = 20 mM) is approximately the same as that for ATPase. Thus, the ATPase depends on the Na+ and K+ concentrations as required by the model of Fig. 7. The similar Km confirm that the transport http://www.albany.edu/~abio304/text/chapter_20.html (15 of 22) [1/9/2003 12:14:18 PM]
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and ATPase activity are coupled. The apparent Michaelis-Menten kinetics of Fig. 8 are probably coincidental, since the ATPase generally shows sigmoidal kinetics (Robinson, 1970); nevertheless, the details of the kinetics do not alter the interpretation. Table 5 Comparison of Cation Fluxes and ATPase Activitiesa
Temperature (oC)
Cation flux (average) (10-14mol cm-2s-1)
Na+,K+-ATPase activitya (10-14mol cm-2s-1)
Ratio Cation/ATP
Human erythrocytes
37
3.87
1.38±0.36 (4)
2.80
Frog muscle
17
985
530±94 (4)
1.86
Squid giatn axon
19
1, 200
400±79 (5)
3.00
Frog skin
20
19, 700
6,640±1,100 (4)
2.97
Toad bladder
27
43, 700
17,600±1,640 (15)
2.48
Electric eel
23
86, 100
38,800±4,160 (3)
2.22
Tissue
noninnervated membrane
_____________ 2.56±0.19
Sachs organ
Reproduced from S.L. Bonting and L.L. Caravaggio, Arch. Biochem. Biophys. 101: 37-46, 1963 with permission. a
Means ±standard errors and number of determinations in parentheses.
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Fig. 8 (a and b) Effect of varying Na+ and K+ concentrations on the ATPase activity of a preparation of human erythrocyte membranes. Isotonicity was maintained by maintaining Na+ and K+ constant. (c) Influence of cell Na+ content on rate of active transport in intact cells. The millimoles of hemoglobin correspond to millimolar concentrations. The sum of the active transport rates of sodium plus potassium in samples taken at 2-h intervals is plotted against the mean Ma content. External K+ was always more than
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30 mM and was not rate limiting. The different symbols indicate three different experiments. Passive transport corrections were less than 7% of the corresponding active rates. All the results plotted for the last experiment were arbitrarily multiplied by 1.5 to obtain a smooth continuation of the curve obtained in the first experiments with fresher cells. This was done because cells stored in the cold for a long period have regularly had a lower pumping capacity than fresher cells. Reproduced by permission from R. L. Post, et al., Journal of Biological Chemistry, 235:1789-1802. Copyright ©1960 American Society of Biological Chemists, Inc.
One of the significant features of the model of Fig. 7 is the asymmetry of the transport. In this model, the Na+ combines with the energized carrier at the inner surface of the membrane, whereas the K+ combines with a different form of the transporter at the outer surface. Experiments already discussed do show some asymmetry. As mentioned, ouabain inhibits the reactions only when placed on the outside of the cells; injected into axons, it is ineffective. The opposite is true of ATP: it must be present internally to be utilized by the system. The asymmetry of ATP hydrolysis is consistent with the model (step 1 of Fig. 7). We have seen that it is possible to vary the internal, as well as the external environment of resealed red blood cell ghosts (see Fig. 3 and Table 6). Therefore, whether the ATPase system is asymmetric in relation to the Na+ and K+ can be tested in a forthright manner. The internal and external environments can be varied in relation to the Na+ and K+. The results of such experiments are shown in Table 6 (Whittam, 1962). The amount of ATP hydrolyzed is measured by following the increase in Pi. Since the system is rather impure, the ATPase activity that is irrelevant to transport must be ignored. This is done by using ouabain in parallel experiments; the Pi liberated in the presence of ouabain and, presumably due to nonspecific ATP hydrolysis, is subtracted from the total formed in its absence. This difference, shown in column 5 of Table 6, represents the ATPase sensitive to ouabain and, therefore, presumed to be associated with transport. This view is reasonable, since the effect of ouabain has approximately the same constants in relation to either the transport or the ATPase activity. In addition, we have seen that ouabain blocks the ATPase of the neuronal membranes (Table 4) as well as the ATP-energized Na+ translocation of erythrocyte ghosts (Fig. 4). The variation in external K+ is shown in column 4 of Table 6. The ATPase activity (column 5) depends heavily on the external K+ concentration. On the other hand, the external Na+ level does not seem to matter, since KCl or choline chloride medium (items 2, 3, and 5) is as effective as Na+ media (items 1 and 4). As shown in part B, the ATPase activity depends on the internal Na+ (shown in column 1). Compare for example, 4a and 5c in column 5. Table 6 Dependence of Na+ and K+ on Ouabain-Sensitive (0.1 mM) ATPase
(1)
(2)
(3)
(4)
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(5)
20. The Cell Membrane: Transport of Ions
Cations inside (mM) Na+
K+
Na+ + K+
K+in medium (mM)
∆Pi liberated (mM/liter ghosts per hour )
a. 149
18
167
0
0.4
10
1.5
0
0.5
10
1.4
Main solute (140-150 mM)
A 1. NaCl
Oubain sensitive
b. 2 . Choline chloride
c. 115
20
135
d. 3. KCl B 4. NaCl
5. Choline chloride
e. -------
-------
------
150
1.9
a. 100
75
175
10
1.9
b. 83
14
97
10
1.7
c. 41
79
120
10
1.2
a. 80
16
96
10
1.6
b. 84
82
166
10
1.6
c. 14
81
95
10
1.0
Whittam (1962) Reproduced by permission from Biochemistry Journal 84: 110-118, copyright ©1962 The Biochemical Society.
In summary, all available evidence indicates that the Na+/K+ ATPase is present in most, if not all, plasma membranes and they support the model of Fig. 7. The molecular organization of the transport ATPases and their topological arrangement will be discussed in the next chapter. V. SECONDARY TRANSPORTS http://www.albany.edu/~abio304/text/chapter_20.html (19 of 22) [1/9/2003 12:14:18 PM]
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Several transport systems of the plasma membrane are powered by the hydrolysis of ATP. These are considered primary transports. In vertebrates, the primary transport is generally the Na+-K+ discussed in this chapter. However, the transports of many other solutes against their electrochemical gradient are coupled to the favorable flux of other solutes in the direction of their gradients (see Chapter 12 and above). The transport systems, coupled in this manner, are frequently referred to as secondary transports. Secondary transport systems play a preeminent role in both prokaryotes and eukaryotes. The primary transports store the energy needed by the secondary transports in the form of a favorable gradient and indirectly pay the cost of the secondary transports. There are many secondary transports of significance. In the plasma membrane of most cells, the efflux of Ca+ is coupled to the influx of Na+. Along with the Ca+-ATPase system, this transporter helps in keeping the Ca2+ concentration in the cytoplasm vanishingly low, so that Ca2+ can be used as a sensitive intracellular signal (see Chapter 7). In the renal proximal tubules, the transport of anions, such as dicarboxylates and sulfate, is also powered by the Na+ gradient (e.g., see Pajor et al., 1998). There are multiple pathways for the transport of amino acids in non-polar cells, such as Ehrlich cells or mouse fibroblasts (see Guidotti and Gazzola, 1993). Similar pathways were found in the intestine (e.g., Stevens, 1993), kidney (Schwegler et al., 1993) and in other tissues. These include Na+-dependent and independent pathways. The transmembrane Na+-gradient has been shown to drive the Na+-dependent pathways. The glutamate-transporter (see Worrall and Williams, 1994) is of particular importance since glutamate is the primary excitatory neurotransmitter in the mammalian brain. In mammals, the transport of glucose in the intestinal and kidney epithelial cells is also powered by the Na+-electrochemical gradient. The Na+, K+-ATPase of the basolateral membrane of epithelial cells provides the appropriate electrochemical gradient by pumping Na+ out of the cells and K+ into the cells. In enterocytes, the transport of peptides is linked to H+. In this case, the pH gradient is provided by the Na+/H+-exchanger in the brush-border membrane that pumps H+ out. The latter is also eventually powered by the Na+, K+-ATPase. The Na+-glucose transporters (SGLTs) and the peptide transporters (PEPTs) (see Steel and Hediger, 1998) are discussed in the rest of this section. Na+-Glucose Transporters (SGLTs) Glucose transport mediated by SGLT1 has a stoichiometry of 2 Na+: 1 glucose and is electrogenic (see Wright et al., 1994). The system was studied with electrophysiological techniques with voltage clamping using two electrodes (see Chapter 22). The importance of these transport systems is shown by human transport defects. A defect in SGLT1 produces familial glucose-galactose malabsorption (GGM) with subsequent severe diarrhea when on a http://www.albany.edu/~abio304/text/chapter_20.html (20 of 22) [1/9/2003 12:14:18 PM]
20. The Cell Membrane: Transport of Ions
diet containing glucose and galactose. In addition, an increase in Na+and glucose reabsorption by SGLT1 and SGLT2 is probably responsible for the pathogenesis of kidney disease in diabetes mellitus. SGLT1 in the brush-border intestinal membrane is responsible for the osmotic reabsorption of water and may actually be involved in the transport directly (Meinild et al., 1998). The study of mutants either present in the human population or produced in vitro in cultured cells, has been able to pin-point domains involved in substrate binding and cotransporter binding (e.g., see Panayotova-Heiermann et al., 1997). SGLTs, as well as the peptide transporters, have been sequenced. Both SGLTs (Hediger et al., 1987) and H+-peptide transporters (Fei et al., 1994) have twelve domains that are most likely to be transmembrane. H+-Peptide-Transporters The hydrolysis of proteins in the gastro-intestinal tract produces a rich mixture of peptides and amino acids. These are transported in the intestinal and renal-brush border membranes. The transporters involved can transport almost any di- and tri-peptide, regardless of amino acid composition or charge but cannot transport individual amino acids or larger peptides (see Meredith and Boyd, 1995). They can also transport therapeutically active compounds (e.g., amino penicillin, cyclacillin, etc.) and facilitate their absorption. Studies with vesicles from the membranes of intestinal and renal tissues have led to the identification of an electrogenic H+-oligopeptide transporter. PepT1 has been shown to be coupled to H+ movement (e.g., Fei et al., 1994). A similar protein, PepT2 (Liu et al., 1995) is present in a number of tissues including kidney (but not intestine). The transport follows 1:1 stoichiometry for neutral or cationic dipeptides. The stoichiometry was found to be 2:1 (charge to substrate) for anionic dipeptides (Steel et al., 1997). For cationic or anionic dipeptides the transport is electrogenic (e.g., Fei et al., 1994). SUGGESTED READING Inesi, G.(1994) Teaching active transport at the turn of the twenty first century: recent discoveries and conceptual changes, Biophys. J. 66:554-560 http://www.biosci.umn.edu/biophys/OLTB/BJ/Inesi.pdf Adobe Acrobat from www.adobe.com is required for reading pdf files. (Medline) Pedersen, P. L. and Carafoli, E. (1987) Ion motive ATPases. Part I. Ubiquity, properties and significance to cell function, Trends Biochem. Sci. 12:146-150. Pedersen, P. L. and Carafoli, E. (1987) Ion motive ATPases. Part II. Energy, coupling and work output, Trends Biochem. Sci. 12:186-189.
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Stein, W. D. (1986) Transport and Diffusion Across Cell Membranes, Chapter 6, Academic Press, New York. Stekhoven, F. S. and Booting, S. L. (1981) Transport adenosine triphosphatase: properties and functions. Physiol. Rev. 61:1-76. REFERENCES Search the textbook
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Chapter 20: References
Back to Chapter 20
REFERENCES Atkinson, A., Gatenby, A. D. and Lowe, A. G. (1971) Transport ATPase-subunit structure analyzed, Nature New Biol. 233:145-146.(Medline) Bonting, S. L. and Caravaggio, L. L. (1963) Studies on sodium potassium activated adenosine triphosphatase. V. Correlation of enzyme activity with cation flux in six tissues, Arch. Biochem. Biophys. 101:37-46. Bonting, S. L., Simon, K. A. and Hawkins, N. M. (1961) Studies on sodium-potassium-activated adenosine triphosphatase. I. Quantitative distribution in several tissues of the cat, Arch. Biochem. Biophys. 95:416-423. Caldwell, P. C., Hodgkin, A. L., Keynes, R. D. and Shaw, T. I. (1960) The effects of injecting "energyrich" phosphate compounds on the active transport of ions in the giant axons of Loligo, J. Physiol. (London) 152:561-590. Christian, J. H. B. and Waltho, I. A. (1962) Solute concentrations within cells of halophilic and nonhalophilic bacteria, Biochim. Biophys. Acta 65:506-508. Craig, W. S. and Kyte, J. (1980) Stoichiometry and molecular weight of the minimum asymmetric unit of canine renal sodium and potassium ion-activated adenosine triphosphatase, J. Biol. Chem. 255:62626269.(Medline) Cummins, J. and Hyden, H. (1962) Adenosine triphosphate levels and adenosine triphosphatases in neurons, glia and neuronal membranes of the vestibular nucleus, Biochim. Biophys. Acta 60:271-283. Fei, Y.J, Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K., Boron, W.F. and Hediger, M.A. (1994) Expression cloning of a mammalian proton-coupled oligopeptide transporter, Nature 368:563-566.(Medline) Freytag, J.W. (1983) The (Na+, K+) ATPase exhibits enzymic activity in the absence of the glycoprotein subunit, FEBS Lett. 159:280-284.(Medline) Glynn, I. M. (1957) The ionic permeability of the red cell membrane, Prog. Biophys. Mol. Biol. 8:241307. Guidotti, G.G. and Gazzola, G.C. (1993) Amino acid transporters: systematic approach and principles of http://www.albany.edu/~abio304/ref/ref20.html (1 of 4) [1/9/2003 12:14:21 PM]
Chapter 20: References
control, in Mammalian Amino Acid Transport: Mechanisms and Control (Kilberg, M.S. and Häussinger, D. ed.), Plenum Press, New York and London, pp.3-29. Hediger, M.A., Coady, M.J., Ikeda, T.S. and Wright, E.M. (1987) Expression cloning and cDNA sequencing of the Na+/glucose co-transporter, Nature 330:379-381.(Medline) Hoffman, J.F. (1962) Cation transport and structure of the red-cell plasma membrane, Circulation 26:1201-1213. Hokin, L. E. (1981) Reconstitution of "carriers" in artificial membranes, J. Membr. Biol. 60:7793.(Medline) Jorgensen, P. L., Hansen, O., Glynn, I. M. and Cavieres, J. O. (1973) Antibodies to pig kidney (Na+-K+)ATPase inhibit the sodium pump in human red cells provided they have access to the inner surface of the cell membrane, Biochim. Biophys. Acta 291:795-800.(Medline) Kyte, J. (1971) Purification of the sodium- and potassium-dependent adenosine triphosphatase from canine renal medulla, J. Biol. Chem. 246:4157-4165.(Medline) Kyte, J. (1974) Properties of the two polypeptides of sodium- and potassium-dependent adenosine triphosphatase, J. Biol. Chem. 247:7642-7649. Lai, Y. F. and Thomson, J. E. (1971) The preparation and properties of an isolated plant membrane fraction enriched in (Na+-K+)-stimulated ATPase, Biochim. Biophys. Acta 233:84-90.(Medline) Liu, W., Liang, R., Ramamoorthy, S., Fei, Y.J., Ganapathy, M.E., Hediger, M.A., Ganapathy, V. and Leibach, F.H. (1995) Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney, Biochim Biophys Acta 1235:461-466.(Medline) Lubin, M. (1964) Cell potassium and the regulation of protein synthesis. In, The Cellular Functions of Membrane Transport (Hoffman, J. F., ed.), pp. 193-209. Prentice-Hall, Englewood Cliffs, N. J. Lubin, M. and Ennis, H. L. (1964) On the role of intracellular potassium in protein synthesis, Biochim. Biophys. Acta 80:614-631. McDonough, A. A., Geering, K. and Farley, R. A. (1990) The sodium pump needs its beta subunit, FASEB J. 4:1598-1605.(Medline) McLennan, D. H. (1969) Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum, J. Biol. Chem. 245:4508-4515.
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Chapter 20: References
Meinild, A., Klaerke, D.A., Loo, D.D., Wright, E.M. and Zeuthen, T. (1998) The human Na+-glucose cotransporter is a molecular water pump, J. Physiol. (London) 508:15-21.(Medline) Meredith, D. and Boyd, C.A. (1995) Oligopeptide transport by epithelial cells, J. Membr. Biol. 145:112.(Medline) Mullins, L. J. and Brinley, F. J., Jr. (1967) Some factors influencing sodium extrusion by internally dialyzed squid axons, J. Gen. Physiol. 50:2333-2355.(Medline) Nakamoto, R.K. and Slayman, C.W. (1989) Molecular properties of the fungal plasma-membrane [H+]ATPase. J. Bioenerg. Biomembr. 21:621-632. (MedLine) Ohta, H., Matsumoto, J., Nagano, K., Fujita, M. and Nakao, M. (1971) The inhibitions of Na+, K+activated adenosine triphosphatase by a large molecule derivative of p-chloromercuribenzoic acid at the outer surface of the human red cell, Biochem. Biophys. Res. Commun. 42:1127-1133.(Medline) Pajor, A.M, Sun, N., Bai, L., Markovich, D. and Sule, P. (1998) The substrate recognition domain in the Na+/dicarboxylate and Na+/sulfate cotransporters is located in the carboxy-terminal portion of the protein, Biochim. Biophys. Acta 1370):98-106.(Medline) Panayotova-Heiermann, M., Eskandari, S., Turk, E., Zampighi, G.A. and Wright, E.M. (1997) Five transmembrane helices form the sugar pathway through the Na+/glucose cotransporter, J. Biol. Chem. 272:20324-20327.(Medline) Post, R. L., Merritt, C. R., Kinsolving, C. R. and Albright, C. D. (1960) Membrane adenosine triphosphatase as a participant in the active transport of sodium and potassium in the human erythrocyte, J. Biol. Chem. 235:1796-1802. Robinson, J.D. (1970) Interactions between monovalent cations and the (Na+K+)-dependent adenosine triphosphatase, Arch. Biochem. Biophys. 139:17-27.(Medline) Sachs, G., Chang, H. H., Rabon, E., Schackman, R., Lewin, M. and Saccomani, G. (1976) A nonelectrogenic H+ pump in plasma membranes of hog stomachs, J. Biol. Chem. 251:76907698.(Medline) Scarborough, G. A. (1980) Proton translocation catalyzed by the electrogenic ATPase in the plasmamembrane of neurospora, Biochemistry 19:2925-2931.(Medline) Schwegler, J.S., Sibernagl, S. and Tamarappoo, B.K. (1993) Amino acid transport in the kidney, in Mammalian Amino Acid Transport: Mechanisms and Control (Kilberg, M.S. and Häussinger, D., ed.), http://www.albany.edu/~abio304/ref/ref20.html (3 of 4) [1/9/2003 12:14:21 PM]
Chapter 20: References
Plenum Press, New York and London, pp. 233-260. Steel, A. and Hediger, M.A, (1998) The molecular physiology of sodium- and proton-coupled solute transporters, News Physiol. Sci. 13:123-130. Steel, A., Nussberger, S., Romero, M.F., Boron ,W.F., Boyd, C.A and Hediger, M.A (1997) Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1, J. Physiol. (London) 498:563-569.(Medline) Steinbach, H. B. (1963) Comparative biochemistry of the alkali metals. In, Comparative Biochemistry (Florkin, M, and Mason, H. S., eds.), Vol. 4, Part B, pp. 677-720. Academic Press, New York. Stevens, B.R. (1993) Amino acid transport in the intestine, in Mammalian Amino Acid Transport: Mechanisms and Control (Kilberg, M.S. and Häussinger, D., ed.) Plenum Press, New York and London, pp. 149-163. York. Tosteson, D. C. and Hoffman, J. F. (1960) Regulation of cell volume by active cation transport in high and low potassium sheep red cells, J. Gen. Physiol. 44:169-194. Whittam, R. (1962) The asymmetrical stimulation of a membrane adenosine triphosphatase in relation to active cation transport, Biochem. J. 84:110-118. Worrall, D.M. and Williams, D.C. (1994) Sodium ion-dependent transporters for neurotransmitters: a review of recent developments, Biochem. J. 297:425-436.(Medline) Wright, E.M., Loo, D.D., Panayotova-Heiermann, M., Lostao, M.P., Hirayama, B.H., Mackenzie, B., Boorer, K. and Zampighi, G. (1994) 'Active' sugar transport in eukaryotes, J. Exp. Biol. 196:197212.(Medline)
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21. Transport of Ions: Mechanisms and Models
21. Transport of Ions: Mechanisms and Models I. Coupling Between ATP Hydrolysis and Transport II. Synthesis of ATP by Transport ATPases III. Models of Ion Transport and Structure Suggested Reading References Back to List of Chapters Examining simple models and possible alternatives sometimes can provide insights into biological processes. This approach has proved very useful in sorting out the data on ion transport and their possible interpretation, and it provides the perspective of this chapter. Section I examines data obtained in a study of Na+, K+-ATPase and, for discussion, uses the model represented in Fig. 1 based on the experiments presented in Chapter 20. This model undoubtedly will require extensive modification and elaboration, but it is a useful summary. Very similar data are available from studies of the Ca2+-ATPase and a similar model could also be drawn for the transport of Ca2+. Section II examines some of the characteristics of the phosphorylation of ADP by inorganic phosphate, catalyzed by transport ATPases in the absence of ionic gradients. These phenomena may reveal some new features of the ATPases and perhaps have some bearing on our understanding of the synthesis of ATP by the ATP synthase of mitochondria, chloroplasts and bacteria. Section III concentrates on possible molecular mechanisms of ion transport and discusses the information gained from knowledge of the amino acid sequences and the reconstruction of the structure of the Ca2+-ATPase. I. COUPLING BETWEEN ATP HYDROLYSIS AND TRANSPORT The evidence reviewed in Chapter 20 unmistakably demonstrates that the active efflux of Na+ and the influx of K+ are coupled to the hydrolysis of ATP. As suggested in step 1 of Fig. 1, the coupling between the translocation of the ions and the hydrolysis of ATP may result from the required phosphorylation of the transporter molecule. The incubation of membrane preparations with ATP labeled with [32P] in its terminal position, labels the membranes. The phosphate, and not the whole ATP molecule, is incorporated since [14C]ATP does not label the membranes. Table 1 (Post et al., 1965) summarizes the incorporation of [32P] into kidney plasma membranes as a function of the cation present in the medium. When Na+ is present, the incorporation is highest, 97 pmoles/mg protein, compared to the incorporation in its absence (between 14 and 29 pmoles). Even in the presence of Na+, the incorporation may not seem http://www.albany.edu/~abio304/text/21part1.html (1 of 8) [3/5/2003 8:24:03 PM]
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very large. This is because the Na+,K+-ATPase is a minor component of the cell membrane (see below). Much higher values can be obtained for membrane fragments containing the Ca2+-ATPase, which represents a very large proportion of the total protein of the sarcoplasmic reticulum. Actually, in both cases the amount of [32P] incorporated corresponds to one per ATPase molecule. In step 1 of the model of Fig. 1, the phosphorylation of X produces Y~P. Different letters, X and Y, are used to denote the two forms because they have very different properties. Y is able to bind Na+ (step 2) and transfer it to the external membrane interface (step 3), from which it is released (step 4). X-P is generated from Y~P (step 5) and it binds K+ (step 6), transfers it to the internal membrane interface (step 7), and releases it to the cell's interior (step 8) with hydrolysis of X-P.
Fig. 1 Early model of the functioning of the Na+,K+-ATPase. The step corresponds to the following: step 1, the phosphorylation of the ATPase indicated as Y; step 2, the binding of Na+ to Y~P; step 3, the movement of the binding group from the cytoplasmic side of the membrane to the outside; step 4, the release of Na+; step 5, the hydrolysis of Y~P to form a different form of Y, X; step 6, the binding of K+; step 7, its displacement to the cytoplasmic side of the membrane; and step 8, its release into the cytoplasm.
Table 1 Effect of Monovalent Cations on Labeling of Kidney Membranes After Incubation with Mg2+ and [32P]ATP
Addition
Labelling (pmol 32P/mg protein)
None
26
Li+
20
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Na+
97
K+
16
NH4+
14
Rb+
18
Cs+
14
Tris+
19
Reproduced with permission from R.L. Post et al., J. Biol. Chem. 240:1437-1445. Copyright ©1965 The American Society for Biochemistry and Molecular Biology.
This scheme suggests that the formation of Y~P requires the presence of Na+, as shown by the results in Table 1. Other univalent cations cannot substitute. Fig. 2 shows the dependence of the phosphorylation expressed as % of the maximum (ordinate) on the concentration of Na+ (abscissa). The phosphorylation is related to transport, as shown by the inhibition of a large portion of the Na+-dependent phosphorylation by ouabain (Post et al., 1965) which blocks the transport of Na+ and K+. In Chapter 20 we saw that ouabain is an inhibitor of the Na+,K+-ATPase. The scheme also predicts that K+ would favor the hydrolysis of Y~P, as shown by the experiment represented in Fig. 3 (Post et al., 1965). In this experiment the membranes were first labeled with radioactive [32P]ATP. Then after the addition of unlabelled ATP, they were incubated in the presence of K+. Although the [32P] is released even in the absence of K+, the release is sharply accelerated when K+ is present. The rates of phosphorylation and dephosphorylation are comparable to those of the ATPase activity (e.g., Kyte, 1974) which, in turn, correspond very closely to the moles of ions being transported, as shown in Table 6 of Chapter 20. The nature of the phosphorylated ATPase has also been examined in relation to its sensitivity to ADP. The increased hydrolysis favored by K+ also appears in the results of the experiment of Fig. 4 (curve 1), carried out with the same protocol as in Fig. 3, but with the addition of K+ or ATP after a 5 min incubation (Post et al., 1965). In this experiment, K+ decreases the radioactivity as expected (curve 1), whereas the addition of ADP (curve 2) has no effect, suggesting that the phosphorylated ATPase is no longer in a high-energy form. In its high energy form, the phosphorylation of the enzyme would be reversible. The results are different when the ATPase has first been treated with N-ethylmaleimide (NEM), which http://www.albany.edu/~abio304/text/21part1.html (3 of 8) [3/5/2003 8:24:03 PM]
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reacts with sulfhydryl groups. Treatment of the ATPase with NEM, blocks the ATPase activity but not the phosphorylation. As shown in Fig. 5 (Post et al., 1965), the NEM-treated ATPase is not sensitive to K+ but is sensitive to ADP. These results suggest that the ATPase may be present in two distinct forms: a form with a high and another with a low phosphate group transfer potential, the latter corresponding to a K+-sensitive form. NEM blocks the conversion of the high energy form to the low-energy form. This scheme is consistent with the following reactions: Nai+ + E1 + ATP ↔ E1~P.Na+ (1) E1~P.Na+↔ E2-P + Nao+ (2) E2-P + Ko+ ↔ E2-P.K+ (3) E2-P.K+↔ E2 + K+i + Pi (4) where E represents the transporter molecule. The subscripts are used to distinguish the various molecular configurations of the enzyme; E1 and E2 correspond to the Y and X of Fig. 1, respectively.
Fig. 2 Effect of ouabain on the sensitivity of the [32P]-labeled intermediate to the concentration of sodium ion. The concentration of ouabain was 2.5 x 10-4 M, and that of Mg-ATP was 0.1 mM. Incubation was for 12 s at 23C. The results are the average of two experiments. Reproduced with permission from R. L. Post, et al., Journal of Biological Chemistry, 240:1437-1445. Copyright ©1965 The American Society for Biochemistry and Molecular Biology.
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Fig. 3 Influence of K+ on the rate of breakdown of the [32P]-labeled intermediate. Kidney membranes were stirred with 0.04 mM Mg-ATP labeled with [32P] for 2 min at 8.5C in the presence of 16 mM Na+ in a volume of 1.0 ml. ( ) K+ absent; ( ) K+ present at 0.04 mM. Then 0.1 ml of 20 mM unlabeled (Tris) ATP was added to reduce the specific activity of the labeled ATP to 2% of its initial value. After the time intervals on the horizontal axis the reaction was stopped with acid. The solid line indicates exponential disappearance with a time constant of 21 s. The dashed line is similar, with a time constant of 4 s. Reproduced with permission from R. L. Post, et al., Journal of Biological Chemistry, 240:1437-1445. Copyright ©1965 The American Society for Biochemistry and Molecular Biology.
The estimates of size of the molecule, together with estimates of the turnover number of the transport ATPase [i.e., moles of product x (moles of enzyme x minutes)-1], permit a number of interesting approximations. The turnover number was calculated to be about 12,000, based on the phosphate hydrolyzed. 1 mmol of Pi per hour is hydrolyzed from the ATP by 1 liter of cells. If we assume that there are 1.1 x 1013 cells per liter, there must be 1.3 x 10-22 moles of enzyme per cell. Multiplied by Avogadro's number (the number of molecules in one mole) this value corresponds to about 80 transporter molecules per cell. Assuming that the volume of each transporter molecule is 3.2 x 10-19 cm3, the total volume of transporter per cell is 80 X (3.2 x 10-19) = 2.6 x 10-17 cm3. The red blood cell surface area is about 1.55 X l0-6 cm2 and its thickness is approximately 5 nm, therefore, the volume of the membrane is about 0.78 x 10-12 cm3. Thus, the transport ATPase occupies about 0.0003% of the membrane volume, an extremely tiny portion of the cell membrane. In the red cell ghost, the Na+,K+-ATPase has been found by a cytochemical electron microscopic method (Charnock et al., 1972) to be distributed evenly over the membrane surface. However, other membranes are quite different. The Ca2+-ATPase is the major protein http://www.albany.edu/~abio304/text/21part1.html (5 of 8) [3/5/2003 8:24:03 PM]
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present in the sarcoplasmic reticulum. In addition, the distribution may not be even, the Na+, K+-ATPase of polar cells such as epithelial cells, is present only on one surface, the apical surface.
Fig. 4 Sensitivity of the phosphorylated intermediate of the native enzyme to ADP and K+. The ATPase was labeled with [32P]ATP. At zero time the radioactivity of the ATP was chased using a 100-fold excess of unlabeled ATP. From Post et al. (1969). Reproduced from The Journal of General Physiology, by copyright © permission of the Rockefeller University Press.
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Fig. 5 Sensitivity of the phosphorylated intermediate of the Na+,K+-ATPase to ADP and K+ after treatment with N-ethylmaleimide. From Post et al. Reproduced from The Journal of General Physiology, by copyright permission ©1969 of the Rockefeller University Press.
II. SYNTHESIS OF ATP BY TRANSPORT ATPases As we saw in Chapter 10, when ion pumps are run in reverse, ATP can be synthesized from ADP and Pi. These findings have certain implications related to the model of Fig. 1. ATP can be synthesized only if the phosphorylated form of Y(Y~P) is a high-energy form (high phosphate group transfer potential); i.e., the G for its hydrolysis is sufficiently low to support the synthesis of ATP from ADP. However, as described above, the evidence indicates that the usual phosphorylated form of the ATPase is hydrolyzed with the addition of K+, but not ADP. As already noted, a possible explanation is that there are two phosphorylated forms of the transporter molecule: a high-energy form involved in the transport of Na+ and a low-energy form that interacts with K+ (X-P). Furthermore, since X-P is a low-energy form, it should be possible to phosphorylate the molecule with Pi in the absence of Na+, and this was found to be the case (Post et al., 1965; Schoot et al., 1977; Sen et al., 1969). In the formulation of Fig. 1, Y~P would then be the precursor of X-P. The Na+,K+-ATPase is present in two forms, E1 and E2, which differ in conformation, as shown by a variety of techniques means such as exposure of regions of the molecule at the membrane surface to tryptic digestion (see below). The Y~P and X-P would then correspond, respectively, to the phosphorylated forms of E1 and E2 (Jorgensen and Petersen, 1979). Digestion of the http://www.albany.edu/~abio304/text/21part1.html (7 of 8) [3/5/2003 8:24:03 PM]
21. Transport of Ions: Mechanisms and Models
enzyme phosphorylated with either ATP or Pi, produces identical electrophoretic patterns (Bontig et al., 1979; Siegel et al., 1969). However, it is not necessary to have an ion gradient to synthesize ATP in the case of either the Na+,K+ATPase (Post et al., 1974) or the Ca2+-ATPase (Knowles and Racker, 1975). The in vitro synthesis is carried out in two steps. First, the ATPase is phosphorylated; we saw that this can be done in the case of the Na+,K+-ATPase by incubation with Pi. Then ATP is synthesized when ADP is added in the presence of a high concentration of Na+. Obviously, this proceeds only for a single turnover. The sequence of events perhaps can be understood best by examining the reactions in some detail. If K+ is ignored, the reactions would be as shown in Eqs. (5) to (7): E2 + Pi↔ E2-P (5) E2-P + Na+ ↔ E1P.Na+ (6) E1P.Na+ + ADP ↔ E1 + ATP + Na+ (7) These reactions represent the reverse of the normal sequence of active transport. The passage from Eq. (5) to Eq. (7) would be highly improbable unless the Na+ concentration was raised sufficiently, which is predictable from the law of mass action. However, as discussed more fully in Section III, the phosphorylation of the ATPase by ATP, presumably reaction (7) run from right to left, decreases the binding constant of the cation -- and the effect is reversible. When the binding of one component (e.g., the phosphorylation) to a protein capable of undergoing conformational change affects the binding of another (e.g., the cation), the inverse will be true. The nature of the enzyme-phosphate bond will thereby be affected by the binding of the cation (Weber, 1972, Weber, 1974). Presumably, the binding of Na+ would then convert the low-energy bond into a high-energy bond. The possibility of obtaining ATP from the reverse of ion transport can be explained by the considerations discussed in this section. The high Na+ present on the outside of the cell will permit the formation of the high-energy phosphate [reaction of Eq. (6)]. The phosphorylation of ADP removes the phosphate and the enzyme can be used again for another cycle of phosphorylation.
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21. Transport of Ions: Mechanisms and Models
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III. MODELS OF ION TRANSPORT AND STRUCTURE The simplest model for transport of an ion would include the following steps: (1) binding of the ion by specific binding groups on the transporter molecule at the loading site; (2) movement of the complex from one interface to the other; and, (3) release of the ion at the discharge site of the membrane. This model ignores a membrane potential to avoid complications unnecessary for our present discussion. The Ca2+ pump of the sarcoplasmic reticulum imports 2 Ca2+ and exports 1 H+per ATP hydrolyzed; the Na+/K+ pump exports from the cell 3 Na+ and simultaneously imports 2 K+ per ATP hydrolyzed. The model would carry out the net transport of the ion. Active transport, i.e., transport against an electrochemical gradient, could take place in this same model when two other conditions are met: (1) the affinity of the binding group changes from high at the loading interface to low at the discharge interface and (2) the free energy of the sequence of reactions decreases. In a transport ATPase the energy is provided by the coupled hydrolysis of ATP. Models capable of carrying active transport can be constructed without postulating a change in binding constants. However, all transport systems known have been shown to have this feature (see Table 2). For simplicity, in the present discussion we assume that the transport of all ions occurs by the same basic process. This approach is not unreasonable because, as we saw in Chapter 20, there is considerable evidence that the transport functions are analogous for the Na+,K+-ATPase, Ca2+-ATPase, the H+,K+ATPase and the H+-ATPase of plants and Neurospora. Furthermore, the properties of these molecules are very similar. A mechanism of active transport, involving the phosphorylation of the transporter and changes in binding constants, is supported by a variety of observations. The experiments discussed here (Ikemoto, 1976) were carried out with a stop-flow apparatus (Fig. 6), which delivers reactants and enzyme (from syringes shown at A) into the same chamber (B) with very rapid mixing in relation to the time course of the reaction. Then the flow is stopped, also very rapidly. The light absorption of the contents of the chamber can be recorded (E). An oscillosope (D) is required to record very rapid reactions. These experiments used a purified preparation of Ca2+-ATPase from the sarcoplasmic reticulum and the Ca2+ indicator Arsenazo III, which changes color when it binds Ca2+. The record of Fig. 7 represents the light absorption with time. The two sets of panels differ in the time scale: set I shows fast changes (intervals correspond to 50 ms) and set II shows slower changes (intervals represent 5 s). The downward deflections reflect increases in the concentration of Ca2+. The concentration of ATP added is shown at the left in the records. In the control (IA and IA), no ATP was added and no Ca2+ was released. The Ca2+
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released increases with the concentration of ATP added (compare B and D) until the system appears saturated (compare D and E), as would be expected because the amount of ATPase is finite. As shown by the longer time scale in set II, the release is temporary; eventually the Ca2+ is bound again, presumably when all the ATP is hydrolyzed. The results show that the ATPase binds Ca2+ and that activation by ATP reversibly decreases the binding. Fig. 8 shows the level of phosphorylation of the enzyme (determined after rapid filtration, curve 1) compared to the Ca2+ release (curve 2) calculated from Fig. 7. The two panels represent identical results plotted on two different time scales. The changes in phosphorylation of the ATPase precede the release of the Ca2+, suggesting that phosphorylation is responsible for the change in binding constants. These results indicate the Ca2+ is bound more tightly (larger binding constant) before activation. Similar data are available for other transport systems, such as Na+,K+-ATPase (Masui and Homareda, 1982; Yamaguchi and Tonomura, 1980). The binding constants on the two sides of the membrane for different transport systems are shown in Table 2 (Tanford, 1983).
Fig. 6 Stop-flow apparatus.
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Fig. 7 ATPase-coupled changes in Ca2+ binding to purified Ca2+-ATPase of the sarcoplasmic reticulum. From Ikemoto (1976), with permission.
The transport process seems to involve precise stoichiometry. As mentioned, 2 Ca2+ are transported per ATP hydrolyzed. Furthermore, 2 Ca2+ are bound per phosphorylated transporter molecule (Inesi et al., 1980). In the case of active transport, the affinity of the binding groups of the transporter for the ligand, decreases when the transporter molecule is phosphorylated and this lower affinity should represent the state of the transporter on the side with the higher concentration at steady state. A model of active transport involving ion binding sites and shuttling of ions across the plasma membrane is consistent with the data. However, these considerations do not resolve how the binding sites can move from one interface to the other without a major movement of the transporter. Integral proteins have distinct domains corresponding to the two different membrane surfaces. It follows that the transporter molecule does not flip or rotate. Furthermore, the Na+,K+-ATPase continues to function even when anchored at one interface with an antibody (Kyte, 1974). These difficulties could be resolved by proposing that the binding sites do not traverse the whole membrane thickness, but rather move over much shorter distances. This would be possible if the binding sites were inside a channel traversing the membrane. http://www.albany.edu/~abio304/text/21part2.html (3 of 16) [3/5/2003 8:24:10 PM]
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Fig. 8 Relationship between Ca2+ release and rebinding and the formation and decay of the phosphorylated intermediate. ( ) Ca2+ release; ( ) P in enzyme. Reproduced with permission from N. Ikemoto, Journal of Biological Chemistry, 251:7275-7277. Copyright ©1976 The American Society of Biological Chemistry and Molecular Biology.
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Table 2 Binding Constants for Transported Ions
Binding constant, Keq(M-1) Protein
Ion
Uptake side
Discharge side
SR Ca2+ pump
Ca2+
107-108
300
Na+ pump
Na+
4 X 103
<20
Chloroplast FoF1
H+
>108
<106
Na+/ Ca2+ exchange
Ca2+
2 x 105-106
400
Tanford (1983), reproduced with permission from the Annual Review of Biochemistry Vol. 52, copyright ©1983 by Annual Reviews Inc.
There are, in fact, many indications that the transporters can act as channels, at least when reconstituted in artificial membrane systems. Addition of purified Ca2+-ATPase to a bilayer (in this case a bilayer made of oxidized cholesterol) changes its conductivity (Shamoo and MacLennan, 1975). Under some conditions, the Na+, K+-ATPase incorporated into planar bilayers of phospholipid shows electrical conductance transitions typical of channels (Last et al., 1983). The anion transporter (Giebel and Passow, 1960) also behaves like a channel. In this case, the selectivity of the transport system appears to depend on the size of the molecule, suggesting a channel 0.8 to 0.9 nm in diameter. The channel behavior is related to the transport process of the native systems and not some irrelevant coincidence. This is shown by the sensitivity of the channel behavior to inhibitors of transport. HgCl2 inhibits both the Ca2+-ATPase activity and the Ca2+ conductivity in parallel. Ouabain and vanadate, both inhibitors of the Na+/K+ transport, inhibit the Na+, K+-ATPase channel behavior. Evidence of conformational rearrangements comes from many kinds of experiments. The sensitivity of the transporter molecule to proteolytic digestion differs at different stages of transport (e.g., see Chapter 4). Gresalfi and Wallace (1984) have examined the circular dichroism (CD) spectra of purified and membrane-attached Na+,K+-ATPase in its E1 and E2 forms, obtained by introducing either Na+ or K+. The spectra for the peptide backbone (190-240 nm) were consistent with extensive conformational differences between E1 and E2. The changes appear to be reversible when the ion composition is altered. Phosphorylation of the ATPase is accompanied by fluorescence changes of its tryptophan residues (Nakamura et al., 1994), indicating a conformational change. http://www.albany.edu/~abio304/text/21part2.html (5 of 16) [3/5/2003 8:24:10 PM]
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The involvement in transport of rearrangements within the ATPase is also shown by x-ray diffraction studies of packed membranes from the sarcoplasmic reticulum containing Ca2+-ATPase (Blasie et al., 1985). A significant portion of the ATPase juts into the cytoplasmic phase, as also shown by threedimensional reconstruction of negatively stained crystals in sarcoplasmic reticulum membranes (see below). Activation of the ATPase produces a conformational change with a displacement of the structure into the bilayer. Both the binding of Ca2+ (DeLong and Blasie, 1993; Cheong et al., 1996) and the phosphorylation of the enzyme (Blasie et al., 1985, Pascolini et al., 1988) were found to change the conformation of the enzyme as seen using X-ray diffraction. The experiments correlating specific steps in the transport used flash photolysis of caged ATP, a compound which releases ATP in response to a flash of light, assuring rapid and synchronous activation. Important details have been provided by other studies, some of them more recent. Ten transmembrane helices (M1 to M10, shown by the numbers in Fig. 9) have been proposed based on the amino acid sequence (MacLennan et al., 1985) and confirmed by high resolution EM (8-Å, Zhang et al., 1998). The functional portions of the cytosolic domains of the head region of the molecule are distinct. The P domain is involved in the phosphorylation, the N domain contains the nucleotide binding site and the A or actuator domain (also called the transducer domain) is thought to have a special role in the transduction (see Toyoshima et al., 2000; Toyoshima and Nomura, 2002). These are represented in the diagram of Fig. 9 and discussed below. Fig. 9 was drawn from models derived from X-ray diffraction data (Toyoshima et al., 2000). The more recent crystallographic study (Toyoshima and Nomura, 2002) with a resolution of 3.1 Å has provided data on the Ca2+-ATPase in its E2-state (with no Ca2+, but protonated with 2 H+).The conformation of E2 was found to differ from that of E1 (with 2 bound Ca2+) as follows. In E1 the three domains (P, N and A domains) are widely separated. In E2 they form a compact structure as shown in Fig. 9. In addition, six out of the ten transmembrane segments also undergo conformational changes when assuming the E2 conformation. These changes might be required for the release of the Ca2+ into the SR lumen by opening a channel for the passage of Ca2+ and the permitting the counter-transport of 2 H+ to cytoplasm in exchange for 1 Ca2+ per 1 ATP hydrolyzed .(see fig. 2 of Green and MacLennan, 2002) as indicated in the model of Fig. 16 below. The structure deduced for the Ca2+-ATPase allowed the construction of an atomic homology model of the H+-ATPase of Neurospora by comparing it to an 8 Å map of the Neurospora proton pump derived from electron microscopy (Kühlbrandt et al., 2002). The model, shows the probable path of the proton through the membrane and indicates that the nucleotide-binding domain rotates by approximately 70o to deliver ATP to the phosphorylation site of the ATPase. This model differs somewhat from that proposed for the Ca2+-ATPase.
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align="center"> Fig. 9 Schematic representations of the Ca2+-ATPase in the Ca2+-binding configuration, based on the reconstruction of Toyoshima et al. (2000). The red P in the P-domain represents the phosphorylated site. The yellow oval represent the site of nucleotide binding in the N-domain. The ten helices traversing the membrane are represented by cylinders and the two red dots represent the bound Ca2+.
In summary, it appears that the transport of ions proceeds by binding the ions to specific sites. These sites are probably present in a channel of the transporter that traverses the membrane. The translocation is associated with some movement of the binding sites, so that the sites are exposed first to one, and then to the other side of the membrane and major rearrangments of the large cytoplasmic domains of the transporter. How can this information be put together in a single model? The presence of a conventional channel would only allow passive flow in the direction of the gradient and could not carry out transport against an electrochemical gradient. For this reason, the models generally considered propose alternating access (see Fig. 10), in which a small conformational change (in this case a rotation) exposes the binding sites first to the water phase on one side of the membrane, and then to the water phase on the other side (Tanford, 1983). The channel would remain closed at all times, but would alternate using two different http://www.albany.edu/~abio304/text/21part2.html (7 of 16) [3/5/2003 8:24:10 PM]
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"gates", comparable to the gates in a lock connecting two bodies of water of different heights. The movement of the binding groups could increase the distance between them, as represented in the diagram and, therefore, could also account for the change in the affinity for the transported ion. During the working cycle of the alternating access pump, when both "gates" are closed, the ion is unavailable for exchange. The ion in the transporter molecule is said to be occluded. Occlusion suggests the presence of an intermediate position of the binding sites, apart from their location at either the uptake or the discharge site (see Glynn and Karlish, 1990). An alternating access model of the Ca2+-ATPase is shown in Fig.16.
Fig. 10 Representation of the alternate-access model of transport. The structures represent polypeptide chains traversing the phospholipid bilayer of the plasma membrane. The circles indicate the binding sites of the transported ion. The closeness of the binding groups on the left accounts for the high-affinity binding, the separation on the right for the decrease in affinity. The slight rotation of the polypeptides accounts for the access of the binding sites from either the uptake site (left) or the discharge site (right).
Mutational studies have identified amino acids critical for transport (see MacLennan et al., 1997). Sitespecific mutagenesis substitutes amino acids at defined locations in the molecule and delineates the functional role of amino acids or amino acid clusters in the transport. In some of these experiments, mutant DNA was incorporated into COS cells, a transformed simian cell line, using a vector and then assayed for function (see MacLennan, 1990). These studies have identified amino acids critical for transport (see MacLennan et al., 1997). Negatively charged residues in M4, M5, M6 and M8 are thought to constitute high affinity Ca2+-binding site (Clarke et al., 1989). The two Ca2+-binding sites are formed by the juxtaposition of acidic and oxygen containing amino acids next to each other in the middle of the four transmembrane helices (Clarke et al., 1989; see Andersen, 1995 and MacLennan et al., 1997) as represented in Fig. 15. Small changes in the position of the helices forming this cluster would disrupt these binding sites. The study of (Toyoshima et al., 2000) suggests the pathway lined by oxygen atoms, allowing for the in-and-out passage of Ca2+ and shows the disruption of structure of the M4 and M6 helices to provide a Ca2+-binding cavity. In addition, they identified mutation-sensitive carbonyl groups in the M4 helix. http://www.albany.edu/~abio304/text/21part2.html (8 of 16) [3/5/2003 8:24:10 PM]
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The properties common to at least some of the transport systems are summarized in Table 3. Some of these examples correspond to active transport, others do not. For all transport systems, the transporter binds the transported substrate. Furthermore, the transporters have been shown to undergo a conformational change. Channel behavior has been shown, at least under some conditions, for some of the transporters. Table 3 Summary of the Properties of Some Transport Systemsa
Ion or Solute
Anion
Active Transport
Binding demonstrated
Channel Properties
Conformational change of transporter
No
Yesb
Yesc
Yesd
Yese
Yes
Yesf
Yesg
Yesh
Yesi
Yesj
Yes
Exchanger Na+, K+ (ATPase)
(Table 2) Ca2+
Yes
(Table 2)
(ATPase) H+
Yes
Yes (Table 2)
(ATP synthase) Na+-glucose
Yes
(see Chapter 17)
Yes
--
--
Yesk
Yes
--
--
Yesl
(cotransporter) Na+-amino acid (cotransporter)
a
Yes indicates that the phenomenon has been observed for the solute or the transporter b Falke et al. (1984b); c Giebel and http://www.albany.edu/~abio304/text/21part2.html (9 of 16) [3/5/2003 8:24:10 PM]
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Passow (1960); d Falke et al (1984b);e Yamaguchi and Tonomura (1980);f Last et al. (1983); gJorgensen (1975); Karlish and Yates (1978); Koepsell (1972) h Shamoo and MacLennan (1975) i Imamura et al. (1984); j Tanford (1983); k Peerce and Wright (1984);l Wright and Peerce, 1984.
Many years of collected evidence support the alternate access model represented in Fig. 10. This model, adapted to reflect the various experimental findings for the Ca2+-ATPase, is shown in Fig. 11. In this figure, 2 Ca2+ are shown to be bound sequentially. One of these is not readily accessible from either side of the ATPase-channel (occlusion). ATP phosphorylates the ATPase so that the two Ca2+ are released sequentially. In this figure the stripes indicate the Ca2+ which is bound to the ATPase first (reaction 1-2) and the dotted circles represent the second Ca2+ bound in reaction 3. As shown, the first Ca2+ is not readily available from the outside or from inside the vesicle. It will equilibrate slowly with Ca2+ in the medium. However, phosphorylation of the enzyme (reaction 4) allows the sequential discharge of Ca2+ to the inside of the vesicles: the first Ca2+ to be bound is released into the vesicles first (reaction 5); the second Ca2+ to be bound is released second (reaction 7) and corresponds to the Ca2+ which is readily exchangeable with 40[Ca2+] before phosphorylation. An alternate access model for the Ca2+-ATPase highlighting the structural aspects is shown in Fig. 16 (Inesi, 1987).
Fig. 11 Diagram representing the sequential mechanism of calcium binding and translocation upon ATP hydrolysis by SR ATPase. From Inesi, 1987. Reproduced by permission. Copyright ©1987 The American Society for Biochemistry and Molecular Biology.
Inesi (1987) explored details of the Ca2+-ATPase mediated transport of the SR with a pulse chase technique. In one experiment, SR vesicles containing Ca2+-ATPase were first equilibrated with the radioactive isotope [45Ca]2+. This incubation was then followed by a chase with nonradioactive [40Ca]2+. The time course of the release of the labelled Ca2+ at low temperature is shown in Fig. 12 (Inesi, 1987).
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In this figure, bound radioactive Ca2+ in the ordinate is shown as a function of the time after addition of the nonradioactive Ca2+. The total initial binding corresponds to 2 Ca2+ per enzyme molecule. A rapid initial release, over in about 0.2 s, is followed by a much slower one, which cannot be seen with the time scale used. The vastly different rates of release are in agreement with the sequential model of Fig. 11. The faster release corresponds to the Ca2+ which is bound second and readily accessible from the outside medium. La3+ displaces all bound Ca2+, so that in the presence of ATP, any Ca2+ not displaced by La3+ represents Ca2+ which has been occluded or transported into the vesicle. The relationship between translocation and binding was examined in an experiment whose results are represented in Fig. 13. The experimental design is shown diagrammatically on the left side of the figure. Curve A represents results obtained without a chase. [45Ca]2+ was first bound to the ATPase of the vesicles and ATP added subsequently. La3+ was added at the various times indicated in the abscissa. The Ca2+ translocated into the vesicle is first very rapid, corresponding to the translocation of Ca2+ initially bound to the ATPase. This is followed by a slower transport that represents the Ca2+ subsequently transported into the vesicle. When ATP is added simultaneously to a chase with [40Ca]2+, the amount transported (curve B) corresponds exactly to that bound (2 Ca2+/enzyme); no additional translocation of the radioactive Ca2+ can take place because of the chase. As indicated by Fig. 12, a chase of 0.2 s with [40Ca]2+ removes the molecule of Ca2+ that was bound second by the ATPase. The transport of the remaining Ca2+ ion (the first to be taken up) can, therefore, be followed by introducing ATP after a 0.2 s chase with the non-radioactive Ca2+ (curve C). After the 0.2 seconds chase, only half of the radioactive Ca2+ was transported into the vesicle (curve C). This indicates that the Ca2+ which occupies the position closer to the outside, is transported first into the vesicle, as predicted from a sequential model of Fig. 11.
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Fig. 12 Isotopic exchanges of bound Ca2+. From Inesi, 1987. Reproduced by permission. Copyright ©1987 The American Society for Biochemistry and Molecular Biology.
Fig. 13 Quench-flow measurements of ATP-dependent calcium uptake. From Inesi, 1987. Reproduced by permission. Copyright ©1987 The American Society for Biochemistry and Molecular Biology.
The Ca2+ uptake of the initial burst (Fig. 13A) may include Ca2+ that is not exchangeable and is trapped in the ATPase, i.e. occluded. A different experimental design can differentiate between bound Ca2+ and occluded Ca2+. When ADP is added in the presence of a Ca2+-chelator [ethylene glycol-bis-(βaminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA)], the phosphorylation of the transporter is reversed. ADP is phosphorylated and the occluded Ca2+ is released into the medium. Only one single cycle of the enzyme is possible because there is no Pi present. In contrast, the Ca2+ transported into the vesicles would be retained (and would not be released by La3+). The results of this experiment are shown in Fig. 14, which shows the radioactive Ca2+ uptake in the ordinate. The time shown in the abscissa represents the time of addition of ADP + EGTA which is then followed by the addition of La2+. In curve A, the preparation is preincubated in [45Ca]2+. Then [40Ca]2+ and ATP are added simultaneously. In this case, the Ca2+ uptake after the ADP+EGTA addition represents the transported Ca2+ (amount taken up + amount occluded). At the earlier times of addition of ADP + EGTA, 4 to 5 nanomoles of Ca2+ are taken up per mg, compared to 9 to 10 without the ADP + EGTA treatment (Fig. 14A). Therefore, approximately half of the original Ca2+ taken up is in the occluded form. When ATP is added after the 0.2 s of [40Ca]2+ chase (which removes the more external Ca2+) (Fig. 14B), half of the Ca2+ uptake has already become ADP + EGTA insensitive. This shows that the insensitive Ca2+ (released into the vesicles) is the one that was bound first (see Fig. 11). These results elaborate and support the alternatingsite model and indicate a sequential release of the Ca2+.
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Much the same information is available for the Na+, K+-ATPase from entirely different experiments. As we have seen, 3 Na+ and 2 K+ bind to separate sites of the protein. First they become occluded (i.e., trapped inside the transporter) and then are released to the other side (see Post et al., 1972; Beaugé and Glynn, 1979). In the absence of K+, Na+ is still translocated (Garrahan and Glynn, 1967) and the translocation is electrogenic (Fendler et al., 1985; Nakao and Gadsby, 1986). The electrical signal during the ion pumping corresponds to the movement of the ions across the channel that traverses the membrane (e.g., Hilgemann, 1994) and is associated with charge movements. The rate of these electrogenic reactions is dependent on the membrane potential, so that enzymes conformations can be shifted. High speed voltage jumps can be used to initiate this redistribution. Three phases are apparent (Holmgren et al., 2000), reflecting the de-occlusion of the three ions. The results indicate that three are released one at a time, in order.
Fig. 14 ADP reversal of ATP-induced calcium translocation. From Inesi, 1987, reproduced by permission.
Analyses of the phosphorylating reactions were also carried out. Either ATP or Pi can phosphorylate the enzyme. The ATP phosphorylation depends on high affinity Ca2+ binding. In contrast, the phosphorylation by Pi is blocked by Ca2+. The Ca2+ occlusion was studied on detergent solubilized SR vesicles in the presence of CrATP. CrATP allows occlusion without the hydrolysis of ATP and it also stabilizes the Ca2+-enzyme complex. A HPLC-molecular sieve procedure was used (see Chapter 1) to separate the proteins from free Ca2+. Mutations at the sites thought to bind Ca2+, prevented occlusion (Vilsen and Andersen, 1992, Andersen and Vilsen, 1994).
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Summary of amino acid substitution introduced into the predicted Ca2+-binding domain. Glu309, Glu771, Asn796, Thr799, Asp800, and Glu908 are thought to be in the transmembrane segments M4, M5, M6 and M8 respectively. From Clarke et al., 1990b. Reproduced by permission.
As already discussed, there is considerable evidence that the ATPase pumps require a channel-like structural arrangement. Modeling of the four helices thought to be involved in Ca2+ binding and which are amphiphilic, show that polar and charged residues are predominantly in one face of each helix with the hydrophobic residues in the opposite face. The hydrophilic components could therefore form hydrophilic clusters in the internal surfaces, thereby forming a channel. The hydrophobic residues, on the other hand, could interact with the bilayers providing the transmembrane arrangement. Present information (e.g., Inesi et al., 1992; Toyoshima et al. 2000) indicates that the Ca2+-binding domain and the catalytic domain are separated by 50 Å. This spatial arrangement would require that any interaction would be indirect, via a conformational change. We have seen that conformational changes have been demonstrated (see Fig. 9). A possible mechanism for the transport of Ca2+ involving the http://www.albany.edu/~abio304/text/21part2.html (14 of 16) [3/5/2003 8:24:10 PM]
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transmembrane helices is indicated in Fig. 16 (MacLennan, 1990). The shift from E1 to the E2 form accompanying phosphorylation, would shift the negatively charged binding groups from the outer to the inner interface. Furthermore, the conformational shift would disrupt the arrangement of the high affinity binding groups to produce low affinity binding sites.
Fig. 16 Model illustrating the possible mechanism of Ca2+ transport by the Ca2+-ATPase. In the E1 configuration, high affinity Ca2+-binding sites are accessible to the cytoplasmic Ca2+. ATP hydrolysis induces the E2 configuration, in which the access of the binding groups from the cytoplasmic side is blocked and their configuration of the binding groups is disrupted. The disruption results in a low affinity binding. From MacLennan, 1990, reproduced by permission.
SUGGESTED READING Inesi, G., Zhang, Z., Sagara, Y. and Kirtley, M.E. (1994) Intracellular signaling through long-range linked functions in Ca2+ ATPase, Biophys. Chem. 50:129-138. (Medline) MacLennan, D.H., Rice, W.J. and Green, N.M. (1997) The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases, J. Biol. Chem. 272:28815-28818. (MedLine) Stein, W.D. and Lieb, W.R. (1986) Transport and Diffusion Across Cell Membranes, Chapter 6, pp. 475-
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612. Academic Press, New York. Tanford, C. (1984) The sarcoplasmic reticulum calcium pump. Localization of free energy transfer to discrete steps of the reaction cycle, FEBS Lett. 166:1-7. (Medline) General Reviews Inesi, G.(1994) Teaching active transport at the turn of the twenty first century: recent discoveries and conceptual changes, Biophys. J. 66:554-560. http://www.biosci.umn.edu/biophys/OLTB/BJ/Inesi.pdf Adobe Acrobat from www.adobe.com is required for reading pdf files. Lauger, P. (1984) Channels and multiple conformational states: interrelations with carriers and pumps, Curr. Top. Membr. Transport 21:309-326. REFERENCES Search the textbook
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Chapter 21: References
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REFERENCES Andersen, J.P. and Vilsen, B. (1994) Amino acids Asn796 and Thr799 of Ca2+-ATPase of sarcoplasmic reticulum bind Ca2+ at different sites, J. Biol. Chem. 269:15931-15936. (MedLine) Beaugé, L.A. and Glynn, I.M. (1979) Occlusion of K ions in the unphosphorylated sodium pump, Nature 280:510-512. (Medline) Blasie, J. K., Herbette, L. G., Pascolini, D., Skita, V., Pierce, D. H. and Scarpa, A. (1985) Time resolved x-ray diffraction of sarcoplasmic reticulum membrane during active transport, Biophys. J. 48:9-l8. (Medline) Bontig, S. I., Schuurmans Stekhoven, F. M. A. H., Swarts, H. G.P. and dePont, J. J. H. H. M. (1979) The low-energy phosphorylated intermediate of Na+,K+-ATPase. In Na,K-ATPase, Structure and Kinetics (Skou, J. C., and Nφrby, J. G., eds.), pp. 317-330. Academic Press, New York. Charnock, J. S., Trebilcock, H. A. and Casley-Smith; J. R. (1972) Demonstration of transport adenosine triphosphatase in the plasma membranes of erythrocyte ghosts by quantitative electron microscopy, J. Histochem. Cytochem. 20:1069-1080. (Medline) Cheong, G.W., Young, H.S., Ogawa, H., Toyoshima, C. and Stokes, D.L. (1996) Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum, Biophys. J. 70:1689-1699. (MedLine) Clarke, D.M., Maruyama, K., Loo, T.W., Leberer, E., Inesi, G. and MacLennan, D.H. (1989) Functional consequences of glutamate, aspartate, glutamine and aspargine mutations in the stalk section of the Ca2+ATPase of sarcoplasmic reticulum, J. Biol. Chem. 264:11246-11251. (Medline) Clarke, D.M., Loo, T.W. and MacLennnan, D. (1990a) The epitope for monoclonal antibody A20 (amino acids 870-890) is located in the luminal surface of the Ca2+-ATPase of sarcoplasmic reticulum, J. Biol. Chem. 265:17405-17408. (Medline) Clarke, D.M., Loo, T.W. and MacLennnan, D. (1990b) Functional consequences of alterations to amino acids located in the nucleotide binding domain of the Ca2+-ATPase, of sarcoplasmic reticulum, J. Biol. Chem. 265:6262-6267. (Medline)
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DeLong, L.J. and Blasie, J.K. (1993) Effect of Ca2+ binding on the profile structure of the sarcoplasmic reticulum membrane using time-resolved x-ray diffraction, Biophys. J. 64:1750-1759. (Medline) Fendler, K., Grell, E., Haubs, M. and Bamberg, E. (1985) Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes, EMBO J. 4:3079-3085. (Medline) Garrahan, P.J. and Glynn, I.M. (1967) The behaviour of the sodium pump in red cells in the absence of external potassium, J. Physiol. (London) 192:159-174. (MedLine) Giebel, O. and Passow, H. (1960) Die permeabilität der eythrocytemembran für organische anionen, Pfluegers Arch. 271:378-388. Glynn, I. M. and Karlish, S. J. D. (1990) Occluded cations in active transport, Annu. Rev. Biochem. 59:171-205. (Medline) Green, N.M. and MacLennan, D.H. (2002) Calcium callisthenics, Nature 418:598-599. Gresalfi, T.J. and Wallace, B.A. (1984) Secondary structural composition of the Na/K-ATPase E1 and E2 conformers, J. Biol. Chem. 259:2622-2628. (MedLine) Hilgemann, D.W. (1994) Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches, Science 263:1429-1432. (Medline) Holmgren, M., Wagg, J., Bezanilla, F., Rakowski, R.E., De Weer, P. and Gadsby, D.C. (2000) Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase, Nature 403:898-901. (Medline) Ikemoto, N. (1976) Behavior of Ca2+ transport sites linked with the phosphorylation reaction of ATPase purified from the sarcoplasmic reticulum, J. Biol. Chem. 251:7275-7277. (Medline) Inesi, G. (1987) Sequential mechanism of calcium binding and translocation in sarcoplasmic reticulum adenosine triphosphatase, J. Biol. Chem. 262:16338-16342. (Medline) Inesi, G., Kurzmack, M., Coan, C. and Lewis, E. (1980) Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles, J. Biol. Chem. 255:3025-3031. (Medline) Inesi, G., Lewis, D., Nikic, D. and Kirtely, M.E. (1992) Long range intramolecular linked functions in calcium transport ATPase, in Advances in Enzymology. Meister, A., ed.. Wiley and Sons, New York, pp. 185-215. (Medline)
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Inesi, G., Zhang, Z., Sagara, Y. and Kirtley (1994) Intracellular signaling through long-range linked functions in Ca2+ ATPase, Biophys. Chem. 50:129-138. (Medline) Jorgensen P. L., and Petersen, J. (1979) Protein conformations of the phosphorylated intermediates of purified Na+,K+-ATPase studied with tryptic digestion and intrinsic fluorescence as tools. In Na+,K+ATPase Structure and Kinetics (Skou, J. C., and Nφrby, J. G., eds.), pp. 143-155. Academic Press, New York. Knowles, A. F. and Racker, R. (1975) Formation of adenosine triphosphate from Pi and adenosine triphosphate by purified Ca2+-adenosine triphosphatase, J. Biol. Chem. 250:1949-1951. (Medline) Kühlbrandt, W., Zeelen, J. and Dietrich, J. (2002) Structure, mechanism, and regulation of the Neurospora plasma membrane H+-ATPase, Science 297:1692-1696. (MedLine) Kyte, J. (1971) Purification of the sodium- and potassium-dependent adenosine triphosphatase from canine renal medulla, J. Biol. Chem. 246:4157-4165. (Medline) Kyte, J. (1974) The reactions of sodium and potassium ion activated adenosine triphosphatase with specific antibodies, J. Biol. Chem. 249:3652-3660. (Medline) Kyte, J. (1981) Molecular considerations relevant to the mechanism of active transport, Nature 292:201204. (Medline) Last, T. A., Gantzer, M. L. and Tyler, C. D. (1983) Ion-gated channel induced in planar bilayers by incorporation of (Na+,K+)-ATPase, J. Biol. Chem. 258:2399-2404. (Medline) MacLennan, D. H. (1990) Molecular tools to elucidate problems in excitation-contraction coupling, Biophys. J. 58:1355-1365. (Medline) MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1985) Amino-acid sequence of a Ca2+ + Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence, Nature 316:696-700. (MedLine) MacLennan, D.H., Rice, W.J. and Green, N.M. (1997) The mechanism of Ca2+ transport by sarco(endo)plasmic reticulum Ca2+-ATPases, J. Biol. Chem. 272:28815-28818. (MedLine) Masui, H. and Homareda, H. J. (1982) Interaction of sodium and potassium ions with Na+,K+-ATPase. I. Ouabain-sensitive alternative binding of three Na+ or two K+ to the enzyme, J. Biochem. 92:193-217.
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Nakamura, S., Suzuki, H. and Kanazawa, T. (1994) The ATP- induced change in tryptophan fluorescence reflects a conformational change upon formation of ADP-sensitive phosphoenzyme in the sarcoplasmic reticulum Ca2+-ATPase, J. Biol. Chem. 269:16015-16019. (Medline) Nakao, M. and Gadsby, D.C. (1986) Voltage dependence of Na translocation by the Na/K pump, Nature 323:628-630. (Medline) Pascolini, D., Herbett, L.G., Skita, V., Asturias, F., Scarpa, A. and Blasie, J.K. (1988) Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to the E1P at 16 resolution via time-resolved X-ray diffraction, J. Biophys. 54:679-688. (Medline) Peerce, B.E. and Wright, E.M. (1984) Sodium-induced conformational changes in the glucose transporter of intestinal brush borders, J. Biol. Chem. 259:14105-14112. (Medline) Post, R.L., Hegyvary, C. and Kume, S. (1972) Activation by adenosine triphosphate in the phosphorylation kinetics of sodium and potassium ion transport adenosine triphosphatase, J. Biol. Chem. 247:6530-6540. (Medline) Post, R. L., Sen, A. K. and Rosenthal, A. S. (1965) A phosphorylated intermediate in adenosine triphosphate-dependent sodium and potassium transport across kidney membranes, J. Biol. Chem. 240:1437-1445. Post, R. L., Kume, S., Tobin, T., Orgutt, R., and Shu, A. K. (1969) Flexibility of an active center in sodium plus potassium adenosine triphosphatase, J. Gen. Phys. 54:306s-326s. Post, R. L., Taniguchi, K. and Toda, G. (1974) Synthesis of adenosine triphosphate by Na+,K+-ATPase, Ann. N.Y. Acad. Sci. 242:80-91. (Medline) Schoot, B. M., Schoots, A. F. M., dePont, J. J. H. H. M., Schuurmans Stekhoven, F. M. A. H., and Bonting, S. L. (1977) Studies on (Na+-K+) activated ATPase. XVI. Effects of N-ethylmaleimide on overall and partial reactions, Biochim. Biophys. Acta 483:181-192. (Medline) Sen, A., Tobin, T. and Post, R. L. (1969) A cycle for ouabain inhibition of sodium- and potassiumdependent adenosine triphosphatase, J. Biol. Chem. 244:6596-6604. (Medline) Shamoo, A. and MacLennan, D. H. (1975) Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca2+ Mg2+)-ATPase of sarcoplasmic reticulum, J. Membr. Biol. 25:6574. (Medline) Siegel, G. J., Koval, G. J. and Albers, R. W. (1969) Sodium- potassium-activated adenosine http://www.albany.edu/~abio304/ref/ref21.html (4 of 5) [3/5/2003 8:24:14 PM]
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triphosphatase, J. Biol. Chem. 244:3264-3269. (Medline) Tanford, C. (1983) Mechanism of free energy: coupling in active transport, Annu. Rev. Biochem. 52:379409. (Medline) Tokoshima, M., Sasabe, H. and Stokes, D.L. (1993) Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane, Nature 362:469-471. Toyoshima, C., Nakasako, M., Nomura,H. and Ogawa, H. (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 Å resolution, Nature 405:647-655. (MedLine) Toyoshima C. AND Nomura, H. (2002) Structural changes in the calcium pump accompanying the dissociation of calcium, Nature 418:605-611. (MedLine) Vilsen, B. and Andersen, J.P. (1992) CrATP-induced Ca2+ occlusion in mutants of the Ca2+-ATPase of the sarcoplasmic reticulum, J. Biol. Chem. 267:25739-25743. (Medline) Weber, G. (1972) Ligand binding and internal equilibria in proteins, Biochemistry 11:864-878. (Medline) Weber, G. (1974) Addition of chemical and osmotic energies by ligand protein interactions, Ann. N.Y. Acad. Sci. 227:486-496. (Medline) Wright, E.M. and Peerce, B.E. (1984) Identification and conformational changes of the intestinal proline carrier, J. Biol. Chem. 259:14993-14996. (Medline) Yamaguchi, M. and Tonomura, Y. (1980) Binding of monovalen cations to Na+,K+-dependent ATPase purified from porcine kidney, J. Biochem. 88:1365-1375. (Medline) Yu, X., Carroll, S., Rigaud, J.L., and Inesi, G. (1993) H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes, Biophys. J. 64:1232-1242. (MedLine) Zhang, P., Toyoshima, C., Yonekura, K., Green, N.M., and Stokes, D.L. (1998) Structure of the calcium pump from sarcoplasmic reticulum at 8-Å resolution, Nature 392:835-839. (MedLine)
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22. Excitation and Conduction I. Neurons: Units of Conduction II. Ionic Origins of the Resting Potential III. Dynamics of the Membrane Potential A. Ionic Basis of Depolarization B. Membrane Mechanisms: Voltage Clamping C. Molecular Mechanisms and Channels IV. Electrogenic Pumps V. Transmission of Excitation Between Cells A. The Synapse B. Neurotransmitters: Discharge and Recovery Involvement of Ca2+ Role of the cytoskeleton and the synapsins Membrane fusion Recycling and recovery Nitric Oxide VI. Plasticity of Synapses Potentiation and depression Basic aspects of potentiation The mammalian system Cell adhesion molecules Increase in synaptic connections VII. Neurotrophins Suggested Reading References Back to List of Chapters Signals between cells and within cells are necessary for most physiological functions. These signals are composed of chemical and electrical events of some complexity. In one way or another, signaling frequently makes use of the electrochemical gradient across the cell membrane. This is the case not only for excitable cells, the primary topic of this chapter, but also for http://www.albany.edu/~abio304/text/22part1.html (1 of 28) [1/9/2003 12:15:12 PM]
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other cells. For example, the entry of Ca2+ from either the medium or internal stores, driven by the Ca2+ electrochemical gradient, has a role in the release of secretory products and in the release of neurotransmitters, which transmit signals between nerve cells. Electrical signals are also coupled to the release of Ca2+ needed for the contraction of muscle or less organized contractile systems (seeChapter 23). Resting membrane potentials are present in most cells, and even in some plant cells dynamic electrical changes take place on stimulation (discussed later in this chapter). The precise physiological role of these electrical events in plants is still not clear. Nerve cells (and muscle cells) are specialized for conducting signals through the transmission of an electrical event (the impulse or action potential). A variety of stimuli are effective in initiating impulses. Perhaps the most direct way to elicit a nerve impulse in the laboratory is by means of an electrical current of an intensity above a critical level (the threshold). Once initiated, the nerve impulse can be propagated without loss of intensity over the entire length of the nerve cell, which can be 1 m long or even longer. Although generally similar events underlie the conduction of impulses, even in nerves, the speed of conduction varies widely. Some mammalian nerve fibers can conduct with speeds as high as l00 m/s (over 200 miles per hour!), while others are much slower, with a conduction rate as low as 0.1 m/s. The response to stimulation of nerves can be relatively direct and readily observable. One stimulating event in a motor nerve may result in one contraction of a striated muscle. However, the response may also be complex and subtle. This is particularly true where nerve cells interact, as in ganglia or in the central nervous system, or where the effector cell responds in a complex manner (e.g., smooth muscle does not respond with a single contraction). I. NEURONS: UNITS OF CONDUCTION Many kinds of cells can propagate an electrical event or an electrical impulse. However, nerve cells (neurons) function primarily to transmit impulses. Some of the kinds of neurons encountered in the cerebellum are shown in Fig. 1 (Mugnaini and Floris, 1994). In general, different shapes are related to different functions of these neurons, such as, where they receive inputs and send their output signals. Many neurons have a characteristic stellate shape. The cell has many small branches, the dendrites, and a long process, the axon. Generally, the cell bodies are present in ganglia or in the central nervous system. The axon, a nerve fiber, is the portion that conducts the nerve impulse over long distances.
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Fig. 1 Cells present in a section of mammalian cerebellar cortex shown to illustrate the variety of sizes and shapes of neurons and associated cells. PC = Purkinje cell: this is an inhibitory output neuron. Arrows= granular cells: the only excitatory cells present, GC = Golgi cell: these are inhibitory cells that can shut the granular cell excitatory output, LC = Lugaro cells: their function is not clear at this time; pa and fa are astrocytes (from Mugnaini and Floris, 1994). Reproduced by permission.
As shown in Fig. 1, neurons assume many shapes and forms. The size and morphology of the cell body (soma) of neurons vary widely. In the mammalian retina alone, a minimum of twenty two distinct cell types have been found (MacNeil and Masland, 1998). Globulus cells of invertebrates can be smaller than 3 µm in diameter. However, neurons can also be huge: for example, in gastropods they can be larger than 800 µm and therefore visible with the naked eye. Even within the same organism the variation in size and shape is surprising. Generally, the smaller cells have very little cytoplasm and larger cells have a good deal of it. The size of the cell is only roughly correlated with the size of the axon that originates from it. Fig. 2 shows a leech segmental ganglion stained by immunofluorescence using an antibody to the neurotetrapeptide FMRFamide (Phe-Met-Arg-Phe-NH2) (Kuhlman et al., 1985). The neurons, which occupy specific positions, differ not only in size or morphology but also in their capacity to interact with the antibody. In Fig. 2B, the neuron that was indicated in Fig. 2A by the arrow has been microinjected with the water-soluble fluorescent dye Lucifer Yellow. Diffusion of the dye inside the cell permits tracing the various extensions corresponding to the same cell. At specialized junctions, the synapses, a nerve impulse in one cell can be communicated to another cell, such as another neuron. Synapses are usually on the surface of the dendrites or on the cell body but occasionally may be on the axon of the cell receiving the impulse. There are also synaptic connections between nerve fibers and effector organs, such as muscle and glands. The mechanism of synaptic transmission may be quite different depending on the synapse, as discussed later in the chapter. In some neuronal synapses, the transmission is electrical through specialized junctions (the gap junctions), resembling the transmission in the nerve fibers themselves (electrical synapses). In others, the transmission is carried out by the release of a neurotransmitter, which then interacts with postsynaptic
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cells and may have an excitatory or an inhibitory effect (the chemical synapses).
Fig. 2 Leech segmental ganglion stained with an antiserum directed against the neuropeptide FMRFamide. Primary antiserum is visualized by indirect immunofluorescence. (A) Demonstration of several cell bodies with an antigen sensitive to the antibody. (B) The same preparation but after intracellular injection of the water-soluble dye Lucifer Yellow. The arrow in (A) indicates the neuron (the penile evertor motor neuron) that is microinjected with Lucifer Yellow. The bar represents 0.1 mm. Courtesy of Ronald C. Calabrese.
In invertebrates, the cell bodies are on the outside layer of the ganglia (the rind), whereas axons, synapses http://www.albany.edu/~abio304/text/22part1.html (4 of 28) [1/9/2003 12:15:12 PM]
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and dendrites are in the core of the ganglia. In the vertebrate central nervous system, the cell bodies are in the gray matter and the axons predominantly in the white matter. In the vertebrate central nervous system, in portions that are considered more primitive (e.g., in the cerebellum), the gray matter is frequently on the inside of the tissues; in higher brain regions (e.g., in the cerebral cortex), the gray matter is on the outside. Many invertebrates and some primitive vertebrates have giant axons. In some organisms, each giant axon is formed from a single cell. In others, it is formed by the fusion of axons from many separate cells. Giant axons such as those of the squid, can be as much as 1 mm in diameter. Their size and hardiness, when excised, have made them a favorite experimental preparation. In ganglia and in tissues of the central nervous system, specialized cells (neuroglia) form sheaths around one or more cells and also act as packing between the cells. They probably have an important maintenance role. In vertebrates, an insulating layer of structured lipoprotein, called myelin, surrounds the larger axons. The myelin layer is interrupted every millimeter or so by deep constrictions or breaks, called the nodes of Ranvier, where impulse conduction takes place. In some axons, the covering is simpler and may consist of a single layer of glial cells. In most vertebrate nerves, the axons are held together in bundles that are enclosed in sheaths. A single bundle of nerves may contain fibers from neurons with very distinct functions. For example, some of the axons may be from motor neurons, which control striated muscle contraction, and others may be from sensory neurons, which transmit information from a receptor. Similar arrangements can also occur in invertebrates, but these animals usually have far fewer nerve fibers. In most cells, there is an electrical potential difference (the resting potential) between the internal cytoplasm and the external environment. Generally, the inside is negative relative to the outside; the cell membrane is said to be polarized. In excitable cells, this potential is poised to permit the release of a nerve impulse at a speed unmatched by most other cellular events. This nerve impulse, once initiated, is propagated along the axon without decrement. For purposes of discussion, we shall regard a nerve impulse as the discharge of the stored (resting) electrical potential (a depolarization). As we shall see, it is actually much more complex than this and is due to a transient reversal of polarization (the inside becomes positive). Resting potentials and changes therein can be examined fruitfully in terms of ionic gradients, ionic channels and ionic movements across the membrane, although some of the mechanisms underlying these events still remain obscure. II. IONIC ORIGINS OF THE RESTING POTENTIAL When two different concentrations of a solution of the same salt come in contact with each other, there is a net flow of both ions from the area of high concentration to that of low concentration. If the mobilities http://www.albany.edu/~abio304/text/22part1.html (5 of 28) [1/9/2003 12:15:12 PM]
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of the two component ions differ significantly, the cation and the anion tend to separate. The electrical potential produced by the separation opposes the diffusional forces and the actual amount of separation is limited and, in effect, the potential accelerates the diffusion of the slower ion. The potential generated by the diffusional events is the diffusion or junction potential. The charge of the more dilute phase corresponds to that of the more mobile ion, which moves ahead of the oppositely charged ion. For the passage of 1 equivalent of charge from one phase (phase 1) to the other (phase 2), the change in free energy is ∆ G = t+ RT ln a+2/a+2 + t- RT ln a-1/a-1 (1) where a refers to the activity of the ion in question and generally can be approximated by the concentration. The subscripts 1 or 2 refer to the phase, t is the transference number defined in terms of the mobility of the ions (u), t+ = u+/(u+ + u-) (1a) t- = u-/(u+ + u-) (1b) and the subscript signs (+ and -) refer to the charge of the ion. As discussed in Chapter 12, the electrical potential corresponds to ∆ G/zF. The diffusion potential, ∆Ψd, will therefore take the form ∆Ψ d = t+ RT/zF ln a+2/a+1 + t-RT/zF ln a-2/a-1 (2) a+2~ a-2 and a+1~ a-1 because in each phase, the concentration of the cation must be virtually the same as that of the anion. Therefore, for monovalent ions, the relationship can be written in a simpler form: ∆ Ψ d = (t+ + t-) RT/F ln a2/a1 (3) or ∆ Ψ d = (1-2t-) RT/F ln a2/a1 (4) When the mobility of one of the ions is much greater than that of the other, only the faster ion needs to be considered. Supposing that u approaches 0, then t approaches 0 [Eq. (1b)] and 1 - 2t approaches 1, so that ∆Ψd is now independent of the mobilities. This point becomes rather significant in relation to biological potentials. Where the mobility of one ion surpasses that of the other, Eq. (4) becomes (5), the familiar
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Nernst equation, which we have encountered before. ∆ Ψ d = RT/zF ln a2/a1 (5) =-58 mV log10 a2/a1 for T=20oC The resting potentials of most cells are governed by the principles that have been outlined. Generally, the permeability of the membrane to K+ is far greater than the permeability to the other ions. In frog sartorius muscle, the permeability to K+ is 100 times greater than to Na+, and in the freshwater alga, Nitella, it is 52 times greater. Therefore, Eq. (5) can be used and a in the equation corresponds approximately to the concentration of K+. In some freshwater cells, such as the algae Nitella and Chara, the internal concentrations of both Na+ and K+ are much higher than those in the surroundings. However, in most complex multicellular organisms the total ionic concentrations of intra- and extracellular fluids are nearly equal. Nevertheless, there are steep gradients of ions, notably K+ (high on the inside) and Na+ (high on the outside). The efflux of K+ and the influx of Na+ is compensated by the transport activities of the cell membrane. In the steady state, the internal ionic composition is maintained by the balance between the movement of ions in the direction of the electrochemical gradient and the active transport of the ions against the electrochemical gradient, so that the internal [K+] remains high and the internal [Na+] low (see Chapter 20). Clearly, the cell membrane plays a fundamental role in the maintenance of the resting potential. However, as already discussed, a potential difference between two phases does not require the presence of a membrane. When one of the ionic components is restrained (e.g., is part of the cell structure), the mobile ions follow a Donnan distribution and the potential may still be considerable (see Collins and Edwards, 1971) even in the absence of a membrane. The ionic compositions of the internal and external media of some cells have been studied extensively. The values for frog sartorius muscle are represented in Table 1 (Conway, 1957). It is possible to predict the magnitude of the potential between the external and internal phases, the resting potential, by means of Eq. (5), remembering the higher mobility of K+. At l8oC, this value is calculated to be 102 mV, with the inside negative. The potential can be measured directly by inserting an electrode with a tip of microscopic dimensions (a microelectrode) into the muscle fibers. With an external concentration of about 2.5 mM K+, the measured value is 80 to 92 mV, which is not very different from the calculated value. It is possible to study the resting potential over a wide range of external K+ concentrations. This has been done by placing the muscle fiber in various concentrations of KCl or a K+ salt of a nonpermeable anion (e.g., acetate). The results of such experiments yield the relationship represented in Fig. 3 (Conway, http://www.albany.edu/~abio304/text/22part1.html (7 of 28) [1/9/2003 12:15:12 PM]
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1957). The straight line (line A) corresponds to a slope of 57 mV for a 10-fold change in K+ concentration. The value predicted from Eq. (5) is 58 mV. Thus, the equation has very good predictive value. Similar results are obtained with nerve and other cells as well. The resting potential of the fresh water alga, Chara, measured directly, is - 181 mV (Gaffey and Mullins, 1958). The value calculated from the K+ concentration and Eq. (5) is -184 mV, in good agreement with the actual measurement. Clearly, the results indicate that in general the differential distribution of K+ between the intracellular and extracellular phases is responsible for the resting potential. Table 1 Intrafiber Compositiona
Constituent
Muscle
Plasma
Concentration in fiber water
K Na Ca Mg Cl HCO2
83.8 23.9 4.0 9.6 10.7 11.6
2.15 103.8 2.0 1.2 74.3 25.4
124 3.6 4.9 14.0 1.5 12.4
Phosphate Sulfate Phosphor-creatine Carnosine Amino acids Creatine Lactate ATP Hexose monophosphate Glucose Protein Urea Water (g/kg) Interspace water (g/kg)
5.3 0.3 23.7 11.0 6.8 5.3 3.1 2.7 1.7 0.5 1.5 1.6 800 127
3.1 1.9
7.3 0.4 35.2 14.7 8.8 7.4 3.9 4.0 2.5
6.9 2.1 3.3
3.9 0.6 2.0 954
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Source: Conway (1957). Reproduced by permission. a Frog muscle and plasma (moles/kg), except where noted
Fig. 3 Mean Em values of frog sartorii immersed in Ringer-Barkan fluid with addition of external KCl. ( ) Averages of results after overnight immersion at 0-3, then brought to room temperature. (
)
Immediate results from isotonic mixtures with acetate ion replacing chloride and bicarbonate. ( ) Average of observations taken immediately with Ringer Conway fluid containing 2.5 mM K+. The vertical line C at 1.11 on the abscissa. Reproduced with permission from E.J. Conway, Physiological Review, 37:84-132, Copyright ©1957 The American Physiological Society.
III. DYNAMICS OF THE MEMBRANE POTENTIAL The resting potentials of nerve and muscle fibers depend on the permeability properties of the plasma membranes to K+. The permeability of cells can be studied by following the entry or exit of the ions with time, usually using radioactive isotopes. In addition, the electrical resistance of the membrane is related to the permeability of the membrane to ions. This resistance can be determined by measuring the voltage produced by a current pulse between a microelectrode inserted into the cytoplasm and a reference electrode in the medium. An analogous, but more complex method, allows calculation of the resistance of the membrane by the passage of alternating currents through cell suspensions. The resistance of either the external medium or the cytoplasm is much lower than that of the cell membrane. The resistance
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measurements, therefore, indicate the presence of a specialized structure of high electrical resistance and, hence, low permeability to ions at the surface of the cells. Many experiments support the view that the cell membrane has an important role in excitation. Most of the axoplasm can be squeezed out from one end of a cut giant axon and be replaced with an artificial medium, such as a KF solution. Yet the excitability and the potentials of the axon remain undisturbed as long as the membrane remains undamaged. A diagrammatic representation of the potential difference across the membrane (+ on the outside and inside), the resting potential, is shown in Fig. 4a. A nerve impulse, or action potential, involves in part a depolarization, represented in Fig.4b. Nerve conduction corresponds to this depolarizing event and the propagation of depolarization.
Fig. 4 Polarized (a) and conducting (b) fibers. The large arrows represent the direction of the propagation after artificial depolarization. The small arrows indicate the passage of ions in the longitudinal direction (1) and across the cell membrane (2).
The action potential can be initiated by an electric current at the site of the negatively charged electrode (cathode) placed at the outer surface of the nerve fiber. These events can be observed most readily with appropriate electrical amplification and recording equipment, such as amplifiers and an oscilloscope. A stimulus must be above a minimal value (the threshold) to elicit an action potential. It is possible to study in detail what happens to the membrane potential with stimuli that vary in intensity above and http://www.albany.edu/~abio304/text/22part1.html (10 of 28) [1/9/2003 12:15:12 PM]
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below the threshold (Fig. 5a) (Hodgkin, 1939). The results for squid axon represent the potentials recorded at the cell surface at the site of an external cathode (negatively charged electrode, curves above the axis) and at the anode (positively charged electrode, curves below the axis). The ordinate is in units relative to the action potential taken as unity. At very low intensity, the pattern is the same at the two stimulating electrodes. Naturally, the potentials are opposite in sign. There will be an enhancement of the potential difference between the inside and the outside at the anode. The system is said to be hyperpolarized. Under the cathode, a partial depolarization will take place. However, at higher intensities (curves 6, 7, 8, etc.) the curves representing the potential at the cathode change in shape. There is a depolarization beyond the direct electrode effect that is greater in both magnitude and duration. The responses of the nerve to these depolarizations can be shown by subtracting the direct effect of the stimulus (which mirrors the anodal response) from the total depolarization. This difference is shown in Fig. 5b. At sufficiently high depolarization, the potential is unstable and can give rise to an action potential (e.g., curves 10-12). The action potential is self-sustained, since it is independent of the input of the stimulating current, and it is also self-propogated. It turns out to be the same in magnitude at all points along the fiber.
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Fig. 5 (a) Electrical changes at stimulating electrode produced by shocks with relative strengths, successively from above, 1.00 (upper 6 curves), 0.96, 0.85, 0.71, 0.57, 0.43, 0.21, -0.21, -0.43, -0.57, 0.71, and -1.00. The ordinate scale gives the potential as a fraction of the propagated spike, which was about 40 mV in amplitude. The 0.96 curve is thicker than the others because the local response had begun to fluctuate very slightly at this strength. The width of the line indicates the extent of the fluctuation. (b) Responses produced by shocks with strengths, successively from above, 1.00 (upper 5 curves), 0.96, 0.85, 0.71, and 0.57; obtained from curves in (a) by subtracting anodic changes from corresponding cathodic curves. Two of the anodic curves necessary for this analysis were recorded but are not shown in (a). Ordinate, as in (a). Reproduced from A. L. Hodgkin, Proceedings of Royal Society Series B., 148:1-37, with permission. Copyright ©1958 The Royal Society, London.
A propagated wave of depolarization (or action potential) can be recorded from a nerve or muscle following each stimulation above threshold, provided that the interval between the stimuli is greater than the refractory period. This is the period during and immediately after an action potential when new action potentials cannot be elicited because the channels that allow Na+ to enter are inactivated (see Section IIIC). An action potential recorded from an electrode inside the squid axon is shown in Fig. 6. The zero level on the scale represents the point at which there is no potential difference between the inside and the outside of the fiber; i.e., there is no potential difference across the membrane. The initial level (-50 mV) is the resting potential, where the inside is negative in relation to the outside. The upward swing that follows is the action potential, or spike. In addition to involving a depolarization, the action potential reverses the polarity of the fiber (Fig. 6). Then the resting potential is reestablished rapidly after a period of hyperpolarization (Hodgkin, 1951) and the nerve can be stimulated again. Therefore, a process must exist that repolarizes the fibers very rapidly. A discussion of the ionic basis for the depolarization phenomenon and the repolarization follows. Analogous events take place in muscle.
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The depolarization underlying the nerve impulse (the action potential or spike) causes a flow of current from the depolarized areas to the adjacent polarized areas. This current flow depolarizes the polarized region, setting up an action potential there. In this way, an action potential is propagated, or conducted, along an axon. The heavy arrows of Fig. 4b represent the direction of propagation of the action potential and the broken line represents the change in membrane resistance. Normally, the action potential is conducted in a single direction since it originates from the cell body. Because the portion behind the depolarized area is in the refractory state, the impulse cannot travel backward. When elicited artificially in the middle of the fiber, the action potential progresses in either direction. A. Ionic Basis of Depolarization Depolarization might be explained partially by proposing that the permeability of the membrane to all ions increases during the action potential so that the flux of ions causes the electrochemical gradient to collapse. Measurements of resistance do support this view. The resistance during the peak of the action potential is at best a small fraction of the resting resistance. However, an increase in the permeability to all ions would cause the membrane to move toward 0 mV and would not account for the overshoot, which is typically to +50 mV. Moreover, experiments in which different external ions substitute for the usual medium indicate that Na+ has to be present to allow an action potential. Is the influx of Na+ sufficient to account for the current of the action potential? The number of moles of ion required for a given change in potential is calculated with Eq.(6). Moles = (C ∆Ψ m )/F (6) where C is the membrane capacitance in farads, or amount of charge (coulombs) across the membrane per volt, ∆Ψm, is the maximum change in membrane potential during the rising phase of the impulse, and F is Faraday's constant (96,500 coulombs per mole of monovalent ion). The action potential of the axon corresponds to about 100 mV and the membrane capacitance is about 1.5 µF/cm2. Therefore, the influx of Na+ cannot be less than 1.6 x 10-12 mol/cm2. This can only be a minimum value, as it ignores the possibilities of accompanying leakage of K+ or entrance of Na+ during the falling phase of the action potential.
Fig. 6 Resting and action potential in giant squid axon at 18.5oC. Reproduced with permission from A.L. http://www.albany.edu/~abio304/text/22part1.html (13 of 28) [1/9/2003 12:15:12 PM]
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Hodgkin, Biological Review of Cambridge Philosophical Society, 26:339-409.
The quantity of Na+ taken up per stimulus is so small that it cannot be detected; however, it can be calculated when the total uptake resulting from repeated stimuli is divided by the number of impulses. In the giant axon of the squid, Loligo, 3.5 x 10-12 mol/cm2 of Na+ is taken up per nerve impulse. Therefore, the entry of Na+ suffices to account for the action potential. Table 2 summarizes similar results obtained for other systems. These results imply that the membrane permeability to Na+ during the rising phase of the action potential is greater than the permeability to other ions, and the relationship between action potential and [Na+] should be quantitatively predictable from Eq. (5) using the appropriate [Na+]. This is shown more clearly by Eq. (7), where [Na+ext] is the external Na+ level, which is changed experimentally, and [Na+st] is the normal value in the extracellular medium. ∆Ψ ext - ∆Ψst = (58 mV) log10 (Na+st)/(Na+ext) (7) Eq. (7) assumes that the internal Na+ level is not changed by this manipulation of the external medium. Results for different tissues are shown in Fig. 7 (Hodgkin, 1951). The measured membrane potentials (ordinate) at each Na+ concentration (abscissa) are represented by the circles. Curve 1 of each graph represents the resting potential, which is hardly affected by the Na+ concentration. Curve 2 represents the results calculated from Eq. (7). The predictions are close, although significant deviations do occur for the squid giant axon. These deviations may result from the approximations assumed in deriving Eqs. (5) and (7). All the results support the idea that Na+ is responsible for carrying the depolarizing current during the rising phase of the action potential. Action potentials also take place in freshwater algae, such as Nitella and Chara. The functional significance of these potentials is not clear, although they seem to be related to the movements of the cytoplasm. There is no external cation to carry the current. In this case the current is carried by the efflux of Cl-. However, the measured efflux is well in excess of the calculated value. This excess may result from unrelated efflux of K+ and Cl- during the slow action potential of Chara. For nerve and muscle, the reversal of polarity and the Na+ permeation can be summarized as in Fig. 8. The small arrow shown perpendicular to the surface indicates the Na+ influx, the large arrows, the direction of the action potential. As mentioned, the depolarization of the action potential is followed almost immediately by repolarization of the axon (Fig. 6). In fact, the whole cycle of depolarization and repolarization generally takes place in 1 ms or so. Repolarization would be accomplished most rapidly by removal of the excess positive charge that has entered the nerve or muscle cell. This could be done most simply by a rapid efflux of K+ in the
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direction of the electrochemical gradient. The efflux of K+ shown in Table 2 (column 2) is of the same order of magnitude as the influx of Na+, as required by the fact that the two should represent equal but opposite phenomena. The changes in permeability for specific ions are likely to be the result of opening and closing of protein-lined channels, as discussed in Section IIIC. The diagram of Fig. 9 summarizes the ionic exchanges accompanying the action potential and the repolarization by the K+ efflux. Table 2 Na+ and K+ Exchanges During Nerve Excitation
Material
Carcinus maenas Carcinus maenas Sepia officinalis Sepia officinalis Sepia officinalis Loligo pealli Loligo pealli Loligo pealli
(1)
(2)
Na+ influx/impulse (10-12mol/cm2)
K+ efflux/impulse (10-12 mol/cm2)
------------3.7 3.8 3.5 4.5 4.4
1.7 2.5 3.4 4.3 3.6 3.0 -------------
aHodkin
Reference
a b c d e f g
and Huxley (1947) bKeynes (1951a) cWeidmann (1951) dKeynes (1951b) eKeynes and Lewis (1951) fRothenberg (1950) gGrundfest and Nachmansohn (1950) Reproduced by permission.
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Fig. 7 Relation between sodium concentration in external solution and potential difference across resting and active membrane. (a) Squid giant axon (from work of Hodgkin and Katz); (b) frog sartorius muscle (from work of Nastuk and Hodgkin); (c) frog myelinated nerve (from work of Huxley and Stampfli). Abscissa: sodium concentration on logarithmic scale (dashed line shows concentration in Ringer's fluid or seawater). Ordinate: potential difference across membrane (outside potential minus inside potential) at rest (1) and at crest of action potential (2). The solid line is drawn with a slope of 58 mV for a tenfold change in sodium concentration. The points in these curves were obtained by adding the original author's values for the resting potential or for the reversed potential difference across the active membrane. Reproduced with permission from A. L. Hodgkin, Biological Review of Cambridge Philosophical Society, 26:339-409.
Both Na+ and K+ can shift across the nerve membrane with action potentials. How can the gradients for these ions be maintained? In part, the answer rests on the fact that each impulse does not change the concentration of the ions significantly. We have seen that many stimuli are needed to detect any change. The K+ loss associated with one stimulus corresponds to about 4 X 10-12 mol/cm2 (Table 2). Since the axon is about 500 µm in diameter, the loss per liter of axoplasm is approximately 1 X 10-5 mol, less than 1 part in 10,000. Although the loss is small, eventually work must be carried out to restore the internal K+ http://www.albany.edu/~abio304/text/22part1.html (16 of 28) [1/9/2003 12:15:12 PM]
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and Na+ levels. In Chapter 20, we discussed a transport system responsible for pumping Na+ out and pumping K+ in. It is this system that maintains the internal Na+ and K+ concentrations. Although the Na+ currents generally underlie the action potential in most animal tissues, there are exceptions. Crustacean muscle depends on Ca2+ for conduction. This is most readily demonstrable in muscles in which the K+ channels have been blocked (e.g., with TEA). The involvement of Ca2+ fluxes was demonstrated with [45Ca] using giant muscle fiber from a barnacle in which the internal Ca2+ was reduced. During induced action potentials the Ca2+ was found to correspond to 2-6 pmoles/µF, where 0.5 pmoles/µF are needed to depolarize the membrane by 100mV (Hagiwara and Naka, 1964). B. Membrane Mechanisms: Voltage Clamping The behavior of the membrane of an axon we examined so far can be outlined simply : 1 The unequal distribution of K+ is responsible for the resting potential. 2 A small depolarization beyond a critical value leads to a change in permeability to Na+, which, in turn, leads to Na+ flow into the fiber. This influx is responsible for the reversal of the resting potential. The initial depolarization could be the result of an electrical stimulus or the depolarization of an adjoining area. 3 The immediate recovery of the resting potential is the consequence of an increase in the permeability to K+, causing movement of K+ in the direction of the electrochemical gradient.
Fig. 8 Diagram representing the membrane potential during the action potential (center) and at rest. The large arrows represent the direction of the action potential (the nerve is stimulated in the middle). The smaller arrows represent the direction of the ion movement.
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Fig. 9 Diagram representing the ionic basis of propagation of the action potential followed by recovery. The large arrows represent the direction of the action potential (the nerve is stimulated in the middle). The smaller arrows represent the direction of the ion movement.
If this model is correct, it seems possible to manipulate the electrical potentials of the axons and to test these premises independently. The potential difference between the inside and the outside can be set at any level by means of an external electrode, a microelectrode implanted in an axon, and appropriate electronics. In the voltage clamp technique, the membrane potential is kept constant at any desired level by passing a current equal and opposite to that generated by the flow of ions across the membrane. With this method, it has been possible to test whether the potentials trigger the changes in flux necessary for the polarization-depolarization cycles. Increases in the potential difference (hyperpolarization) cause an inflow of current. This is expected from the passive resistive properties of the membrane. A small decrease in the potential causes an outflow of current, as expected. A decrease in the potential difference beyond a critical value has a very different effect (Fig. 10) (Hodgkin, 1958). Initially there is a large inward flow of current (dashed line, Fig. 10a); however, it is followed quickly by an outward current (full line), which in the absence of a clamp, would repolarize the nerve. The inward current should correspond to the Na+ influx that would take place during depolarization (in the absence of a clamp). Experimental tests of this point show that this current is, in fact, critically dependent on the presence of Na+; replacement of the Na+ in the external fluid by choline (which does not penetrate), blocks this event completely (Fig. 10b). The outward current that follows is likely to be carried by K+, which normally would repolarize the nerve. A test of whether K+ is involved can be carried out by measuring the current flow and the K+ efflux simultaneously. The results of this experiment are shown in Fig. 11 (Hodgkin and Huxley, 1953). The efflux of K+ and the outflow of http://www.albany.edu/~abio304/text/22part1.html (18 of 28) [1/9/2003 12:15:13 PM]
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current, correspond quantitatively. These potentials can be illustrated in a rather convenient form as shown in Fig. 12a (Narahashi et al., 1964). The inflowing current (the Na+ current) and the outflowing current (the K+ current) can be plotted as a function of the voltage at which the membrane potential is clamped. The current can be corrected for leakage, by assuming that the leakage current is a linear function of the potential (dashed line) and subtracting it from the total current. A meaningful application of this system is shown in Fig. 12b, where the Na+ passage is blocked by the toxic drug tetrodotoxin. Tetrodotoxin is a toxin extracted from certain organs of the puffer fish (the poison of the fugu fish of James Bond and more recently Homer Simpson fame). The Na+ current is blocked completely, whereas the K+ current remains unaffected. In contrast to tetrodotoxin, tetraethylammonium interferes with the passage of K+ and, hence, with the recovery of the resting potential after stimulation (not shown). The results we have examinedm support the idea that the phenomenon of excitation can be explained entirely by underlying ionic currents. However, the ionic currents must be a reflection of events occurring in the structure of the membrane, and these events are beginning to be studied in detail. C. Molecular Mechanisms and Channels Resting potential, action potential and the recovery of the resting potential all depend on changes in the permeability of the membrane to Na+ and K+. What accounts for these changes? The lipid portion of the membrane is not likely to allow the passage of ions at the high rates that have been measured. The activation energy for such passage is of the order of 250 kJ/mol (Parsegian, 1969). In contrast, passage through channels would lower the activation energy for K+, to about 20 kJ/ mol (Frankenhaeuser and Moore, 1963). Furthermore, it is difficult to visualize the lipid components being regulated to vary the passage of ions in response to a membrane potential. For these reasons, the presence of protein-lined channels has been considered for some time.
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Fig. 10 Membrane currents associated with depolarization of 65 mV in presence and absence of external sodium ions. The change in membrane potential is shown at the top; the lower three records give the membrane current density; 11o C, outward current and internal potential shown upward. Reproduced from A. L. Hodgkin, Proceedings of Royal Society Series B, 148:1-37, with permission. Copyright © The Royal Society, London.
Fig. 11 Relation between membrane current density and potassium efflux when a Sepia axon is depolarized. The axon was depolarized by an applied current for periods of 60 to 600 s. Vertical lines show ±2 x SE; the horizontal line is drawn at a level corresponding to complete suppression of the average http://www.albany.edu/~abio304/text/22part1.html (20 of 28) [1/9/2003 12:15:13 PM]
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resting efflux. Reproduced from A. L. Hodgkin and A. F. Huxley, Journal of Physiology, 121:403-414, with permission. Copyright ©1953 The Physiological Society, Oxford, England.
Fig. 12 (a) Current-voltage relations before treatment with tetrodotoxin. Circles refer to peak Na+ current corrected for leakage current, triangles refer to steady-state K+ current corrected for leakage current, and squares refer to leakage current; l, designated component of the membrane current (inward direction negative); EM, membrane potential; EH holding potential. (b) Current-voltage relations during treatment with tetrodotoxin 3 x 10-5 g/ml. Reproduced from the Journal of General Physiology, 47:965-974, ©1964, by copyright permission of the Rockefeller University Press.
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Membrane channels are integral proteins that span the lipid bilayer. As we discussed throughout this book, channels have far-reaching roles in cell function. Ion channels have an important role in hormone secretion, visual transduction, transepithelial ion transport and the activation of contractile mechanisms. Ion channels are frequently present as oligomers and they operate to regulate the passage of ions. The channels which concern us at this time are involved in the propagation of the electrical impulses in excitable cells and for postsynaptic responses to neurotransmitters, which are discussed later. In the latter case, the receptors that are part of the ion channels bind the neurotransmitter. Upon binding, the channels favor an open configuration. Ion channels display specificity for certain ions, saturation kinetics in relation to ion concentration, competitive inhibition by analogs and conformational changes (to go from the open to the closed configuration) - all characteristic of enzyme activity such as that of transport proteins. However, they also exhibit behavior that differs from that of enzymes. The rate of ion transport is orders of magnitude higher than enzyme turnover and the temperature dependence of the ion passage is very low (Latorre and Miller, 1983). Furthermore, saturation kinetics is exhibited only at extremely high concentrations. As we shall see, proteins that have the appropriate characteristics have been isolated and functional channels reconstituted into bilayers. Channels have been studied in their native state by patch-clamping. Patch refers to the fact that, by this technique, a "patch" of membrane is studied independently of the rest of the membrane. Clamping refers to maintaing the voltage electronically at a fixed value, that is, voltage clamped. With patch-clamping, a small heat-polished pipette is sealed against the cell membrane. The pipette is filled with an electrolyte solution. In effect, a small patch of membrane has been isolated and can be studied electrically independently of the rest of the membrane. This tight seal between pipette and membrane has been referred to as a giga seal (giga meaning a billion, referring to the fact that the pipette and the sealed patch, together, have a resistance in the range of 109 to 1011 ohms). When the voltage is clamped, a recording of the current permits the study of a single channel in the membrane (Hamill et al., 1981). Each deflection from the base line represents the opening of a channel. The relationship between voltage and current can be studied over a wide range of clamped voltages. In the neurotransmitter-regulated channels of the synapse, discussed later in this chapter, the effects of agonists (activators) and antagonists (blockers) can be studied at the level of the simplest unit. The relationship between current passing through a single channel and clamped voltage is shown in Fig. 13 (Hamill et al., 1981). The larger the voltage, the larger the currents seen as a deflection from the base line. The relationship between current deflections and voltage is linear, reflecting a constant conductance of individual channels. Note that at any given voltage, the levels of current in all openings are constant and, therefore, characteristic of those channels. Conductance is the reciprocal of resistance and is expressed in siemens, S, (1/ohms)(formerly called mhos). S = ohms-1. Typical ion channels range in conductance between 5 to 100 pS. The patches can also be isolated mechanically on the micropipette, either an inside-out or right-side out (i.e., outside-out) configuration. Alternatively, when the pipette is still attached to the cell, the patch can be ruptured, providing a direct connection between the cytoplasm and the fluid inside the pipette.
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Maintaining voltage at a selected constant values by voltage-clamping, provides a means of examining the behavior of channels at these voltages. From the characteristics of the action potential and the curves depicted in Fig.12, we would expect the Na+ channels to open at depolarizing voltages. Similarly, we would expect the K+ channels to open during repolarization. This is found to be the case. The time spent in an open conformation is longer.
Fig. 13 Single-channel current recordings when the patch is clamped at the indicated voltage. Reproduced from O. P. Hamill, et al., European Journal of Physiology, 391:85-100, with permission. Copyright ©1981 Springer-Verlag, Heidelberg.
The ability to open or close in response to voltage changes across the membrane is known as voltage gating. Voltage gating is presumably the result of conformational changes in the proteins constituting the channels. Many different kinds of channels that differ in ionic specificity have been recognized. Most of these are voltage gated. At any one time, regardless of voltage, some channels will be open, others closed. The behavior of a sector of the membrane will be a summation of the activity of individual channels. The whole cell current (macroscopic) after a voltage pulse, is represented at the top of Fig. 14 (Aldrich, 1986) for a bovine chromaffin cell of the adrenal medulla in tissue culture. These cells secrete epinephrine and norepinephrine and are excitable. The diagrams below (a to c) represent hypothetical voltage clamp records for individual channels, where each of the three lines represents the current recorded for a single channel. The individual channels that are specific for Na+ open in response to a depolarization. Three different states have been reported: resting states (R), which are present at hyperpolarizing voltages (no ions go through); open states (O), which allow the passage of Na+ and which have opened as the result of an initial depolarization; and inactivated states (I), which are closed and cannot be opened with further depolarization. The latter correspond to the state during the refractory period. The Na+ current represented at the top of Fig. 14 can be generated by the behavior of the channels shown http://www.albany.edu/~abio304/text/22part1.html (23 of 28) [1/9/2003 12:15:13 PM]
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under the curve and summarized by the schemes shown at the right in the figure. Figure 14a (top) corresponds to a long-maintained opening; more channels open in the rising current phase and close in the falling phase. This effect could result from a fast transition to the open state (0) followed by a slow transition to both the resting (R) and inactivated (I) states. Figure l4b shows how short-lived open states could generate the same pattern. In this case, the rate of opening and returning to the resting state must be high and the rate of conversion to the inactive state low; otherwise repeated opening and closing would not be possible. The open state would nevertheless predominate when the current passage is maximal. Similarly, the pattern could be explained by Fig. l4c, in which the opening is short-lived as the result of slow transitions from the open to the resting state and a rapid inactivation. The last case appears to be correct (Aldrich et al., 1983). Much is now known about the proteins constituting the Na+-channel (see Catterall, 2000). Most recently a detailed 3D-reconstruction using cryoelectron microscopy and tomographic analysis (Sato et al., 2001) has been carried out. For a more complete discussion see addendum
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Fig. 14 Three possibilities for sodium channel gating that predict identical macroscopic sodium currents but different single-channel behavior. Reproduced from R. W. Aldrich, Trends in Neurosciences, 9:82-85, with permission. Copyright ©1986 Elsevier Science Publishers, England.
The appropriate behavior for the K+ channels has also been shown using patch clamping of internally perfused giant squid axons. Since the repolarization of the axon after the action potential depends on an increase in K+ permeability, we would expect the frequency of opening the K+-specific channels to increase with depolarization. The results of Fig. 15 (Conti and Neher, 1980) show exactly this behavior. In Fig. 15a, the voltages at which the patches are clamped are listed at the left of each record. Depolarization increases from the bottom of the figure to the top. The frequency of opening is minimal when the axon is polarized (see lower two records). It increases with depolarization, and then the channels close again. The increased conductance at constant voltage, is an indication of the number of channels that are open. Figure 15b compares the variances observed experimentally (the points) to those calculated from theoretical considerations. The agreement between the points and the line shows that the observed current changes correspond to random open-close transitions of the same channels.
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Fig. 15 (a) Recordings of patch current at different membrane potentials. From Conti and Neher (1980).
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(b) Variance as a function of voltage. The points are experimental points and the line has been calculated from the observed probability of opening on the assumption that there are seven channels in the patch. Reproduced by permission from Nature 285:140-143, copyright ©1980 Macmillan Magazines Ltd.
K+-channels are very diverse (e.g., see Wei et al., 1990). Four different voltage-gated K+-channels have been found in Drosophila , fourteen in rats and more are likely to be present. The Drosophila Shaker channel is an integral membrane protein of 70,200 daltons containing seven potential membranespanning sequences (Tempel et al., 1987) and probably assembling into a channel containg four subunits (see MacKinnon, 1991; Doyle et al., 1998). Several of these channels function during the action potentialrecovery cycle: they open following depolarization (e.g., brought about by the action potential) and then close again (known as inactivation). In Shaker-type K+-channels, the inactivation gate, responsible for the corresponds to the channel's cytoplasmic amino terminal. The data suggest that inactivation is part of a sequential process (Zhou et al., 2001). First the gate binds to the cytoplasmic channel surface and then penetrates the pore, blocking its opening. Until recently, K+ channel subunits were found to have one pore forming P domain with the characteristic TXGYG sequence (Yellen et al., 1991; Heginbotham et al., 1994). The P domain was identified after sitedirected mutagenesis (see Chapter 1) identified in the Drosophila Shaker channel an amino acid residue that specifically affects the affinity for intracellular tetraethylammonium (TEA) ( Yellen et al., 1991; Heginbotham et al., 1994).We have seen that TEA blocks K+-channels. The single P-domain channels studied include the voltage-gated K+ (Kv) (e.g., Abbott et al., 2001), the inward rectifying (Kir) (e.g., Krapivinsky et al., 1995) and KCNQ channels (e.g., Schroeder et al., 2000). The KCNK channels comprise a family of potassium-selective leak channels possessing two pores. KCNK2 is involved in maintaining the resting potential (see Goldstein et al., 2001). The channel becomes voltage dependent during the rise of the action potential thereby promoting recovery of the resting potential. The phosphorylation of the channel by protein kinase A reversibly switches it into a voltage dependent channel ( Bockenhauer et al., 2001). The large-conductance K+ (BKCa) channels (see Vergara et al., 1998) are activated by membrane depolarization or intracellular Ca2+. They are involved in the regulation of neuronal excitability, secretion, and vascular tone. In most tissues, their stimulation results in a non-inactivating hyperpolarizing K+ current that reduces excitability. These channels have four identical α subunits coded by the Slo gene. Although only one gene codes the pore forming α subunit of the channel, the properties can be quite different mostly because of alternative splicing (e.g., Tseng-Crank et al., 1994; see Shipston et al., 2001 ). The splice variants depend on the particular cell-type and cause significant changes in excitability and cell function (e.g., Tian et al., 2001). In addition, in some tissues the activity can also be modified by binding to the β subunits (see Toro et al., 1998; Gribkoff et al., 2001) and phosphorylation (see Levitan, 1999) via the cAMP-dependent protein kinase (PKA). The phosphorylation activates the channels in some tissues (e.g., smooth muscle and some neurons) and deactivates them in others (e.g., http://www.albany.edu/~abio304/text/22part1.html (27 of 28) [1/9/2003 12:15:13 PM]
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endocrine cells of the anterior pituitary). The stress axis regulated exon (STREX) of the α subunit plays a particularly significant role. STREX codes a 59 residue segment responsible for Ca2+ and voltage sensitivity. STREX is present in excitable cells. In other tissues, the splice variants are determined by physiological conditions (presence or absence of hormones, depolarization of the cell, etc.) (e.g., see Xie and McCogg, 1998; Xie and Black, 2001). IV. ELECTROGENIC PUMPS The electrical potential across the plasma membrane of many cells can be considered a K+ diffusion potential. However, the transport of ions can generate potential. They are said to be electrogenic. One turnover of the Na+ pump is responsible for the uptake of 3 Na+ for each 2 K+ extruded (Chapter 20). Therefore it should be electrogenic. Although in these cells the primary determinant of the resting potential is the relatively high permeability to K+, hyperpolarizations have been observed in a number of cases (e.g., snail ganglion cells and striated muscle) during periods of rapid Na+ extrusion. These potentials have been considered to be the result of the transport mechanism. In some fungal and plant cells, electrogenic pumps play an important role in maintaining the resting potential. In Neurospora hyphae, part of the resting potential across the membrane responds to K+ concentration and part responds rapidly and reversibly to interference with metabolism by inhibitors of respiration, such as azide or carbon monoxide (Slayman, 1965). In this case (Slayman and Tatum, 1965), an electrogenic pump is the major component. The pump, a 100 kDa protein, is a H+-ATPase of the Ptype (see Nakamoto and Slayman, 1989; Scarborough, 1996). The structure of the ATPase has been studied using electron crystallography (Auer et al., 1998).
Go to Part 2 REFERENCES Search the textbook
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V. TRANSMISSION OF EXCITATION BETWEEN CELLS Excitable cells can communicate with each other, generally through specialized regions of contact, the synapses. In vertebrates, the synaptic connections form a network of great complexity. It has been estimated that there are 4 X 1011 synapses per gram of guinea pig cerebral cortex. Many of the complexities of neural behavior are thought to be a reflection of the organization of neuronal networks and the properties of the synapses. The convergence of different terminals can produce complex effects because many excitatory and inhibitory influences are summed and integrated. Furthermore, the various connections and their properties are dynamic and continuously modified by experience. These dynamics have implications in sensation, memory, and learning. A very intriguing interaction between neurons and glial cells which may have implications in the modulation and transmission of signals is discussed in Chapter 7. A. The Synapse Typically a vertebrate central nervous system synapse connects a presynaptic axon terminal to a postsynaptic dendrite. The synaptic cleft of about 200-300 Å separates the two. The synaptic gap is filled with electron-dense material, containing cell adhesion molecules (CAMs) (see Chapter 6) discussed below. Presynaptic terminals are filled with vesicles containing neurotransmitters either glutamate at most excitatory synapses and GABA or glycine at inhibitory ones. The junction resembles the adherens junction of epithelial cells (see Chapter 11). One of the most studied synapses is that between nerve and striated muscle, the neuromuscular junction, represented diagrammatically in Fig. 16 (Whittaker, 1968). At this junction the nerve terminal expands into a baglike arrangement: nerve and muscle are separated by a space. Transmission occurs by the release of a transmitter, acetylcholine (Ach), at the presynaptic terminals. Ach induces a depolarization of the specialized portion of the muscle membrane, the end-plate. Presumably, the transmitter attaches to receptor sites on the end-plate. These are chemically gated channels, which open upon binding Ach. The application of Ach to the junction with a micropipette, generates postjunctional potential changes. The postjunctional potentials induced by the action potential of the nerve fiber can be blocked by certain drugs that compete with Ach for binding to the receptors. Curare, the poison used on arrow tips by South American Indians, acts in this manner. At rest, there is an occasional release of small packets or quanta of Ach from the undisturbed cells. This release produces the small postjunctional potentials (miniature
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end-plate potentials) (Fatt and Katz, 1952; Miledi, 1966). The size of these potentials can be calculated to correspond to the release of 1,000 to 10,000 molecules of acetylcholine. It has been suggested that they correspond to the release of the contents of acetylcholine-containing vesicles that are visible with the electron microscope (for a discussion, see Auerback, 1972). These vesicles, the synaptic vesicles, have been isolated and shown to contain acetylcholine.
Fig. 16 (a) Diagram of a motor end-plate showing axoplasm (ax) and myelin (my) of motor nerve, and saclike terminals (arrows) lying in gutter-like depressions of the mitochondrion-rich muscle sarcoplasm (sarc). The terminals are protected by teloglia (tel). Muscle nuclei (m.n.) and myofibrils (mf) are also diagrammed. (b) Tracing of an electron micrograph of a portion of the nerve terminal similar to that between the arrows in (a). Note the highly folded postsynaptic membrane extending into the muscle sarcoplasm, the fingerlike projections of teloglia, and the numerous vesicles and mitochondria (Mit.) in the terminal cytoplasm. Reproduced with permission from V. P. Whittaker, Proceedings of the National Academy of Sciences, 60:1082, 1084, 1968.
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Graded end-plate potentials can be produced by applying electrical currents locally to a nerve-muscle preparation, in which the nerve and the muscle are rendered unresponsive with tetrodotoxin (Katz and Miledi, 1967a; Katz and Miledi, 1967b). Conversely, the end-plate potential can be blocked by hyperpolarization of the nerve terminal by means of a current pulse delivered locally by a microelectrode. The neurotransmitter release requires external Ca2+. Therefore, the results suggest a scenario in which the action potential arriving at the presynaptic terminal opens voltage gated Ca2+channels. The influx of Ca2+ triggers the release of neurotransmitter. Neuronal synapses also have specialized regions. The part of the cell through which incoming events influence the synapse is called the presynaptic terminal. The presynaptic terminal stimulates the postsynaptic cell. The synaptic membranes may be tightly apposed, with only a small space between them forming a gap junction (Pappas and Bennett, 1966). Gap junctions have been discussed in some detail in Chapter 4. Presynaptic and postsynaptic cells are connected by channels. In gap junctions transmission occurs by purely electrical events. Other junctions resemble the neuromuscular junction. The distance between the membranes is greater and the transmission is thought to require a neurotransmitter. Fig. 17 (Pappas and Bennett, 1966) shows an electron micrograph of a section of the spinal cord of the toadfish, in which the two kinds of synaptic endings are present side by side. The two types of synapses are represented diagrammatically in Fig. 18 (Whittaker, 1968).
Fig. 17 Electron micrograph of a section from the spinal cord of the toadfish. Profiles of two synaptic endings can be seen separated by a glial cell process (G). The two synaptic endings form contact on a neuronal cell body (N). In the chemically transmitting synapse, vesicles (V) are clustered close to the presynaptic membrane. The pre- and postsynaptic membranes are distinctly separated by a 20-nm space http://www.albany.edu/~abio304/text/22part2.html (3 of 8) [1/9/2003 12:15:19 PM]
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(arrow) at the chemically transmitting synapse. At the electrical synapse (at the left) the apposing membranes are very close and no space is discernible. M, Mitochondria. Bar corresponds to 714 nm. Reproduced from Pappas and Bennett (1966), with permission.
By inserting a stimulation electrode into one kind of cell (i.e., presynaptic or postsynaptic) and a recording electrode into the other, one can follow the various electrical events. The results confirm the presence of two distinct kinds of synapses. In gap synaptic junctions, both depolarizations and hyperpolarizations can be transmitted from one cell to the other in either direction (Bennett et al., 1967). Consequently, presynaptic cells can be stimulated from a postsynaptic action potential. The gap junction synapses also lack neurotransmitter vesicles, in agreement with the notion that in these cases the model of transmission is electrical. The presynaptic terminals of the chemical synapses are full of vesicles that correspond to the synaptic vesicles isolated from brain and containing neurotransmitter, supporting the idea that the transmission is mediated by a neurotransmitter. In contrast to the gap junction, postsynaptic potentials cannot stimulate presynaptic cells. However, the postsynaptic potentials can be evoked electrically even when the presynaptic action potentials are blocked by tetrodotoxin or tetraethylammonium (Katz and Miledi, 1967a). As discussed above, tetrodotoxin blocks the Na+ channels, whereas tetraethylammonium blocks the passage of K+ needed for repolarization (Katz and Miledi, 1967c). Therefore, presynaptic electrical events are not directly involved in stimulating the postsynaptic cell. Small spontaneous postsynaptic depolarizations have been observed and are thought to reflect a continuous subthreshold release of packets of neurotransmitter. Certain pharmacological agents can also block these synapses by preventing neurotransmitter binding. Much of this evidence suggests a similarity to neuromuscular transmission. Although acetylcholine is a synaptic neurotransmitter, many other chemicals have been implicated. At some junctions, the transmitters may be noradrenaline, dopamine, 5-hydroxy-tryptamine or serotonin (the monoamines). Some of the drugs that produce hallucinations (e.g., LSD and mescaline) and some tranquilizers (e.g., thalidomide) are thought to produce their effects by their structural similarity to neurotransmitters. There is some evidence that different synapses on the same cell may involve different transmitters (Gerschenfeld et al., 1967). Glutamate is the major excitatory neurotransmitter in mammalian brain, although it can also be inhibitory acting via an inhibitory post-synaptic potential (Fiorillo and Williams, 1998). There are several kinds of postsynaptic receptors that respond to glutamate. Two of these are ligand-gated ion channels, the NMDA and AMPA receptors. The initials are based on pharmacological ligands: N-methyl-D-aspartate and αamino-3-hydroxy-5-methyl-4-isoxasole propionate, respectively. (see Bettler and Mulle, 1995). In contrast, the metabotropic receptors belong to the seven transmembrane receptor family. Their action is much slower because they are mediated by GTP-binding proteins and they depend on second messengers. The NMDA receptors become activated only when the glutamate signal arrives after the postsynaptic neuron has been activated by another signal. Therefore, NMDA receptors are well suited to respond to http://www.albany.edu/~abio304/text/22part2.html (4 of 8) [1/9/2003 12:15:19 PM]
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closely timed signals, supposedly a feature important for learning (see Section D, below). The AMPA receptors are ligand-gated cation channels responsible for the fast component of excitatory postsynaptic currents in the central nervous system (see Hollmann and Heinemann, 1994). They have been implicated in long term potentiation and depression (see Section D, below). The NMDA receptors have a double function (Hayashi et al., 1999). In addition to acting as ion channels, they act as signal transducers (see Chapter 7) by interacting with the Lyn tyrosine kinase, a protein of the Src-family non-receptor proteins. After activation, Lyn activates the pathway involving the mitogen-activated protein kinase (MAPK) and it increases the expression of brain derived-neurotrophic factor (BDNF) mRNA. Therefore, AMPA receptors can act as signals that may contribute to the expression of BDNF with consequent effects on synaptic plasticity (see Section D, below). The NMDA-glutamate receptors are activated by membrane depolarization and the simultaneous binding of ligand. They promote the post-synaptic influx of Ca2+ (MacDermott et al., 1986). A role of these receptors in forming synapses during development has been shown using specific antagonists, such as D,L(-)-2-amino-5-phosphonovaleric acid (APV). γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter which is bound postsynaptically to either GABAA, GABAB or GABAC receptors (see Macdonald and Olsen, 1994; Lukasiewicz, 1996). The GABAC receptors are almost exclusively in the retina of adult vertebrates on bipolar cell axon terminals. The GABAA receptor forms a ligand-gated Cl-channel (Barnard et al., 1987). Its inhibitory effect results from the hyperpolarization that follows opening of the channel (from the Cl--diffusion potential, see Section II). GABAB receptors act more slowly through a GTP-binding protein, which activates or inhibits adenyl cyclase and activates adjacent K+-channels and closes voltage gated Ca2+ channels. The effects are slower, but longer lasting. There are also GABAB presynaptic receptors usually in axons that release GABA. Their stimulation decreases the release of neurotransmitters, possibly through effects on Ca2+ or K+ channels (Thompson et al., 1993). The GABA receptors are probably held in place by interactions with the microtubules. The microtubule associated protein 1B (MAP-1B) interacts specifically with the GABAC receptor subunit ρ1 subunit, but not with GABAA receptors (Hanley at al., 1999). A cytoplasmic protein, GABAA-receptor-associated protein (GABARAP), binds to the γ2 subunit of the receptor. GABARAP has sequence similarity with subunits of MAP proteins and has a putative tubulin-binding motif (Wang et al., 1999).
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Fig. 18 Characteristics of a chemical synapse (left) and an electrical synapse (right). Note in the latter synapse the absence of both synaptic vesicles and cleft and the invasion of the postsynaptic elements by action currents, which in the chemical synapse are short-circuited by the low-resistance cleft. Reproduced with permission from V. P. Whittaker, Proceedings of the National Academy of Sciences, 60:1082, 1084, 1968.
Excitatory neurotransmitters depolarize the postsynaptic membrane. As might be expected from our previous examination of depolarization, this process involves the opening of channels. In this case, however, the opening depends on binding of the neurotransmitters; i.e., the channels are chemically gated. Postsynaptic depolarization has been studied with patch clamping techniques primarily in cells in culture or freshly dissociated neurons, because the native structures are enveloped in protective sheets. One of the most versatile techniques involves the reconstitution of extracted channel proteins in bilayers or in the lipid at the tip of a patch pipette. This approach has been used extensively with many proteins, including Na+ and K+ channels and acetylcholine-activated channels. The receptors have been isolated most commonly from the electric organ of the ray Torpedo. The acetylcholine-activated channels increase the permeability of the membrane to a variety of ions when http://www.albany.edu/~abio304/text/22part2.html (6 of 8) [1/9/2003 12:15:19 PM]
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they bind acetylcholine as well as other agonists. The four distinct glycoproteins are present as a pentamer (Montal et al., 1986) with the composition α2 βγ δ. The activation of single channels reconstituted into a bilayer is shown in Fig. 19 (Montal et al., 1984).
Fig. 19 Single acetylcholine receptor channel currents activated by different cholinergic agonists in planar lipid bilayers. Reproduced from Biophysical Journal, 45:165-174, ©1984, by copyright permission of Rockefeller University Press.
Postsynaptic membrane receptors are anchored to structures connecting them to the cytoskeleton. In the nervous system, the assembly of these structures is thought to have a role in neuronal plasticity (see Section D, below). A common theme is the attachment of the receptor to an adaptor protein which attaches directly or indirectly to cytoskeletal elements. Acetylcholine receptors (AchR) and sodium channels (NaCh) clustering at the neuromuscular junction (see Colledge and Forhner, 1998) depend on agrin. Agrin is a large heparan sulfate proteoglycan, a protein synthesized and secreted by motor neurons, that becomes incorporated in the synaptic basal lamina. AchRs are attached to the adaptor protein rapsyn of 43 kDa. In contrast, the NaChs are associated with syntrophins which bind to dystrophin complexes. Dystrophin, in turn, is connected to cortical actin and a transmembrane complex that interacts with the extracellular matrix. Agrin also mediates the formation of immunological synapses (see Trautmann and Vivier, 2001). As in the case of the neuromuscular junction, the immunologivcal synapse involves signaling between cells via surface receptors and these receptors are clustered. Agrin is found at T-cell receptor sites during activation of primary immune cells. Furthermore, activated purified agrin triggers lipid raft clustering (Khan et al., 2001) and the clustering of antigen-specific T cell receptors http://www.albany.edu/~abio304/text/22part2.html (7 of 8) [1/9/2003 12:15:19 PM]
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Gepheryn plays a role similar to that of rapsyn, but in certain inhibitory synapses where glycine is the neurotransmitter. Gephyrin binds to glycine receptors (GlyRs) (see Meyer et al., 1995) and mediates the binding to tubulin (Kirsch and Betz, 1995). Gene targeting mutations in mice showed that gephyrin is required for synaptic clustering of GlyRs and in non-neuronal tissue molybdoenzyme activity (Feng et al., 1998). Gene targeting was carried out by deleting the upstream sequences responsible for initiating transcription and translation. Glutamate receptors also bind to adaptor proteins (see O'Brien et al. 1998). The latter, in turn, bind to the cytoskeleton by attaching to α-actinin-2 (an actin binding protein) (e.g., Wyszynski et al., 1997).
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B. Neurotransmitters: Discharge and Recovery This section will focus on the mechanisms of discharge and recovery of neurotransmitters. The discharge is by exocytosis as in other secretory events (see Chapter 10). However, synaptic transmission, whether at an electrical or a chemical synapse, must be very quick: in the millisecond range. In a chemical synapse, the speed of discharge is needed for rapid communication. The quick recovery is also essential to permit continued excitability. Other secretory events are similar to that of neurotransmitter. However, in these cases, speed is not as important. Therefore, presynaptic terminal of chemical synapses must differ in some way. What are these differences? In synaptic transmission, the vesicles in close apposition to the presynaptic membrane are thought to be in a rapidly releasable pool (see Fig. 17). Vesicles that are a short distance away are thought to be held in reserve and be able to replace quickly the vesicles that have been discharged. Recent studies using total-internal-reflection microscopy (see Chapter 1) allowed following the process of exocytotic discharge of single vesicles in the goldfish retinal bipolar neurons (Zenisek et al., 2000), where the synaptic vesicles were labeled with a fluorescent lipid. The results are in agreement with the scenario of two different pools of vesicles. In these experiments the synaptic terminal attaches tightly to a coverslip. The brightness of a fluorescently labeled vesicle increases as it approaches this site. At the fusion, the fluorescence is incorporated into the plasma membrane. The fusion was found to occur at specific spots in the membrane probably corresponding to Ca2+-channel clusters. Vesicles close to the membrane fused quickly. In contrast, vesicles held 20 nm away from the cell membrane were allowed to advance to the exocytotic sites and could fuse with the plasma membrane 0.25 s later The recovery from the neurotransmitter discharge, although much slower than the exocytotic events, is nevertheless relatively quick occurring in about 10 s (Borges et al., 1995; Stevens and Tsujimoto, 1995; Rosenmund and Stevens, 1996). Ca2+ and the synaptic vesicles' attachment-detachment from cytoskeletal elements are thought to play an important role in all these events. Involvement of Ca2+ As in other kinds of exocytosis, Ca2+ plays a role at the synapse. Presynaptic depolarization discharges neurotransmitter only when Ca2+ is present. Chemicals that block Ca2+ channels (e.g., Cd2+ or Mn2+) also block chemical synapses. The role of presynaptic Ca2+ can also be demonstrated more directly. Electrophysiological measurements of Ca2+-current have shown that Ca2+ influx is followed by http://www.albany.edu/~abio304/text/22part3.html (1 of 26) [1/9/2003 12:15:29 PM]
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neurotransmitter release within 200 µsec (Llinas et al., 1981). One of the pieces of evidence is shown in Fig. 21, discussed later. In this figure, the middle trace corresponds to the Ca2+ current which precedes the postsynaptic response, well within the time needed for synaptic transmission. Other data involving chelators agree with this notion. The intracellular presynaptic microinjection of the slowly binding chelator EGTA is ineffective in blocking neurotransmitter release (Adler et al., 1991). However, the fastbinding chelator BAPTA (1,2-bis (2-amino phenoxy) ethane-N,N,N,N-tetraacetic acid), which has similar binding constants, blocks synaptic transmission. The rapid response of the presynaptic processes suggests that the machinery for the release must be already in place. These experiments also suggest the Ca2+ concentration that triggers release must be 100 µM or more (Adler et al., 1991). The resting concentration of Ca2+ is below the µM range. Many effects of Ca2+ are thought to be triggered by concentrations in the range of 1-5 µM. However, Ca2+ concentrations as high as 100 µM are entirely possible in microdomains of the presynaptic processes. The photoemission of the protein aequorin (see Chapter 1) when injected into nerve terminals indicates Ca2+ concentrations in this range (Llinas et al., 1992). A high local concentration could be explained by the proximity of the system delivering the Ca2+ to the vesicles. Freeze-fracture studies indicate that vesicle fusion takes place close to or at the location of intramembranous particles (Heuser, et al., 1974) that are thought to correspond to the Ca2+ channels. Other observations also indicate that the association between Ca2+ flux and neurotransmitter release is an intimate one. Measurements of Ca2+-current and appearance of neurotransmitter using patch-clamping techniques in the study of chick ciliary ganglia suggest that the opening of a single Ca2+-channel is sufficient for the release of a quantum of acetylcholine (Stanley, 1993). Role of the cytoskeleton and the synapsins Synaptic vesicles are attached to the cytoskeleton. Quick-freeze, deep-etch EM (Hirokawa et al., 1989, Gotow et al., 1991) found it to consist mainly of microtubules and actin filaments. The actin filaments form a network frequently associated with the presynaptic plasma membrane and extending to the cortical areas devoid of microtubules (Landis et al., 1988; Hirokawa et al., 1989). The attachment of the vesicles to the actin is mediated at least in part by proteins of the synapsin family (Valtorta et al., 1992, Greengard et al., 1993), derived from differential splicing of the transcript of two synapsin genes (one for synapsin I and the other for synapsin II). Synapsins are present in virtually all nerve terminals, where they are exclusively associated with the cytoplasmic surface of the synaptic vesicle membrane. Synapsins could be recognized using immunogold EM techniques, or from their distinctive morphology with a head region 14 nm in diameter and a filamentous portion 33 nm long. Single synapsin molecules were found to cross-link actin filaments to other actin filaments and microtubules throughout the head region. The tail region was found to bind to the vesicles. The connecting portion was found to be about 30 nm in length. Longer and thinner strands (about 100 nm in length) were also found associated with vesicles and the plasma membrane.
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Does the association of the vesicles to the cytoskeleton play a role in transmitter release? Many biological processes are regulated by the phosphorylation and dephosphorylation of proteins (e.g., Chapters 13). Phosphorylation-dephosphorylation cycles of the synapsins are thought to function in determining the availability of the vesicles loaded with neurotransmitter. The phosphorylation of synapsin was found to increase under all conditions that promote Ca2+-dependent release of neurotransmitter, as summarized in Table 3 (Nestler and Greengard, 1984). The properties of synapsin Ia and Ib are summarized in Table 4 (Nestler and Greengard, 1984). Part A of the table summarizes the physicochemical properties, part B, the protein kinase specificity and part C the site of their occurrence. The phosphorylation is catalyzed by either cAMP or Ca2+ -dependent protein kinases. As indicated in Table 4, synapsin I is phosphorylated by Ca2+-calmodulin dependent protein kinase II (CaM kinase II). This latter phosphorylation is very rapid during physiological activity of neurons. Phosphorylation causes a conformational change in the synapsin I molecule (Benfenati et al., 1990). A variety of experiments suggests that unphosphorylated synapsin constrains the vesicles so that they fail to provide neurotransmitter; this constraint is removed by phosphorylation. Therefore, phosphorylation would be the trigger for neurotransmitter release. Micromanipulation experiments support this view. Microinjection of unphosphorylated synapsin I into neurons inhibits neurotransmitter release (Llinas et al., 1985). In contrast, injection of the phosphorylated form has no effect. In addition, the introduction of CaM kinase II either has no effect or increases neurotransmitter release. We have seen that Ca2+ is involved in the release of the vesicles. Therefore, it would seem possible that Ca2+ triggers the phosphorylation of the synapsin, most likely by acting via CaM kinase II. Several experiments support this view. Table 3 Physiological and pharmacological regulation of synapsin From Nestler and Greengard, 1984. Reproduced by permission.
1. In synaptosomes and slices of nervous tissues, depolarizing agents and cyclic AMP increases the phosphorylation 2. In specific anatomical regions of central and peripheral nervous system, the relevant neurotransmitter (serotonin, dopamine, norepinephrine, adenosine) increase state of phosphorylation 3. In isolated peripheral nervous tissue and in posterior pituitary 4. In whole animals, convulsants increase and depressants decrease state of phosphorylation in cerebrum 5. In whole animals, neurotransmitters and hormones increase total amount in specific brain regions http://www.albany.edu/~abio304/text/22part3.html (3 of 26) [1/9/2003 12:15:29 PM]
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The relationship between internal Ca2+, synapsin and neurotrasmitter release was shown in experiments of Llinas et al., (1991) on the squid giant synapse. The amount of presynaptic neurotransmitter release was followed by measuring postsynaptic potential, which is proportional to the concentration of neurotransmitter. The presynaptic potential and the Ca2+ current were also recorded. Fig. 20 (Llinas et al., 1991) shows presynaptic potentials (in this case action potentials) and the postsynaptic potentials, before and after injection of dephosphorylated synapsin I in the presynaptic cell. The numbered curves represent the postsynaptic potentials at the time in minutes indicated by the number. As shown, the presynaptic potential is not affected. In contrast, the postsynaptic potential is almost completely blocked 24 minutes after microinjection. Fig. 21 (Llinas et al., 1991) represents the postsynaptic potential (upper curves) and the presynaptic Ca2+ currents (middle curve) when the presynaptic voltage (lower record) is clamped at 25 mV. The numbers on the left of the figure represent the time after microinjection of dephosphorylated synapsin I. The postsynaptic potentials are inhibited by the microinjection. As indicated, the Ca2+ current is not affected, showing that the defect is between the internal Ca2+ signal (which is normal) and the release of the neurotransmitter. Whereas unphosphorylated synapsin blocks exocytosis of the neurotransmitter, the Ca2+-dependent phosphorylation facilitates release, as shown by the effect of the injection of CaM kinase II. Similar results were obtained in experiments using isolated synaptosomes (vesicles corresponding to neuron terminals) from the rat brain. Synapsin I was introduced into the vesicles by transient permeabilization using freeze-thawing (Nichols et al., 1992). Unphosphorylated synapsin I decreased the K+-induced release of the neurotransmitter glutamate. Increased external K+ depolarizes the vesicle membranes. In contrast, the introduction of CAM kinase II was without effect as expected for a system where the synapsin is already phosphorylated. Other experiments showed that dephosphorylated synapsin I, when injected presynaptically, decreases the spontaneous miniature postsynaptic potentials (mESPs) in goldfish Mauthner axons (Hackett et al. 1990). These potentials correspond to spontaneous quantal release of neurotransmitter as mentioned in Section V, Part A.
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Fig. 20. Potentials from the squid giant axon synapse before and after injection of synapsin I. No significant changes in presynaptic voltages was observed. However, synaptic transmission was competely blocked after 24 min. Reproduced from Llinás et al., 1991, by permission.
Fig. 21 Effect of presynaptic injection of synapsin I. Top: postsynaptic voltage. Middle: presynaptic Ca2+current. Bottom: Constant presynaptic voltage pulses were delivered at regular intervals before and after
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injection. The numbers at left give the time intervals at which the presynaptic responses were recorded. From Llinas et al., 1991, reproduced by permission.
How can synapsin control the fate of synaptic vesicles? The model represented in Fig. 22 (Greengard et al., 1993) summarizes the mechanism that is implicated by current data. Before stimulation, synapsin cross-links the reserve pool of vesicles to other vesicles or actin (1). Upon stimulation, the influx of Ca2+ activates CaM kinase II, which catalyzes the phosphorylation of synapsin I (2). The dissociation vesicles and actin allow the vesicles to become part of the readily releasable pool (3). After exocytotic discharge, the vesicles are recycled (4,5). When a phosphatase dephosphorylates synapsin, the dephosphorylated synapsin again crosslinks actin and the vesicles (6), to reinitiate the cycle.
Fig. 22. Model illustrating how the state of phosphorylation of synapsin I regulates the availability of synaptic vesicles to exocytosis. 1. Resting conditions: synapsin I () cross links actin to vesicles . 2.Activation of CaM kinase II phosphorylates synapsin I () .3. The complex is disrupted. 4. The vesicle fuses with plasma membrane. 5. Retrieval of vesicle. 6. Phosphatase dephosphorylates synapsin I, which can now cross-link the vesicle to the actin. Reproduced with permission from Greengard, P., Valtorta, F., Czernik, A.J. and Benfenati, F. (1993) Synaptic vesicle phosphoproteins and regulation of synaptic function, Science 259: 780-785. Copyright ©1993 American Association for the Advancement of Science.
Table 4 Physicochemical properties, protein kinase specificity, and distribution of Synapsin I. From Netler and Greengard, 1984. Reproduced by permission.
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A. Physico-chemical Properties
Molar proportion Molecular weight Isoelectric point Stokes radius Sedimentation coefficient Frictional ratio Acid soluble Amino acids Other structural features
Synapsin Ia 1 86,000 10.3 59
Synapsin Ib 2 80,000 10.2 59
2.9 S 2.2 yes rich in proline and glycine a collagenase-insensitive domain and a proline-rich collagenase sensitive domain
2.9 S 2.2 yes rich in proline and glycine a collagenase-insensitive domain and a proline-rich collagenase sensitive domain
B. Protein kinase specificity Synapsin I undergoes multisite phosphorylation 1. One serine residue (site 1) in the collagenase-insensitive domain of Synapsin I is phosphorylated by cAMP-dependent protein kinase and by Ca2+/calmodulin-dependent protein kinase I. 2. Two serine residues (sites 2 and 3) in the collagenase-sensitive domain of Synapsin I are phosphorylated by Ca2+/calmodulin-dependent protein kinase II. 3. Not an effective substrate for cyclic cGMP-dependent protein kinase or Ca2+/phosphatidylserinedependent protein kinase.
C. Distribution 1. Present in nervous system (both central and peripheral) 2. Within nervous system, present only in neurons 3. Within neurons, concentrated in presynaptic terminals 4. Within terminals, associated with synaptic vesicles 5. Present in virtually all synapses http://www.albany.edu/~abio304/text/22part3.html (7 of 26) [1/9/2003 12:15:29 PM]
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6. Not present in adrenal chromaffin cells 7. Appears simultaneously with synapse formation during development
This model is supported by several findings. Synapsin binds to synaptic vesicles with high affinity. The binding is saturable, indicating the presence of specific binding sites. The binding affinity is decreased when synapsin I is phosphorylated by CaM kinase II (Huttner et al., 1983, Schiebler et al., 1986). In resting cells, 96-97% of the synapsin I is bound to vesicles (Schiebler et al., 1986, Benfenati et al., 1991). Synapsin I interacts in vitro with cytoskeletal proteins, including actin (Baines and Bennett, 1985, 1986; Goldenring et al., 1986; Bähler and Greengard, 1987; Petrucci and Morrow, 1987). The binding of synapsin II to actin produces actin bundles in vitro; this bundling is decreased when the synapsin is phosphorylated by CaM kinase II (Bähler and Greengard, 1987, Petrucci and Morrow, 1987). Therefore, synapsin is probably responsible for the trapping of the vesicles in the cytoskeletal network, and synapsin can release the vesicles when phosphorylated in response to an increase in Ca2+. A physiological cycle of this kind is supported by computer simulations (Benfenati et al., 1991). The carboxyl terminal of the synapsin bound to a vesicle protein has been implicated in the phosphorylation-dephosphorylation binding cycle (Benfenati et al., 1989a, 1989b). One of the proteins binding to synapsin has been shown to be the subunit of CaM kinase II (Benfenati et al., 1992), which would be consistent with the rapid response required for synaptic transmission. Myosin IIA and IIB and myosin V are thought to be involved in the passage of vesicles from the reserve zone to the fusion competent vesicles. Myosin V has been implicated in the transport of post-Golgi secretory vesicles (e.g., Govindan et al., 1995), and the tail region of brain myosin V binds to the synaptic vesicle proteins synaptobrevin II and synaptophysin (Prekeris and Terrian, 1997). Myosin II has been shown to be present in presynaptic terminals. The heavy chains of myosin IIA and IIB bind to acid phospholipids, are capable of assembly and may play a role in neurotransmitter release (Mochida et al., 1994). Membrane fusion Neurotransmitter release requires targeting of the vesicles to the presynaptic plasma membrane followed by fusion of the membranes (see Jahn and Südhof, 1999). Membrane fusion and secretion are general processes of cells and many of the components have been found to be very similar, from yeast to mammalian neurons. Many of the details have been elucidated for the synaptic vesicle fusion with the presynaptic plasma membrane. A cell-free system has been developed suited for the systematic study of exocytosis in a neuroendocrine cell line (Avery et al., 2000). In this assay, cells are loaded with acridine orange. Sonication produces flat vesicles with attached vesicles. The release of the dye and the disappearance of labeled vesicles allow http://www.albany.edu/~abio304/text/22part3.html (8 of 26) [1/9/2003 12:15:29 PM]
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following exocytosis This kind of assay could accelerate progress in this area by finding what factors could substitute for cytosol. The formation of vesicles was demonstrated with EM and atomic force microscopy. In vitro systems (for a review see Rothman and Orci, 1992) have provided enough information to allow the formulation of models that suggest what might be taking place in the various cells and organisms. These propose the interaction of several proteins. The N-ethylmaleimide sensitive factor (NSF) and α, β and γ-SNAP (soluble NSF attachment protein) form a 20 S complex required for all fusion events (see Chapter 11). The fusion would require specific receptors both in the vesicle (vSNARE) and the target membrane (t-SNARE). Each receptor would be specific for the vesicle and appropriate target. The proteins isolated by affinity chromatography (Söllner et al., 1993a) using NSF and α-SNAP, were identified as syntaxin a and b, SNAP-25 and synaptobrevin. Syntaxins are associated with the plasma membrane identifying them as t-SNARE. Synaptobrevin is associated with vesicles identifying it as a vSNARE. SNAP-25 is thought to bind to syntaxin. Syntaxin is also intimately connected to Ca2+ channels (Yoshida et al., 1992, Sheng et al., 1994), suggesting that the release of the neurotransmitter from a docked vesicle could occur almost instantaneously. The sequence of events could be as diagrammed in Fig. 23 (Söllner et al., 1993b). The specific association of the components of the vesicle or plasma membrane, only requires the presence of t- and v-SNARES (syntaxin, SNAP25 and vSNARE). In the absence of SNAPs and NSF, the three SNARE proteins form a complex that can bind synaptotagmin. α-SNAP can displace synaptotagmin from its binding site (reaction 1). In this model, synaptotagmin acts as a clamp, preventing the fusion of membranes. When synaptotagmin is displaced by α-SNAP, the complex can bind NSF needed for the fusion reaction (reaction 2). ATP hydrolysis displaces the complex and initiates fusion (reaction 3). We saw that Ca2+ is needed for exocytosis. It might have a role in binding to synaptotagmin so that α-SNAP will be able to bind to the active site releasing the clamp. Contradicting this model, however, the addition of Ca2+ to the in vitro system was not found to release synaptotagmin from the SNARE complex. However, this might indicate that an additional component is still to be found. Synaptotagmins constitute a family of proteins with at least twelve isoforms with a unique amino-terminal domain and a conserved carboxy-terminal domain (see Schiavo et al., 1998; Craxton and Goedert, 1999).
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Fig. 23 Model of vesicle fusion to the plasma membrane during exocytosis. From Söllner et al., 1993b, reproduced by permission.
The release of neurotransmitter from the presynaptic terminals may also be modulated by second messengers. Stanley and Miroznik (1996), using patch-clamp techniques on chick ciliary ganglia, were able to show that the opening of the Ca2+-channels depended on the activation of a GTP-binding protein. The response required intact syntaxin. Recycling and recovery The release of neurotransmitter by exocytosis has to be matched by the recovery of vesicles to be reloaded with neurotransmitter (see Cremona and De Camilli, 1997). The most widely accepted model proposes retrieval by clathrin mediated endocytosis (e.g., see Heuser and Reese, 1973). For a general discussion of endocytosis, see Chapter 9. However, other models have been formulated and the various likely mechanisms are not mutually exclusive. An alternative mechanism that also can coexist with clathrinmediated endocytosis, is the so-called "kiss and run" model (see Fesce et al., 1994). In this model, vesicles never lose their identity and the rapid closure of the temporary fusion pore reconstitutes the http://www.albany.edu/~abio304/text/22part3.html (10 of 26) [1/9/2003 12:15:29 PM]
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vesicle. There are arguments that support this view. There are intermediate structures before the exocytotic pore is open entirely and these are capable of releasing secretory products (Chow et al., 1993; Alvarez de Toledo, 1993) (see also Chapter 10). The exocytotic pore can close again after transient opening (Fernandez et al., 1984) and is capable of flickering between open and closed states (Breckenridge and Almers, 1987). An additional mechanism has been proposed involving internalization via large vacuoles (Artalejo et al., 1995). A role of clathrin-coated vesicles in neuronal endocytosis is supported by genetic studies in invertebrates. The component of the clathrin coats of neuron terminals include clathrin, the clathrin adaptor AP-2 (a heterotrimer composed of α, β, µ and σ subunits) and the protein AP180. In Drosophila, mutation of the α-adaptin gene blocks synaptic vesicle formation in nerve terminals (Gonzalez-Gaitan and Jackle, 1997). Mutations of a gene with homology to the µ subunit of AP-2, contributes to lethality in Drosophila (Petrovich et al., 1993). Several other proteins have been found to be involved in synaptic endocytosis. Endophilin I is a brain tissue protein. It contains an src-homology domain (SH3). This domain binds to the proline rich region of dynamin and two other proteins found at the synapse: synaptojanin and amphiphysin (Micheva et al., 1997; Ringstad et al., 1997). In fact, clathrin, dynamin and endophilin I are required to form vesicles in a system reconstituted from perforated rat neuroendocrine PC12 cells in a mixture containing cytosol and ATP (Schmidt and Hutter 1998). A deletion mutant lacking the SH3 domain of endophilin I cannot interact with dynamin and is incapable of mediating the formation of vesicles (Schmidt et al., 1999). Endophilin I is a lysophosphatic acid acyl transferase which catalyzes the formation of phosphatidic acid (two acyl chains) from lysophosphatic acid (one acyl chain) and arachidonoylCoA. Endophilin I is present in the cytoplasmic leaflet of the plasma membrane. This is an interesting finding because it implicates the complex in controlling the lipid components of the membrane. Furthermore, endophilin I may play a role in the formation of vesicles by altering the membrane curvature to facilitate invagination during endocytosis (see Schmidt et al., 1999). Presumably this would be accomplished by increasing the surface area of the cytoplasmic leaflet (to become the external leaflet of the vesicle). The recycling of neurotransmitter at the presynaptic terminal also differs from the recycling of conventional secretion. Rather than linking endocytosis to retrograde intracellular transport, the presynaptic vesicles recovered by endocytosis remain at the terminal, where they are rapidly reloaded with neurotransmitter. The synaptic vesicles that are synthesized in the cell body are transported to the terminals by the microtubular system and assembled from two different types of precursor vesicles (Okada et al., 1995). The synthesis of neurotransmitter does not take place at the cell body (see Siegel et al., 1994), where other secretory products are produced. Rather, neurotransmitter synthesis involves several biochemical reactions that may occur in different compartments close to or at the terminal. Synaptic organelles, such as mitochondria (e.g., in the production of glutamate), or even the glial cells, may be involved. The reloading of the vesicles with the neurotransmitter requires active transport mediated by V-ATPase. http://www.albany.edu/~abio304/text/22part3.html (11 of 26) [1/9/2003 12:15:29 PM]
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This ATPase resembles the mitochondrial F1-ATPase. In contrast to F1, however, the V-ATPase is not an ATP-synthase. It links neurotransmitter translocation to proton transport (McMahon and Nicholls, 1991). There are four transporters, one each for acetylcholine and glutamate, one for biogenic amines, and another for amino butyric acid (GABA) and glycine (Nelson, 1992, Fordac, 1992). C. Nitric Oxide NO mediates many signals in the peripheral or central nervous system, either as an intracellular second messenger or as a neurotransmitter. NO differs from other neurotransmitters because it does not have an elaborate machinery for its release and can diffuse rapidly to its target. In addition, it has a short half life and can only affect nearby targets. NO binds to the iron of heme groups and can produce conformational changes in proteins, such as guanylyl cyclase. This enzyme contains a heme, responsible for its activation by NO (see Wolin et al., 1982). NO synthase (NOS) synthesizes NO from L-arginine. NOSs are induced by various cytokines and bacterial products and are extensively present in the central nervous system. That NO is a neuronal messenger was shown in experiments in which N-methyl-D-aspartate (NMDA) added to granule cells increased cGMP. NMDA is a ligand for glutamate channel receptors. NO was implicated because the increase was blocked by NOS inhibitors and hemoglobin (Garthwaite, 1988). NO is produced postsynaptically, in response to Ca2+ influx following the activation of the NMDA channel receptors (NMDARs) or release from internal stores. Ca2+ binds calmodulin that, in turn, activates NOS. The newly formed NO then can act as a retrograde signal on the presynaptic process. As a retrograde transmitter, NO increases cGMP synthesis and potentiates synaptic transmission in the brain (Garthwaite et al., 1988; O'Dell et al., 1991; Schuman and Madison, 1994). NO activates cyclicnucleotide-gated channels (CGN) and triggers the release of glutamate (Savchenko et al., 1997). NMDA receptor channels are an unusual kind of glutamate receptors that usually require depolarization to activate Ca2+ fluxes. NO-releasing compounds have been shown to reduce NMDA-induced currents. NO is thought to act as an oxidizing agent on closely spaced cysteine residues of NMDA. In this way, NO acts as a feedback inhibitor. This feedback inhibition may have a role in long-term potentiation (LTP) (see Schuman and Madison, 1994) discussed in the next section. VI. Plasticity of Synapses Transmission of impulses through the neuron by action potentials is largely an all-or-none effect that, within limits, does not change significantly. Events at the synapse are capable of being modified. The efficacy of a synapse does depend on previous experience. This phenomenon goes by several names such as plasticity, and involves neurotransmitter release and responses to the neurotransmitter. This is the topic http://www.albany.edu/~abio304/text/22part3.html (12 of 26) [1/9/2003 12:15:29 PM]
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of this section. The properties of synapses have suggested the involvement of pre- and postsynaptic events in memory and learning. The actual de novo formation of synapses may also come into play and is discussed at the end of this section. The magnitude and significance of this plasticity can only be appreciated if we consider that cortical projections in adult animals are continuously being changed by experience (see Buonomano and Merzenich, 1998). This cortical map reorganization results from synaptic plasticity. For example, sensory deprivation was found to alter short-term synaptic excitatory pathways within the supragranular cortex (Finnerty et al., 1999). Potentiation and depression Learning is the modification of behavior by experience. Memory is the retention of sensory experiences or learning. Since the early discoveries of Ramón y Cajal, the cellular organization of the nervous system has suggested an involvement of events at the neuronal level. The discovery that the strength of synaptic potential (i.e., extent of response following a signal) is plastic and varies as a function of activity and use (Feng, 1941) has suggested possible mechanisms. The increase in strength has been termed potentiation. The opposite phenomenon has been termed depression. Brief periods of high frequency presynaptic activity (elicited by high frequency electrical stimulation called tetanus) can enhance the synaptic potential for minutes or even hours. Similarly, the synaptic potential can be depressed by low frequency presynaptic activity (Lloyd, 1949; for a review see Bear and Malenka, 1994). Certain aspects of this problem have been studied in the large marine snail, Aplysia. Genetic approaches have used Drosophila. The hippocampal region of mammalian brain has also been found useful. This region is responsible for the initial storage of memory (Squire, 1992). In the mammalian system, many observations support the consolidation theory of memory, according to which newly acquired sensory information is transmitted from the cortex to the hippocampus. In the hippocampus learning is established first and then transmitted later, repetitively, to the cortex. In the cortex, memories are stabilized (i.e. consolidated) and available even if the hippocampus is removed (see McGaugh, 1966; Squire 1992). Presumably, the hippocampus is necessary for declarative (recognition) memory: the ability to recollect everyday events and factual knowledge. These memories are distinct from nondeclarative memory in that nondeclarative (implicit) memory such as skills and habits, simple conditioning, and the phenomenon of priming abilities which do not require the hippocampus. The hippocampus is needed temporarily to coordinate events taking place in the neocortex that together represent a whole memory. Long term potentiation (LTP) has been considered a good model for learning (see Bliss and Collingridge, 1993). A single brief tetanus increases synaptic strength but then in a few hours it decays back to the original level. Longer duration tetanus produces an LTP that lasts for 8 hours in hippocampal slices and longer in intact animals (late LTP, LLTP, defined as the potentiation that lasts more than 3 hours). The phenomenon opposite to LTP, produced by low frequency stimulation, has been termed long-term depression (LTD) (see Linden and Connor, 1995). Basic aspects of potentiation
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As we have seen for many biological processes, insights into their mechanism have been provided by the study of simple organisms. This is also the case in the study of synaptic plasticity. Many seminal studies of plasticity have been carried out on the synapse between siphon sensory neurons and gill and siphon motor neurons of Aplysia where the withdrawal reflex, the so-called sensitization is induced by stimulation of the tail. The model (see Bailey et al., 1996) that has emerged from the various studies indicates that the neurotransmitter, serotonin initiates the short-term potentiation by binding to its receptors at the surface of the presynaptic cell. The activation of adenylyl cyclase that follows, produces cAMP which activates cAMP activated protein kinase (PKA). PKA first phosphorylates K+ channels which increases Ca2+ influx and increases the magnitude of the action potential. In addition, the exocytosis of neurotransmitter is also increased. These changes do not require new synthesis of macromolecules. In contrast prolonged stimulation or release of neurotransmitter lead to the transfer of PKA to the nucleus and the activation of transcription factors. Similar observations have been made in Drosophila As just outlined, short term changes do not depend on macromolecular synthesis (Schwartz et al., 1971) either in culture or in the intact animal. In contrast, inhibitors of transcription and translation block long term changes both in animals (Castellucci et al., 1989) and in culture (Montarolo et al., 1986). In Aplysia as well as other invertebrates and vertebrates there is a critical time period for the required protein and RNA synthesis (see Davis and Squire, 1984, Montarolo et al., 1986). One of the consequences of LTP has been found to be synaptic growth (Bailey and Chen, 1988a and b). The steps necessary to produce LTP have been delineated by activating the cascade of events without stimulation. Since stimulation of the presynaptic cell is know to result in the release of neurotransmitter, it would seem that the initial steps in either short or long potentiations must involve neurotransmitter. In fact, in the case of Aplysia, exposure to serotonin appears to be responsible for LTP in the absence of stimulation either in intact animals (Glanzman et al, 1989; Montarolo et al., 1986) or in cultures ( Rayport and Schacher, 1986). In addition, synaptic growth can be induced by microinjection of cAMP (a second messenger for serotonin) in presynaptic cells (Nazif et al., 1991). The level of cAMP increases upon exposure to serotonin and this in turn increases the active form of protein kinase A (PKA). With repeated stimulation, PKA enters the nucleus and phosphorylates cAMP-responsive element binding protein(CREB)-related transcription factors (Bacskai et al., 1993) which act by binding to the cAMP response elements (CRE) (see Chapter 7). Present evidence indicates that CREB1 activates the transcription factors involved in LTP whereas CRB2 (the latter not induced by serotonin) is inhibitory. The synthesis of proteins near synaptic sites (e.g., Aakalu et al., 2001) plays a major role in plasticity (see Jiang and Schuman, 2002). Apparently, the periphery is independent of the cell bodies for this function. As might be expected, the components of the translational machinery have been found in dendrites (e.g., Gardiol ete al., 1999). In agreement with the notion that this machinery plays a role in plasticity, some of the mRNAs present at dendrites are involved in synaptic function (see Jiang and Schuman., 2002). For example, NR1-mRNA which codes for a subunit of NMDAR has been found in dendrites. This receptor is needed of LTP and LTD (e.g., Tsien et al., 1996). Similarly, mRNA which codes the α subunit of the http://www.albany.edu/~abio304/text/22part3.html (14 of 26) [1/9/2003 12:15:29 PM]
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calcium dependent protein kinase II (CaMKII) needed for LTP and mRNA for the brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, have been found in post-synaptic locations. Activation of TrkB is needed for LTP. Studies on RNA distribution in the cytoplasm are discussed in Chapter 2 and those of the events accompanying RNA transport at specific cellular sites in Chapter 10. The mammalian system As already mentioned hippocampus and the cortex have separate roles. This conclusion is supported by experiments in which one copy of the two genes of coding for α-calcium-calmodulin-dependent protein kinase type II (CAMKII) (see below) is inactivated in mice. The mutation does not interfere with learning, although memory is impaired. The mutation also does not interfere with LTP in the hippocampus or the cortex. However, the LTP decays very rapidly in the cortex (Frankland et al., 2001). These experiments suggest that learning takes place in the hippocampus in pre-existing synapses. However, for the consolidation phase, the neurons of the cortex have to make new connections dependent on hippocampal replay. Many of the features just discussed for Aplysia are to be found in the mammalian systems. Inhibitors of transcription and protein synthesis (e.g., Frey et al., 1996; Nguyen et al., 1994 ) block long lasting LTP so that it decays as fast as the LTP produced by a brief tetanus. Therefore, synthesis of proteins is thought to play a role in this consolidation (Nguyen et al., 1994 ). The occurrence in two steps, a short term potentiation and a longer lasting LTP, suggest that a change occurs at the synapse first independent of protein synthesis, then followed by a second effect dependent on protein synthesis. This second effect is necessarily slow, because proteins are synthesized in the cell body and are transported the length of the axon to the synapse only slowly. Two different kinds of LTP have been recognized (Nicoll and Malenka, 1995). One involves the activation of postsynaptic N-methyl-D-aspartate receptors (NMDARs), the other, the mossy fiber LTP, has a main presynaptic component and does not involve NMDARs. The mossy fiber LTP is initiated by a rise in presynaptic Ca2+ and requires the activation of protein kinase A and the GTPase Rab3A as shown in knockout mice mutants (Castillo et al., 1997) and in addition, the RIMα, an active zone protein that binds to Rab3A and which is a substrate for protein kinase A (Castillo et al., 2002; Schoch et al., 2002). The active zone corresponds to the part of the cytoplasm from which synaptic vesicles are discharged. Mossy fiber LTP has been recognized in hippocampus, mossy fiber, cerebellar and corticothalamic synapses. Frey and Morris (1997) examined in hippocampal neurons whether newly synthesized proteins could also consolidate the signal in another synapse of the same neuron. The experiment was carried out as follows. The first synapse was stimulated with repeated tetani. The second synapse was examined to see whether LLTP could be induced by a single tetanus. The wave of protein synthesis induced by the repeated tetani of the first synapse permits consolidation of the LTP of the second synapse. However, it had to be stimulated within 90 minutes of the initial repeated tetani. Therefore, the new proteins are available to all http://www.albany.edu/~abio304/text/22part3.html (15 of 26) [1/9/2003 12:15:29 PM]
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synapses of the same neuron. The results suggest that only a transient change in gene expression is necessary to provide a prolonged response. Apparently the induction of LTP creates a synaptic specific receptor (called "tag" by Frey and Morris) that accepts the gene product. In the presence of the receptor, the material supplied by the gene leads to consolidation of the synaptic enhancement. The complex events that lead to LTD or LTP postsynaptically, are initiated by the activation of NMDARs (activated by tetanus) (see Section A, above). It would therefore seem logical to examine changes in behavior of NMDARs or receptor associated proteins accompanying LTP or LTD. The NMDARs are formed by the assembly of NR1 subunits with one or more NR2 subunits (see Hollman and Heinemann, 1994; Nakanishi, 1994) which assemble into a channel responsible for conducting Ca2+ into the cell. The molecular size of native NMDA receptors is approximately 605-850 kDa, consistent with the assembly of four to five subunits (e.g., Blahos and Wenthold, 1996). There are several possible NR2 subunits (A to D) whose deployment is developmentally and regionally regulated. The subunits determine the properties of the channel. For example, the NR2B subunits (implicated in LTP and learning, see below) permit a higher amount of Ca2+ to enter cells. The NR2 subunits have long carboxy-terminals in the cytoplasm that contain phosphorylative and protein interaction sites (Smart, 1997). The NMDARs initiate the events of synaptic development and plasticity in the central nervous system by allowing the entry of Ca2+ (see Ghosh and Greenberg, 1995). The Ca2+-binding protein, calmodulin (see Gnegy, 2000) and calcineurin have been implicated in these mechanisms (see below). An involvement of NMDARs in LTP and learning is supported by experiments in rats where a drug (D,L2-amino-5-phosphonopentanoic acid, AP5), acting as an NMDAR antagonist, blocks the induction of LTP and interferes with the rats' ease of finding their way around a maze (Morris et al., 1986). Furthermore, deletion (see Chapter 1) of the NMDAR1 gene in the CA1 pyramidal cells of the hippocampus, produced a mouse strain (Tsien et al., 1996) that had lost the NMDAR-mediated synaptic currents and LTPs in the CA1 synapses. The mice also exhibited impaired spatial memory but unimpaired nonspatial learning. These experiments indicate that the NMDA receptors of the CA1 synapses are needed for the acquisition of spatial memories. More recent experiments lend strong support to the involvement of the NMDA receptors. Transgenic mice that overexpress NR2B show an increase in NMDAR mediated current (Tang et al., 1999). Furthermore, they have and enhanced level of LTP and learning (such as fear conditioning to a shock paired to a sound). Although NMDARs have an important role in LTD or LTP, the deployment of AMPAR receptors (AMPARs) (see above) is responsible for the post-synaptic changes in excitability. The AMPARs are hetero-oligomers of three subunits in different combinations (GLUR1-GLUR4). In the adult hippocampus, the combinations of AMPAR subunits are GluR1/GluR2 or GluR2/GluR3. GluR4 is expressed mostly in early development. The number of receptors at the cell surface result from a steady state between insertion and removal, the latter via chlathrin dependent endocytosis (see Chapter 9). The receptors are removed or added to synapses depending on whether these are weakened or strengthened (Carroll et al., 1999; Zamanillo et al., 1999; Shi, et al., 1999). A role of these receptors in LTP or LTD is supported by the decrease in the AMPAR during LTD (Carroll et al., 1999) as shown by immunological http://www.albany.edu/~abio304/text/22part3.html (16 of 26) [1/9/2003 12:15:29 PM]
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microscopic techniques (see Chapter 1). In other experiments, an increase of post-synaptic AMPAR at the synapse accompanying LTP was actually demonstrated (Shi et al., 1999). Neurons of cultured brain slices were transfected with a gene coding for one of the units of the AMPARs, fused to green fluorescent protein (see Chapter 1). Two photons laser scanning microscopy (see Chapter 1) revealed the AMPARs clustering at dendritic spines. After LTP was induced, more AMPARs were inserted at the tips of the spines, supposedly the site of the synapses. In addition, it has been shown (Zamanillo et al., 1999) that the absence of functional AMPARs precludes LTP in at least one kind of neurons. In this study, mice strains were produced lacking one of the four subunits of AMPAR. The other three subunits can form functional receptor. However, LTP cannot be elicited from CA1 neurons generally used in LTP experiments. Since other neurons are able to undergo LTP, it can be argued that the result are the consequence of a reduced supply of AMPARs in the mutant mice. Later work allowed more clear distinctions in hippocampal neurons (Shi et al, 2001). AMPAR GluR1/GluR2 receptors were found to be added to synapses as a function of activity, where GluR1 and PDZ domain proteins interact. These receptors are likely to have a role in plasticity. GluR2/GluR3 receptors at synapses were found to follow a different pattern. They were inserted at post-synaptic membranes that already have AMPARs and replaced existing synaptic receptors continuously, an interaction which required GluR2, NSF and PDZ domain proteins. These receptors were not regulated by activity. How are the events that occur at the synapse orchestrated? Apparently the calcium/calmodulin-dependent protein kinase II (CaMKII) mediate some of the effects of the glutamate receptors. The sequence of events is initiated by the activation of the NMDAR channels. Ca2+ enters the cells via the NMDARs, activating CAMKII . In turn, CAMKII induces the insertion of AMPAR at synapses ( Hayashi et al., 2000b) and increases their conductance (Derkach et al., 1999). CaMKII and the NMDAR are bound together ( Gardoni et al., 1998).The CAMKII binding to NMDAR permits it to be transferred to synaptic sites where it would be most effective and in remaining in an active configuration ( Bayer et al., 2001). CaMKII remains active after the Ca2+ concentration returns to a lower level. The phosphatase calcineurin has been implicated in the molecular basis of LTD and LTP (e.g., Lu et al., 1996; Mansuy et al., 1998; Mulkey et al., 1994). This pathway resembles one that activates a number of genes in immunological responses (e.g., Clipstone and Crabtree, 1992; Hoey et al., 1995) (discussed in Chapter 7). In mammalian hippocampal neurons, calcium entry into L-type voltage gated calcium channels is triggered by depolarization of the postsynaptic potential (see Chapter 7) and the increased Ca2+ has been shown to activate calcineurin. The calcineurin supposedly activates the transcription factor ARc4/NF-AT3, which is then translocated from the cytoplasm to the nucleus (blocked by the immunosuppressive drugs cyclosporin A and FK506) and initiates NF-AT-dependent transcription (see Beals et al., 1997; Graef et al., 1999). Exit from the nucleus occurs when the Ser/Thr kinase glycogen synthase kinase-3(GSK-3) phosphorylates ARc4/NF-AT3 (Graef et al., 1999). These results indicate that gene expression mediated by NF-AT is involved in some manner in LTD and LTP. The cytoplasmic postsynaptic protein, PSD-95, contains domains that participate in protein-protein
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interactions including binding to the carboxy-terminal of the NR2 subunits of the NMDAR (Kornau et al., 1995; Niethammer et al., 1996). For this and other reasons, PSD-95 was thought to have a role in targeting and clustering of the NMDA receptors (e.g., see Kim et al., 1996) and, therefore, potentiation or depression. However, experiments using PSD-95 nul mice (see Chapter 1) challenge this view and suggest a much more subtle role of PSD-95. In PSD-95 nul mice (Migaud et al., 1998) the frequency function of NMDA-dependent LTP and LTD is shifted producing an increase in LTP at different frequencies of synaptic stimulation. The frequency shift is accompanied by severely impaired spatial learning. These findings indicate that PSD-95 does not have a role in the targeting and clustering of NMDA receptors, since these are still functional. However, it plays an important, still to be elucidated role in plasticity. In contrast, the NMDA receptor action has been shown to be intimately involved with the Eph receptors and ephrin system discussed below with other cell adhesion molecules. As in Aplysia cAMP-responsive element-binding protein (CREB) play a role, the involvement of a cAMPresponse element (CRE) (see Chapter 7) is suspected in LTP, because CRE-mediated gene expression occurs in hippocampal neurons during repeated tetani (Impey et al., 1996). This was demonstrated in mice transgenic for a CRE-regulated reporter construct (CRE-lacZ, whose expression can be recognized by the activity of β-galactosidase). CRE-mediated gene expression was increased after late-LTP, but not after decremental LTP. The effect required activation of PKA. Inhibitors of PKA blocked late-LTP and associated gene expression (but not decremental LTP). The signaling required for Late-LTP (but not DLTP) was found sufficient to stimulate CRE-mediated transcription. Furthermore, cAMP is generated at tetanized synapses (Chetkovich and Sweatt, 1993). Mice with a targeted (see Chapter 1) disruptive mutation in the CREB are deficient in long term memory, with normal short term memory and a corresponding deficiency in the induction of the late LTP with repeated tetani in hippocampal slices (Bourtchuladze et al., 1994). In addition, permeable analogs of cAMP produce a non-specific synaptic strengthening that resembles late LTP in hippocampal slices (Frey et al., 1993). Cell adhesion molecules Cell adhesion molecules (CAMs) are responsible for cell-cell and cell-ECM interactions (see Chapter 6). At the synapse they produce structural changes induced by long-term nerve activity (see Fields and Itoh, 1996). As we saw, intracellular-signaling systems are generated by nerve activity. These are thought to have effects on CAM expression. In Aplysia, for example, the presence of CAM at the surface of a motor neuron is affected by the neuropeptide FMFamide (Peter et al., 1994) and 5-Hydroxytryptamine (Mayford et al., 1992). CAMs have been shown to play a role in both short term and lasting plasticity (see Benson et al. 2000). Several kinds of CAMs are found at synapses, including integrins, immunoglobulin superfamily members, cadherins and the brain specific neurexins and neuroligins (see Benson et al., 2000). An involvement of CAMs in post-tetanic potentiation of short duration (E-LTP) has been shown on hippocampal slices by applying antibodies against CAMs (e.g., Tang et al., 1998; Yamagata et al., 1999)
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or the use of synthetic peptides antagonistic to dimerization of cadherins (e.g., Tang et al., 1999) or other means (e.g., Muller et al., 1996). Similarly, N-cadherin has been shown to have a role in L-LTP. In addition to all these findings, learning and memory are impaired by interfering with cell-adhesion function (e.g., see Grotewiel et al., 1998). Neural cell adhesion molecules (NCAMs) knock-out mice show impaired spatial learning (Cremer et al., 1994) CAMs of the immunoglobulin superfamily play a role in development of the nervous system but also in synaptic plasticity in the adult. The receptor-like protein tyrosine phosphatases (RRTPs) have similar properties and their role in the development of neurons has been demonstrated with Drosophila mutants. Three axonal RPTPs were shown to be required for routing and making appropriate neuronal connections (Krueger et al., 1996; Desai et al., 1996). RPTPs may also have a role in plasticity (see Peles et al., 1998). The role of the NMDA receptors in synaptic development and plasticity have been found to involve the Eph family of receptor tyrosine kinases/ephrin system. The Eph family of receptor tyrosine kinases at the surface of a cell bind to ephrin ligands attached to membranes of other cells. The two are important in regulating cell-cell interactions. The signaling between the receptor bearing cell and the ephrin bearing cell is bidirectional (Holland et al., 1996) so that information is exchanged between Eph-expressing and the ephrin-expressing cells. It was recognized early that ephrin and its receptors are regulators of axon pathfinding and neuronal cell migration during embryogenesis (e.g., see Flanagan and Vanderhaeghen, 1998). More recently the two have been found to have a role in angiogenesis, tissues such as specialized epithelia (e.g., Frisén et al., 1999) and as outlined below in neuronal plasticity. The crystal structure of the Eph receptor-ephrin complex has been elucidated (Himanen et al., 2001). The ephrins constitute a family of membrane attached ligands. Ephrins and Eph receptor tyrosine kinases are responsible for activity independent processes during development and are involved in synaptic plasticity.(see Klein 2001). Present thought suggests that in the post-synaptic neuron, NMDA activated by glutamate recruit EphB2 receptors which are in turn activated by ephrinB of the pre-synaptic terminals. The two form a bridge between the presynaptic and the postsynaptic neurons (see Klein, 2001). The action of the receptors is independent of their protein kinases activity since a truncated form of EphB2 lacking kinase activity rescued the EphB2 phenotype in null mutants (Grunwald et al, 2001). A function of the ephrins and Eph receptor system in plasticity is indicated by experiments in which mice lacking EphB2 receptor had reduced LTP at hippocampal CA1 and dentate gyrus synapses. In addition, stimuli known to induce changes in synaptic structure increase EphB2 activity (Henderson et al., 2001). The EphB receptor tyrosine kinases are found in clusters at NMDA receptors. In neurons, ephrinB2 activates EphB and facilitates the NMDA receptor-dependent influx of Ca2+. This effect results in the phosphorylation of tyrosines in NMDA receptors by activating the Src family of tyrosine kinases and increases the activation of NMDAR dependent genes (Takasu et al., 2002). The role of this system in actin polymerization accompanying the formation of growth cones and the extension of other processes important in increasing synaptic connections (see next section) is discussed in Chapter 23). The formation of synapses during development involve signals exchanged between presynaptic and http://www.albany.edu/~abio304/text/22part3.html (19 of 26) [1/9/2003 12:15:29 PM]
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postsynaptic cells, in both directions. We saw that the ephrin-Eph system operates bidredctionally. In addition, one of these signals is retrograde, from post-synaptic to pre-synaptic cells. Wnt proteins, synthesized by the post-synaptic cells have a role in the development of the pre-synaptic processes (Hall et al., 2000). In the cerebellar cortex granule cell of mammals (GC) neurons receive synapses from mossy fibers from other sections of the brain. Cultured GC neurons secrete factors that have profound effects on the structure of mossy fibers (Hall et al., 2000). Conversely, mice with mutations in Wnt-7a exhibit changes in the development of the mossy fibers. The effect is not as pronounced as might be expected, probably because other Wnts are expressed in the cerebellum and may compensate for the defect. A Wnt7 antagonist blocks the changes and these are mimicked by the presence of Wnt-7a. Cultured GC neurons from mice, release factors affecting mossy fiber axons and growth cones before innervation. Wnts are therefore likely to have a significant role in synapse formation. Synapse formation in relation to plasticity is discussed in the next section. Wnt proteins are a family of sixteen or more cysteine-rich molecules of the extracellular matrix. They have a role in determining cell polarity and embryonic patterning (e.g., see Eastman and Grossdl, 1999) and in synapse formation during development and possibly in plasticity. These proteins bind to integral membrane proteins with seven transmembrane segments, the Frizaleds proteins. One of these proteins (GSK3β) is a serine/threonine kinase with multiple targets. GSK3β activity is blocked by binding to Wnt. When active, this kinase phosphorylates microtubular associated proteins and β-catenin. When the kinase is held in check by Wnt, β-catenin is allowed to accumulate. The binding of β-catenin to a family of HMG proteins (LEF-1/TFCs) activates the Wnt target genes. Highmobility group (HMG) proteins are nonhistone nuclear proteins that play an important role in the regulation of chromatin structure and function. Catenins are proteins which link cytoskeletal proteins, such as actin, to integral proteins, such as cadherins, which interact with proteins external to the cell (e.g., see Chapter 11). Increase in synaptic connections The story of synaptic alterations in memory and learning appears to have another layer of complexity in a variety of organisms. In the marine mollusc Aplysia, long term sensitization (for more than three weeks) of the gill withdrawal reflex results in an increase in presynaptic terminals of identified neurons, as seen in serial sections (Bailey and Chen, 1988). New dendritic filapodial extensions or spines have been observed in chicks after learning an avoidance task (Patel et al., 1988), in rats after spatial learning (Moser et al., 1994) and after the induction of LTP in anesthetized rats (Andersen et al., 1996). Dendritic spines are the only known postsynaptic connections at least in the excitatory hippocampal pathway (Andersen et al., 1966). The synapse, or more precisely neurotransmitter receptors are also likely to be the target of hormones (see Wehling, 1997) with possible effects on behavior and learning. Inhibitory and excitatory effects of progesterone on receptors of neurotransmitters have been reported (Valera et al., 1992). Some neurons receive thousands of synaptic inputs through their dendrites. It is well recognized that Na+ and Ca2+ channels (e.g., Johnston et al., 1996; Yute and Tank, 1996) and more recently K+ (Hoffman et http://www.albany.edu/~abio304/text/22part3.html (20 of 26) [1/9/2003 12:15:29 PM]
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al., 1997) are present at dendrites. Accordingly, they are capable of modifying postsynaptic responses and they are likely to play an important role in the propagation and integration of information. How do connections between neurons come about? This question is of great significance not only in memory and learning, but also in the development of the nervous system and in its regeneration. The electrical activity and its temporal and spatial characteristics, shape the synaptic connections in early development (see Katz and Shatz, 1996). Similarly, memory is likely to be connected to similar morphological correlates of activity (see Bailey and Kandel, 1993). Living dissociated neurons in culture exhibit motility at the growing tips (growth cones) and filopodia (see Chapter 23, Section IV C) that can initiate both cell-to-cell contact and the formation of synapses (e.g., Ziv and Smith, 1996, Fischer et al., 1998). This motility may be regulated by neurotransmitters and second messengers (Kater and Rehder, 1995). The use of fluorescent dyes and confocal microscopy (see Chapter 1) on tissue slices confirm the results in cell culture and show extension and retraction of filopodia in whole embryos and brain tissues regulated by electrical activity or the presence of neurotransmitters (e.g., Dailey and Smith, 1996). In a recent study (Maletic-Savatic et al., 1999), neurons in developing rat hippocampal slices were transfected with a virus containing the green fluorescent protein (GFP) gene (Chapter 1, Section II B). The fluorescence was followed using a two-photon laser scanning microscope (see Chapter 1). The results confirm the presence of filopodial protrusions in dendrites and demonstrated that firing action potentials increases filopodia formation (or spines) and probably formation of synapses (as seen with the light microscope). This activity was blocked by an antagonist for NMDA receptors (APV), implicating the latter (see Section A, above). A similar study was carried out on the developing pyramidal neurons of rats where neuronal filapodia in the postsynaptic neurons form connections which depend on sensory experience. The filopodia appeared, disappeared or changed shape rapidly (minutes) (Lendvai et al., 2000). Similar results were obtained by Engert and Bonhoffer (1999) using thin slices cultured for several days. The neurons were traced after the microinjection of a fluorescent dye. An electron microscope study was carried out with mouse hippocampal slices in organotypic cultures after high-frequency stimulation (Toni et al., 1999). The study found transient remodelling of the postsynaptic membrane, followed by an increase in the proportion of axon terminals contacting two or more dendritic spines. Pharmacological blockade of LTP prevented these morphological changes. Calcium-calmodulin-dependent protein kinase II (CaMKII) plays a role in the maintenance of the synaptic architecture (see also below). This is not surprising since CAMKII is intimately involved in synaptic transmission (see above) and CAMKII is present at excitatory synapses (e.g., Lisman et al., 1997; Braun and Schulman, 1996). CaMKII overexpression (e.g., by transfection) stabilizes dendritic structure in maturing neurons. In contrast, CaMKII inhibition increases their dendritic growth (Wu and Cline, 1998). CAMKII is required for LTP (Kennedy, 1988; Lisman, 1994). The incorporation of GluR1-containing AMPARs (see above) into synapses, seems to involved in the plasticity produced by CAMKII and LTP
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(Hayashi et al., 2000). However, the target of CAMKII activity involved in LTP is still to be identified. As indicated in the next section, growth factors (neurotrophins) are essential for the proper functioning of neuronal networks (see Section VI). Indeed, neurotrophins can regulate the morphology of nerve processes (e.g., Ming et al., 1997) and the efficiency of the transmission across synapses (e.g., Sala et al., 1998; Gottschalk et al., 1998). Electrical activity can regulate the synthesis (e.g., Funakoshi et al., 1995) and release (e.g., Wang and Poo, 1997) of neurotrophins. Current evidence indicates that brain-derived neurotrophic factor (BDNF), is transmitted from the pre-to the postsynaptic cells (e.g., Fawcett et al., 2000). Some of the evidence is direct. In these experiments, cDNAs of green fluorescent protein (GFP)tagged BDNF and red fluorescence protein were microinjected into the nucleus of single neurons ( Kohara et al., 2001) . BDNF was found to move in the anterograde and to some extent in the retrograde direction. A transfer of the GFP-BDNF to postsynaptic neurons was found to be activity dependent and probably represents a secretion. In contrast the red-fluorescence protein was not transferred postsynaptically indicating that the translocation of the GFP-BDNF is specific.These observations have obvious implications for neuronal plasticity (see McAllister et al., 1999). Other observations strengthen this conclusion. LTP is reduced in the hippocampus when brain-derived neurophic factor (BDNF) is depleted (e.g., Kang et al., 1997) or in BNDF-knockout mice (e.g., Korte et al., 1995) and the deficit is rescued by exogenous BDNF (Patterson et al., 1996). These observation suggest that neurotrophins mediate at least part of the mechanism of synaptic plasticity (e.g., see Berninger and Poo, 1996; Bonhoeffer, 1996). The neurotrophic activity and the facilitation of BDNF potentiation by presynaptic activity was found to be inhibited by cyclic AMP (cAMP) inhibitors (see Chapter 7), suggesting a possible pathway for the action of BDNF (Boulanger and Poo, 1999). cAMP can trigger many processes including transcription of mRNA for receptors and neurotrophins (see Condorelli et al., 1994) and has been implicated in LTP and LTD in other experiments (see discussion above). An involvement of NMDA receptors in the production of BDNF has been discussed above (see above). Surprisingly, BDNF and neurotrophin-4/5 delivered to cell bodies of various types of neurons have been found to elicit action potentials as rapidly as the neurotransmitter glutamate (Kafitz et al., 1999). The depolarization is the consequence of the opening of sodium channels and the conductance is blocked by a protein kinase blocker of tyrosine kinase Trk receptors. Trk kinase receptors are the signaling receptors for neurotrophins (see Barbacid, 1995; see Chapter 7). These observations suggest a possible positive reinforcement mechanism between neuronal activity, increased neurotrophin secretion, followed by increased neuronal activity. A role of glial cells in the formation of synapses, at least in cell culture, suggests an involvement of these cells in the plasticity of neurons. Glial astrocytes are present at synapses throughout the central nervous system. They serve as support and in the removal of ions and neurotransmitters. In the presence of glial cells, central nervous system neurons were found to produce functional synapses seven times as frequently as in their absence (Ullian et al., 2001). These findings indicate a possible role of glia in the development of the nervous system as well as in learning and memory.
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A plasticity possibly related to all the events described in this section is the apoptotic elimination of some neurons during fetal or early neonatal development (see Chapter 2). At least in the fetal or early neonatal development of rats, pharmacological blockade of NMDA glutamate receptors for only a few hours triggered apoptosis of neurons suggesting that glutamate stimulation of NMDA receptors controls neuronal survival (Ikonomidou et al., 1999). It is now clear that new neurons can be formed in adult mammals at least in regions involved in smell and memory (e.g., see Kuhn et al., 1996). The possibility that the production of new neurons may be involved in plasticity has to be seriously considered. There is also some evidence that new neurons are formed in the hippocampus for animals to be able to associate stimuli that are separated in time (trace conditioning) (Shors et al., 2001). In this study, neuronal divisions were estimated through the incorporation of bromodeoxyuridine into DNA (which requires cell division). Bromodeoxyuridine incorporation was detected using fluorescent microscopy and immunological techniques (see Chapter 1). Treatment with an inhibitor of neurogenesis was found to block trace conditioning but not other forms of learning. VII. NEUROTROPHINS We briefly discussed the role of neurotrophin in LTP (see above. In addition, there are interactions between neurons or neurons and their target cells that areessential for their maintenance of function. Neurons degenerate when their target cellsare damaged. Similarly, post-synaptic neurons degenerate when their presynaptic neuronsare damaged.
What mechanisms could possible explain these effects? Neurotrophins play thecentral role in these interactions. Neurotrophins are growth factors that promote survivaland development of many kinds of neurons. They act on presynaptic neurons by retrogradesignaling by the post-synaptic cell (that can be a post-synaptic neuron). This is shown,for example, in
null mutations unable to synthesize the neurotrophin, or when the target cell is destroyed. Both cases result in losses of presynaptic cells (e.g., Jones et al., 1994). However, adding neurotrophins blocks this effect. A less understood effect is anterograde degeneration. The destruction of a presynaptic cell or cutting its axon results in a degeneration in the postsynaptic neuron. That a similar mechanism is operative is indicated by the prevention of anterograde degeneration by neurotrophins (see Cowan and Clarke, 1970). However, until recently, there was little known about the details of this process. Altar et al., 1997 showed with an immunological method (see Chapter 1) that the neurotrophin, brainderived neurotrophic factor (BDNF), is present in neuronal terminals and, furthermore, it is present in parts of the brain that lack BDNF-mRNA but are innervated by neurons that produce both BDNF and BDNF-mRNA. In addition, interference with axonal transport prevented the presence of the BDNF in the post-synaptic cells (in the striatum) and accumulated the neurotrophin in the cell bodies of the presynaptic neurons (cortical neurons). Obviously, the targeting of neurotrophins plays a very significant role in the maintenance of the nervous http://www.albany.edu/~abio304/text/22part3.html (23 of 26) [1/9/2003 12:15:29 PM]Kamiya, N., and Kuroda, K. (1957) Cell operation in Nitella. II. Behavior of isolated endoplasm, Proc. Jpn. Acad. 33:201-205.
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Rohatgi, R., Ma, L., Miki, H., Lopez, M., Kirchhausen, T., Takenawa, T. and Kirschner, M.W. (1999) The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly, Cell 97:221-231.(MedLine) Rottner, K., Hall, A. and Small, J.V. (1999) Interplay between Rac and Rho in the control of substrate contact dynamics, Curr. Biol. 9:640-648.(MedLine) Roy, S., Coffee, P., Smith, G., Liem, R.K., Brady, S.T. and Black, M.M. (2000) Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport, J. Neurosci. 20:6849-6861. (MedLine) Sakai, H., Mabuchi, I., Shimoda, S., Kuriyama, R., Ogawa, K., and Mohri, H. (1976) Induction of chromosome motion in the glycerol-isolated mitotic apparatus: nucleotide specificity and effects of antidynein and myosin sera in motion, Dev.Growth Diff. 18:211-219. Satir, P. (1968) Studies on cilia. III. Further studies on the cilium tip and a "sliding filament" model of ciliary motility, J. Cell Biol. 39:77-94.(MedLine) Saxton. W. M., Stemple, D. L., Leslie, R. J., Salmon, E. D., Zavortink, M., and McIntosh, J. R. (1984) Tubulin, dynamics in cultured mammalian cells. J. Cell Biol. 99:2175-2186.(MedLine) Scales, S.J. and Scheller, R.H. (1999) Lipid membranes shape up, Nature 401:123-124.(MedLine) Schmidt, A. and Huttner, W.B. (1998) Biogenesis of synaptic-like microvesicles in perforated PC12 cells, Methods: A Companion to Methods in Enzymology 16:160-169.(MedLine)9 Schmidt, A., Wolde, M., Thiele, C., Fest, W., Kratzin, H., Podtelejnikov, A.V., Witke, W., Huttner, W.B.and Soling, H.D. (1999) Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid, Nature 401:133-141.(MedLine) Schroeder, T. E. (1963) Actin in dividing cells: contractile ring filaments bind heavy meromyosin, Proc. Natl. Acad. Sci. USA. 70:1688-1692. Schvartz, I., Seger, D. and Shaltiel, S. (1999) Vitronectin, Int. J. Biochem. Cell Biol. 31:539544(MedLine) Shah, J.V., Flanagan, L.A., Janmey, P.A. and Leterrier, J.F. (2000) Bidirectional translocation of neurofilaments along microtubules mediated in part by Dynein/Dynactin, Mol. Biol. Cell 11:3495-3508. (MedLine) Sharp, D.J., Yu, W. and Baas, B.W. (1995) Transport of dendritic microtubules establishes the http://www.albany.edu/~abio304/ref/ref23.html (13 of 17) [3/5/2003 8:26:34 PM]
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nonuniform polarity orientation, J. Cell Biol. 130:93-104.(MedLine) Sharp, D.J., Yu, W., Ferhat, L., Kuriyama, R., Rueger, D.C., and Baas, P.W. (1997) Identification of a microtubule-associated motor protein essential for dendritic differentiation, J. Cell Biol. 138:833843.(MedLine) Small, J.V., Herzog, M., Anderson, K.A (1995) Actin filament organization in the fish keratocyte lamellipodium, J. Cell Biol. 129:1275-1286.(MedLine) Smilenov, L.B., Mikhailov, A., Pelham, R.J., Marcantonio, E.E. and Gundersen, G.G. (1999) Focal adhesion motility revealed in stationary fibroblasts, Science 286:1172-1174.(MedLine) Stearns, T. and Kirschner, M. (1994) In vitro reconstitution of centrosome assembly and function: the central role of γ-tubulin, Cell 76: 623-637.(MedLine) Stearns, T., Evans, L. and Kirschner, M. (1991) γ-Tubulin is a highly conserved component of the centrosome, Cell 65: 825-836.(MedLine) Suen, P.W., Ilic, D., Caveggion, E., Berton, G., Damsky, C.H. and Lowell, C.A. (1999) Impaired integrinmediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages, J. Cell Sci. 112:4067-4078.(MedLine) Summers, K. E., and Gibbons, I. R. (1971) Adenosine triphosphate-induced sliding of tubules in trypsintreated flagellae of sea urchin sperm, Proc. Natl. Acad. Sci. USA 68:3092-3096.(MedLine) Suter, D.M., Errante, L.D., Belotserkovsky, V. and Forscher, P. (1998) The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate-cytoskeletal coupling, J. Cell Biol. 141:227-240.(MedLine) Svitkina, T.M. and Borisy, G.G. (1999) Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia, J. Cell Biol. 145:100910026.(MedLine) Svitkina, T.M., Verkhovsky, A.B., McQuade, K.M. and Borisy, G.G. (1997) Analysis of actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation, J. Cell Biol. 139:397415.(MedLine) Symons, M.H. and Mitchinon, T.J. (1991) Control of actin polymerization in live and permeabilized fibroblasts, J. Cell Biol. 114:503-513.(MedLine)
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interrupted by prolonged pauses, Nature Cell Biol. 2:137-141. (MedLine) Warner, F. D., and Mitchell, D. R. (1981) Polarity of dynein-microtubule interactions in vitro: cross linking between parallel and antiparallel microtubules, J. Cell Biol. 89:35-44.(MedLine) Waterman-Storer, C.M. and Salmon, E.D. (1997) Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling, J. Cell Biol. 139:417-434.(MedLine) Waterman-Storer, C.M. and Salmon, E.D. (1998) How microtubules get fluorescent speckles, Biophys. J. 75:2059-2069.(MedLine) Waterman-Storer, C.M. and Salmon, E.D. (1999) Fluorescent speckle microscopy of microtubules: how low can you go? FASEB J. 13 Suppl 2:S225-230.(MedLine) Waterman-Storer, C.M., Desai, A., Bulinski, J.C. and Salmon, E.D. (1998) Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells, Curr. Biol. 8:12271230.(MedLine) Waterman-Storer, C.M., Worthylake, R,A,, Liu, B.P., Burridge, K. and Salmon, E.D. (1999) Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts, Nature Cell Biol. 1:4550.(MedLine) Weber, K. (1976) Biochemical anatomy of microfilaments in cells in tissue culture using immunofluorescence microscopy. In Contractile systems in Non-Muscle Tissues (Perry, S.V., ed.), pp. 5166. Elsevier/North-Holland, New York. Weber, K., Pollack, R., and Bibring, T. (1975) Antibody against tubulins: the specific visualization of cytoplasmic microtubules in tissue culture cells, Proc. Natl. Acad. Sci. USA 72:459-463.(MedLine) Weiner, O.D., Servant, G., Welch, M.D., Mitchison, T.J., Sedat, J.W. and Bourne, H.R. (1999) Spatial control of actin polymerization during neutrophil chemotaxis, Nature Cell Biol. 1:75-81.(MedLine) Welch, M.D. (1999) The world according to arp: regulation of actin nucleation by the Arp2/3 complex, Trends Cell Biol. 9:423-427.(MedLine) Wessels, D., Soll, D.R., Knecht, D., Loomis, W.F., De Lozannne, A. and Spudich, J. (1988) Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain, Dev. Biol. 128:164-177.(MedLine) Wohlfarth-Bottermann, K.E. (1964) Differentiations of the ground cytoplasm and their significance for the generation of the motive force of ameboid movement. In Primitive Motile Systems in Cell Biology http://www.albany.edu/~abio304/ref/ref23.html (16 of 17) [3/5/2003 8:26:34 PM]
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(Allen, R., and Kamiya, N., eds.), pp. 79-109. Academic Press, New York. Yang, N., Higuchi, O., Ohashi, K., Nagata, K., Wada, A., Kangawa, K., Nishida, E. and Mizuno, K. (1998) Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization, Nature 393:809-812.(MedLine) Zheng, Y., Yung, M.K. and Oakley, B.R. (1991) γ-Tubulin is present in Drosophila melanogaster and Homo sapiens and is associated with the centrosome, Cell 65: 817-823.(MedLine) Zheng, Y.X., Wong, M.L., Alberts, B., and Mitchison, T. (1995) Nucleation of microtubule assembly by a γ-tubulin-containing ring complex, Nature 378: 578-583.(MedLine)
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24. Mechanochemical Coupling: Molecular Basis I. Molecular Basis of Contraction in Striated Muscle A. Myosin and Actin B. Troponin, Tropomyosin and Paramyosin C. Cross-Bridges and Contraction D. Attachment of the Thin Filaments: α-Actinin and Vinculin E. Titin and Nebulin Titin Nebulin II. Mechanisms in Other Muscles III. Movement in Cilia and Flagella IV. Movement in the Cytoplasm A. Microfilaments and Microfilament Bundles The actomyosin system Actins Actin-binding proteins B. Myosins Unconventional myosins and vesicle movement Unconventional myosins and cell movement Sensory role of myosins Myosin and GTP-binding proteins C. Microtubular Motors Cytoplasmic dynein Kinesins Motors and mitosis D. Intermediate Filaments V. Mechanisms of Movement and Comparison of Motors A. The Movement B. Inside the Motors C. Directionality of the Movement VI. Association of Motors with Vesicles VII. Triggering of Contraction Suggested Reading
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Web Resources References Back to List of Chapters At times the extraction and purification of the molecular components of a system leads to a deeper understanding of its intimate mechanisms. Ideally, it should be possible to reassemble the various component molecules and reestablish function. Only then can the significance of the molecular elements of the system be certain and perhaps lead us to decipher the mechanism of the action at the molecular level. The experimental approach to contraction need not be different from the studies carried out with enzymes and multienzyme complexes. Conformational rearrangements of the molecules accompany many enzymatic reactions. Similarly, movement, perhaps in the form of a conformational change, is likely to play a role in the transport across cell membranes. Contraction could be considered a conformational change involving macromolecular assemblies and magnified by a variety of means. The task of elucidating the nature of events underlying contraction may be more complex than those involved in other conformational changes: in most cases intermediate compounds are not readily evident, and more ephemeral structural states may perhaps be involved. The viewpoint is emerging that many processes are similar to the movement mediated by conventional molecular motors (see Vale, 1996). A strong argument can be made that the elongation factor G involved in the movement of ribosomes along the mRNA molecule is similar to that of the motors discussed in this chapter (see Cross, 1996, Rodnina et al., 1996). Skeletal muscle as well as cilia and flagella may be considered the prototypes of cell movement because they were the first to be studied and a good deal is known about them. For this reason, actin and myosin (section I and II) and tubulin and dynein of these systems are addressed first (section III). Analogous molecules have been found to be associated with other forms of cellular movement and are discussed in section IV. I. MOLECULAR BASIS OF CONTRACTION IN STRIATED MUSCLE The study of the molecular mechanism of contraction requires some indication of activity. Since the system transduces chemical energy into mechanical energy (contraction), it could be possible to examine preparations for their capacity to contract. However, in some respects it may be more practical to examine activities that must be associated with mechanical activity, e.g., the Ca2+ dependence of ATPase that is characteristic of the myofibril system or the capacity of components to complex with other macromolecules. Several other useful assays will be described. A. Myosin and Actin The myofibrils contain protein associated with contraction: 55% of this is myosin, 20% actin, 5% tropomyosin, and 3 to 4% troponin. We will have to focus our attention on these components. Two of http://www.albany.edu/~abio304/text/24part1.html (2 of 23) [3/5/2003 8:27:18 PM]
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these, actin and myosin, can be recombined to form actomyosin. The complex can be made into a fiber by releasing it into distilled water or into a dilute solution. The fiber shortens in the presence of ATP. Because the actomyosin molecules are randomly distributed, the shortening occurs in all directions and the thread cannot displace a weight. But when actomyosin is appropriately oriented, by spreading it on a surface, for example, it will contract only longitudinally on the addition of ATP and is thereby capable of lifting weights. This experiment is shown in Fig. 1 (Hayashi, 1952). The contractions are slow -- it takes several minutes to reach the maximal level-in contrast to the very fast contraction of intact muscle. This slowness may be the result of the slow diffusion of ATP into the relatively thick fibers. It is also possible that not all the appropriate components of the system are present. During the contraction, ATP is hydrolyzed, as estimated from parallel experiments carried out under the same conditions. Events thought to correspond only indirectly to the primary events of contraction have also been found useful in studying actomyosin. For example, it has been found that the viscosity of the complex decreases on addition of ATP and then returns to normal, indicating dissociation and reformation of a complex. The viscosity depends on the shape of the macromolecules in solution. The longer the molecule, the higher the viscosity. The dissociation of the rodlike actomyosin produces a decrease in viscosity, because the axial ratios (i.e., the length to the width) of actin and myosin are much lower than the axial ratio of actomyosin. As we shall see, the dissociation of actomyosin and its subsequent reassociation from actin and myosin are likely to be part of the contractile mechanism. Actomyosin gels decrease in volume on addition of ATP (the superprecipitation reaction), a change that has also been used as an index of contractility.
Fig. 1 Percent contraction with load of actomyosin fibers. Each point represents a 15-minute contraction. From (Hayashi, 1952). Reproduced from The Journal of General Physiology, ©1952, 36: 139-151, by copyright permission of the Rockefeller University Press.
As seen with the electron microscope after negative staining, skeletal muscle myosin is a long molecule, about 160 nm in length and 20-40 nm in diameter. A single myosin unit has a molecular mass of about http://www.albany.edu/~abio304/text/24part1.html (3 of 23) [3/5/2003 8:27:18 PM]
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500 kDa and contains two heavy chains of about 230 kDa and four light chains of 16 to 20 kDa. Electron microscopic views of the monomers are shown in Fig. 2 (H.E. Huxley, 1963). When either the pH or the ionic strength is reduced, myosin monomers associate (i.e., polymerize) into thick filaments remarkably similar to the thick filaments of the myofibrils. As we saw in Chapter 23, there is evidence that the thick filaments of striated muscle are made up of myosin. In the fibers that polymerize from myosin monomers, the tail ends of the molecules cling together and the head pieces jut out. The molecules align in the filament in such a way that some of the head pieces face in one direction while others face in the other direction, forming a symmetrical composite. Figure 3a (Huxley, 1963) shows such polymerized filaments. Figure 3b is a diagrammatic representation of how these filaments are probably formed.
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Fig. 2 Myosin monomers seen with the electron microscope. Reproduced with permission from (Huxley, H. E., 1969) The mechanism of muscular contraction. Science 164:1356-1366. Copyright ©1963 by the American Association for the Advancement of Science.
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Fig. 3 (a) Myosin aggregates formed in vitro as seen with the electron microscope after negative staining. (Bar corresponds to 77 nm.) (b) Scheme showing mechanism of formation of fibrous aggregates from myosin monomers. Reproduced with permission from (Huxley, H. E., 1969) The mechanism of muscular contraction. Science 164:1356-1366. Copyright ©1963 by the American Association for the Advancement of Science.
The head portion of the individual myosin molecules at the amino-terminal appears to correspond to bridges between thick and thin filaments seen with the electron microscope in sections of sarcomeres. The head portions can be isolated after partial proteolytic digestion of the myosin as heavy meromyosin (HMM). Their ability to then combine with actin supports the view that they correspond to bridges observed with the electron microscope. The HMM fragment can be further cleaved into two smaller subfragments, S1 and S2. The myosin head pieces seen with the electron microscope after shadowing appear to be formed by two subunits (Slayter and Lowry, 1967). A model incorporating our present knowledge is shown in Fig. 4 (Stebbins and Hyams, 1979). The addition of HMM (or S1) to actin produces an appearance of many neatly arranged arrowheads. This arrowhead configuration could occur only if the HMM fragments were specifically oriented by the thin filaments. It is therefore considered to be a specific test for the presence of actin filaments and indicates that the actin itself has polarity. The ends of the actin have been named after the appearance of the arrowheads, i.e., pointed (P) and barbed (B). The actin with oriented arrowheads, as seen in the negatively stained preparation shown in Fig. 5, is referred to as decorated. The two views shown in the figure represent electron micrographs at slightly different tilts, and they permit visualization of the arrangement in three dimensions using a simple standard or homemade stereoscope.
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Fig. 4 Structure of myosin. Light meromyosin (LMM) and heavy meromyosin (HMM) and the subfragments of HMM, S1 and S2, are shown. The broken lines indicate the cleavage sites of the proteolytic digestion. From H. Stebbins and J. S. Hyams Cell Motility, with permission. Copyright ©1979 Longman Group Ltd., Edinburgh.
Actin is a smaller molecule than myosin; it has a molecular mass of 42 kDa. In its globular, monomeric conformation (G-actin), it is about 5.5 nm in diameter. In this conformation, 1 mole of actin contains 1 mole of ATP and 1 mole of divalent cation, Ca2+ or Mg2+. G-actin polymerizes in a process in which ATP is hydrolyzed to form a long thread of F-actin, a double helix 7-8 nm in diameter (Fig. 6) (Hanson and Lowy, 1963). Both the ADP derived from the ATP and the divalent cation remain attached to the actin. The divalent cation is thought to be involved in regulation of the polymerization rate. Since actomyosin threads formed from the combination of actin and myosin contract, the two components and the complex must have properties that play a fundamental role in contraction. Myosin, for example, hydrolyzes ATP. We have seen that ATP is probably the energy source for contraction. In fact, there is a good correlation between the speed of contraction of various striated muscles and their ATPase activity (Maddox and Perry, 1966).
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Fig. 5 Rabbit actin complexed with heavy meromyosin (HMM), negatively stained with 1% aqueous uranyl acetate. Stereophotographs at +6 and -6 tilt. For three dimensional depth, view with a standard stereoscope or a simple homemade prism stereoscope [see E. G. Gray and R. A. Willis, J. Cell Sci. 3:309326 (1968)]. Toward upper part of filament note clear demonstration of arrowhead orientation in relation to the filament. Electron microscopy by Barry S. Eckert and S. M. McGee-Russell, Department of Biological Sciences, S.U.N.Y. at Albany. Reproduced by permission.
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Fig. 6 (a) Electron micrograph of actin filaments extracted with water-glycerol in the presence of MgATP and EDTA. Negative staining with uranyl acetate. x36,125. (b) Electron micrograph similar to that in (a). However, the preparation corresponds to F-actin. The electron micrograph permits counting the number of globular units per turn of the two-stranded helix. x44,625. From Hanson and Lowy (1963), reproduced with permission.
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each other. What are the minimum requirements of such a model at the molecular level? The events accompanying contraction, according to the sliding-filament model, must be cyclic. At least four events must take place: (1) binding between actin and myosin, (2) the pulling of the actin filaments from the two sides of the sarcomere toward each other, (3) coupling of the hydrolysis of ATP to the appropriate structural changes, and (4) breaking of the bond between actin and myosin. Since a sarcomere shortens more than the distance between side chains or cross-bridges, the cyclic breaking and remaking of bonds must occur over a distance corresponding to several cross-bridges. In frog muscle the shortening corresponds to 30% of the sarcomere length, or about 370 nm, whereas the myosin projections are approximately 40 nm apart. Let us examine some of the properties of the actomyosin system with these steps in mind. As mentioned, it is possible to fragment the myosin molecule by proteolytic digestion. The individual fragments can be separated out and tested for various properties. The ATPase activity resides in only a portion of the myosin molecule, the heavy meromyosin associated with the thicker part of the molecule (Jones and Perry, 1966; Mueller and Perry, 1962). A basic role of the interaction between actin and myosin predicted by the sliding-filament model is supported by the observation that actin and myosin have to be complexed for shortening to occur. It is interesting to note that the complexing ability also resides in the myosin fragment that has the ATPase activity. The actin and the ATP binding sites reside in different parts of the molecule and are nearly 4 nm apart. B. Troponin, Tropomyosin and Paramyosin Myofibril contractility requires the presence of Ca2+. Muscle contraction is initiated by the release of Ca2+ from the sarcoplasmic reticulum. In vertebrates and arthropods, the Ca2+ sensitivity of actomyosin apparently depends on the presence of two proteins, troponin (Tn) and tropomyosin (Tm). In some other organisms, such as mollusks, brachiopods, and some worms (Lehman et al., 1972), a light chain of myosin seems to be directly involved in this control mechanism (Kendrick-Jones, 1974). In annelids (e.g., the sandworm Nereis), both mechanisms are present (Lehman et al., 1972). In vertebrates, the Ca2+-activation of the contractile mechanism depends on binding of Ca2+ to Tn (see Zot and Potter, 1987). Tn and Tm are located periodically along the thin filament of the myofibril (Ebashi et al., 1972; Flicker et al., 1982). Tm dimers are present in a coil-coil configuration. A representation of the possible arrangement of these components in relation to actin is shown in Fig. 7 (Payne and Rudnick, 1989).
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Fig. 7 Probable arrangement of Tn, Tm and actin in the thin filament of a myofibril. Reproduced from Trends in Biochemical Science, vol. 14, Payne, M.R. and Rudnick, S.E., Regulation of vertebrate striated muscle contraction, pp.357-360. Copyright ©1989 with permission from Elsevier Science.
Binding of Ca2+ to the regulatory site on the TnC subunit alters the interactions between Tn and Tm and actin. These structural changes increase the interaction between actin and myosin, increase the ATPase activity of actomyosin, and produce contraction (see Payne and Rudnick, 1989). Supposedly, these effects are reversed when the free Ca2+ concentration decreases. The details on how this is achieved are still not entirely clear. Actin, Tn, and Tm in 7:1:1 molar ratios form the thin filament anchored to the Z-line (Yates and Greaser, 1983). When actin polymerizes, forming a double stranded helix, the Tm molecule polymerizes head-totail so that one Tm molecule spans seven actin monomers. One Tn molecule contains three different subunits (1 TnC:1 TnT and 1 TnI) present one third of the distance from the carboxy terminal end of each Tm molecule. TnC is composed of 159 amino acids and contains four Ca2+ binding sites. Two bind Ca2+ with low affinity and are unable to bind Mg2+. Two others bind both Ca2+ and Mg2+ with high affinity. The four binding sites are thought to correspond to four homologous regions that conform to a helix-loop-helixCa2+ domain referred to as an EF hand (see Collins, 1976). The low affinity sites are those thought to have a regulatory role. Paramyosin is found in certain invertebrate muscles that are capable of maintaining tension over long periods of time with a minimum expenditure of energy (catch muscle). In some muscles paramyosin may represent as much as 30% of the total protein. Paramyosin forms threads 133 nm long and 2 nm in diameter. Intact muscle fibers, probably corresponding to paramyosin, are intermeshed with thin filaments that probably correspond to actin (Hanson and Lowy, 1961). This arrangement is thought to have some role in contraction and the catch phenomenon. C. Cross-Bridges and Contraction As we have seen, the reactions involved in contraction must be cyclic; the association and dissociation involves several cross-bridges. The isometric tension (i.e., tension at constant length) developed during
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contraction at various sarcomere lengths (Gordon et al., 1966), correlates well with the degree of overlap of thick and thin filaments, which in turn allows the maximum number of cross-bridges. Figure 8a (Gordon et al., 1966) shows a diagrammatic representation of the overlap in filaments based on studies with the electron microscope. Figure 8b is a graphical summary of the results. The tension developed is expressed as a percentage of the maximal value (ordinate). The sarcomere length, from Z line to Z line, is represented on the abscissa. The numbered arrows correspond to the numbers in the diagram (Fig. 8a). Comparison of two diagrams shows that maximal tension is developed when the overlap of the crossbridges is maximal. The tension falls when the myofibrils are stretched beyond this length and fewer bridges are in register with the corresponding point of attachment in the actin fibers. The tension also drops sharply when the myofibril is shorter than this optimal value; apparently in this case the thin filaments overlap (Fig. 8a).
Fig 8 (a) Schematic diagram of the filaments. The cross-bridges are represented by the small lines on the thick filaments.
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Fig. 8 Continued (b) Schematic summary of results. T = percent tension. The arrows along the top are placed opposite the striation spacings at which the critical stages of overlap of filaments occur, numbered as in (a). From Gordon et al., Journal of Physiology (London) 184:170-182, with permission. Copyright © 1966 The Physiological Society, Oxford, England.
. The swinging myosin cross bridge hypothesis of muscle contraction (see Lymn and Taylor, 1971) summarized succinctly much of the evidence available until recently. The hypothesis proposed a contractile cycle starting with the binding of myosin to actin through a myosin cross-bridge, the so-called rigor position, (1) in Fig. 9 (Holmes, 1997). Then ATP dissociates the actin-myosin complex by binding to the ATPase site (2), and after the hydrolysis of the ATP, myosin forms a stable complex with ADP and Pi (3). Actin then rebinds at the myosin cross-bridge (4), and the bridge undergoes a conformational change or power stroke. ADP and Pi dissociate from the myosin and the myosin-actin complex is reformed at a new position (1). This model provided a useful framework to explain many observations of molecular events complementing the formulation of the sliding filament mechanism. In general, the details of this model were difficult to demonstrate because in a myofibril only a small fraction of the total myosin heads were involved at one time. However, a change in the conformation of the S1 fragment elicited by ATP was shown by several studies (e.g., Wakabayashi et al., 1992, using X-ray scattering).
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Fig. 9 Lymn-Taylor cycle of swinging cross-bridges. The spheres represent actin monomers. The vertical bar represents the myosin thick filament (M). The nucleotide-free cross-bridge (the myosin head) is shown attached to the actin in the 45o position (in relation to the actin filament), the rigor state (1). The binding of ATP releases the actin from the cross-bridge (2), still in the 45o position. The hydrolysis of ATP puts the cross bridge at the 90o conformation (3). The release of the products of hydrolysis that puts the myosin head in the 45o position (return to 1) is the power stroke (from Holmes, 1997). Reproduced by permission.
In vitro studies of movement of myosin and actin filaments, X-ray crystallography and cryoelectron microscopy have fueled recent advances. In vitro motility assays have shown that only the myosin head (subfragment 1 or S1) is required for in vitro movement on actin filaments (e.g., Kishino and Yanagida, 1988). X-ray crystallographic studies (see Fig. 10) revealed not only the structure of S1 but suggested how movement may take place (Rayment et al., 1993a and b; Dominguez,et al. 1998) (see Fig. 11). S1 contains a globular motor domain (MD) and a long (8.5 nm)α-helical tail region at the carboxy terminal (Fig. 10). The MD contains the ATP and actin binding sites. The tail region binds the light regulatory chains [containing the calmodulin-like essential (ELC) and regulatory light chains (RLC)]. http://www.albany.edu/~abio304/text/24part1.html (14 of 23) [3/5/2003 8:27:18 PM]
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The tail can act as a lever arm capable of magnifying small conformational changes in the MD corresponding to different nucleotide induced states (Rayment et al., 1993b; Xie et al., 1994) (e.g. as shown in Fig. 11). Although we discuss the various myosin together in this section the discussion should be viewed with caution. There are several kinetic, structural and mechanical differences between the various myosins (e.g. see Jontes et al., 1997; Gollub et al., 1996). The systems that have been studied include the brush border myosin-I (BBMI) (see section IVB), rat liver myosin I (the myr-1a gene product), smooth muscle myosin II and striated muscle. In BBMI and smooth muscle there is a conformational change upon binding ADP that does not take place in skeletal muscle myosin. The angular swing induced by binding ADP is greater for BBM-I than for smooth muscle S1 (31o vs 23o). Smooth muscle lacks the rotational component of the movement found in BBM-I. In the in vitro studies, a laser trap system (see Chapter 1) has shown that movement does proceed in steps of 10-20 nm (11±1.2, mean±SD) and forces of up to 7 pN (3.4±1.2 pN, mean±SD) (Finer et al., 1994). 10 nm steps are consistent with the stroke size predicted for a swinging cross-bridge model (Huxley, 1969). The distance between cross-bridges (corresponding to S1) is 14 nm. However, other studies indicate a much shorter working stroke, one of about 4 nm (Molloy et al., 1995b; see also Block, 1995) with a force of about 1.7 pN under isometric conditions. The longer values could be the result of the inclusion of Brownian motion of the actin filament. A new instrument was developed that can manipulate individual myosin S-1 fragments using a scanning probe. Single S-1 fragments could be seen using a fluorescent label. The data of experiments with striated muscle S-1 are consistent with single steps of 5.3 nm. Two to five steps were also found to take place in succession consistent with the completion of a single ATP cycle (Kitamura et al., 1999). In this study, the S-1 fragments were attached to a fluorescent probe and biotinylated at their regulatory chain. A scanning probe was attached to a glass needle mounted on a 3-D piezoelectric scanner. The probe was coated with streptavidin so that it could attach to a single biotinylated S-1 unit. The fluorescent probe at the tip of the scanning probe were visualized with total internal reflection microscopy (Tokunaga et al., 1997) (see Chapter 1). This was accomplished in a relatively simple manner by switching from epifluorescence microscopy to objective-type achieved by translation of a single mirror in the system. Clear images of single molecules were obtained with a fluorescence-to-background ratio of 12, using a conventional high aperture objective. Different results were obtained using the single headed myosins, myr-1 from rat liver and BBMI from chicken brush-border epithelium (Veigel et al., 1999). In these experiments, an actin filament was suspended between two 1 µm plastic beads held by optical tweezers. A myosin molecule bound to nitrocellulose was brought in contact with the filament. Veigel et al. found an initial displacement of 6 nm followed by a 5.5 nm interaction 100-300 ms later. The interval between the two steps was independent of ATP concentration. However, the interval between the second step and the end of the interaction was shorter at higher ATP concentrations. In contrast, with S-1 from skeletal muscle only one http://www.albany.edu/~abio304/text/24part1.html (15 of 23) [3/5/2003 8:27:18 PM]
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5.5 ms step was detected. The substeps may correspond to intermediate steps in the ATP-hydrolysis cycle such as the release of Pi followed by the release of ADP. This view is in agreement with the image reconstruction of BMM-I head attached to actin filaments (Jontes and Milligan, 1997). The data assembled so far is consistent with the model depicted in Fig. 11 (Holmes, 1997). The proposed mechanism predicts that the length of the tail will determine the length of the power stroke. Mutants of Dictyostelium with different lever lengths were produced. In vitro motility tests of this premise found this prediction to be correct, including one case in which the lever was longer than in the wild type (Uyeda et al., 1996). Surprisingly, the α-helical lever can be replaced by the rigid structure of γ-actinin repeats (Anson et al., 1996). These findings suggest a role for the portion of the molecule near the base of the tail in producing the mechanical movement. The electron microscope data shows this to be the hinge region (Fisher et al., 1995a). In addition, many mutations in Dictyostelium myosin that interfere with movement, occur in this region (Patterson and Spudich, 1996). This portion of the molecule, intimately involved in the energy transduction, is referred to as the converter region (see Trayer and Smith, 1997). Crystal structures of S1 with bound analogues of ADP and Pi have provided insight into how ATP hydrolysis could produce mechanical work (e.g., see Fisher et al., 1995b; Smith and Rayment, 1996). Combining F-actin and S1 atomic structures (Rayment et al., 1993b; Schroeder, et al., 1993) permits arriving at a three dimensional model represented in Fig. 10 (Holmes, 1997). Cryoelectron micrograph reconstructions of actin decorated with BB myosin (Whittaker et al., 1995) or smooth muscle (Jontes and Milligan, 1997), are consistent with a rotation of the lever arm away from the rigor position on binding ADP. The movement of the lever arm during the power stroke has been referred to as the swinging lever arm model, represented in Fig. 11 (Holmes, 1997). The precise dimensions of the power stroke could not be arrived by either cryoelectron microscopy or X-ray diffraction because only the the structures of the nucleotide free and transitional conformations had been studied. Dominguez et al. (1998) have reported the crystallographic structure of myosin from chicken gizzard smooth muscle (see Fig. 12). They examined the structure of the motor domain of myosin (MD) and a complex of the motor domain attached to its ELC (MDE) while bound to nucleotide analogs. They found that tail structure shows a 70o rotation as compared to the nucleotide free S1 (Rayment et al. 1993a). This corresponds to a potential displacement of approximately 10 nm during the power stroke. The presence of the ELC changes the position of the converter region and hence that of the lever arm. Most myosins move toward the plus end of the actin filament. Myosin VI moves in the opposite direction (Wells et al., 1999). Cryo-electron microscopy and image analysis of myosin VI found an ADP-mediated conformational change in the lever arm that is in the opposite direction of that occurring in other myosins. The class VI myosins have large insertion at the converter domain, confirming the importance of this sector in the motor function of the myosins. Models can be constructed that can explain simply how direction can be reversed in a myosin that essentially has a conventional structure (see Schliwa, 1999).
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The movement of the lever arm was also followed by a technique devised for measuring motion in protein domains. This method consists in introducing two cysteine residues at a defined location in the protein. The cysteines are then crosslinked to bifunctional rhodamine (Corrie et al., 1998). The polarization of the rhodamine fluorescence can then be used to measure the orientation of the rhodamine dipole. When returned to its native environment the orientation of the protein domain can be calculated from observations on sets of these dipoles. Changes in orientation can be measured in the submillisecond range. Applied to the myosin-light chain domain located in the lever arm region, this approach shows that there is increase in tilt angle and a decrease in twist angle (Corrie et al., 1999). Generally, the findings are in agreement with the crystallographic models. In current models, as indicated in Fig. 9 [(4) to (1)], the force generation is precisely coupled to the release of ADP and Pi from the myosin head. Experiments combining two very different approaches (measurement of nucleotide binding combined to the measurement of the displacement; Ishijima et al., 1998), have indicated that the two need not be coupled. Myosin must be able to store energy so that the working stroke can occur later. Fluorescence was detected from a single molecule of a fluorescent ATP analog bound to myosin (Funatsu et al., 1995). With this technique (total fluorescence reflection) a stationary fluorescent spot indicates binding of ATP (or ADP) to the attached myosin (see Chapter 1). Disappearance of the spot indicates dissociation from myosin of either ATP or the ADP produced by hydrolysis. The simultaneous displacement of an actin filament caused by a single myosin molecule attached to a slide was measured using the optical trap method (see Chapter 1) where beads held in optical traps were attached to the ends of a single actin filament which was held firmly. Binding of the actin filament to myosin is indicated by a reduction in the Brownian motion of the beads. The use of the two techniques simultaneously confirmed that release of actin from striated muscle myosin coincided with ATP binding ([Fig. 9, (1) to (2)]. Free myosin bound ATP (or ADP). The ATP was released when actin and myosin combined [Fig. 9, (4) to (1)] during the working stroke. However, approximately 50% of the time, there was a long delay (several 0.1 s intervals) between the disappearance of the fluorescence (release of the nucleotide) on the one hand, and the decrease in Brownian motion (indicating binding of the myosin to the actin) and the displacement on the other. Presumably, myosin hydrolyses ATP and releases ADP and Pi before attaching to actin but can still carry out the working stroke when it finally binds to actin.
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Fig. 10 The structure of the actin-myosin complex (Holmes, 1997). On the left, five actin monomers in an actin helix (blue and grey strands). On the right, a myosin S1. The 25 kDa domain is green, the 50 kDa upper domain is red and the lower domain is white. Part of the 20 kDa domain is light blue (including the SH2 domain). The SH1 helix, converter domain and carboxy-terminal is dark blue. The regulatory light chain is magenta and the essential light chain is yellow. Reproduced by permission of Current Biology. (Available in the BioMedNet library at: http://biomednet.com/cbiology/cub)
Fig. 11 Swinging lever arm model of movement. Only part of the tail of the myosin head acts as a swinging lever (Holmes, 1997). Reproduced by permission of Current Biology. (Available in the BioMedNet library at: http://biomednet.com/cbiology/cub)
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Fig. 12 Ribbon diagram representing the two different orientations of the lever arm. Beginning of the power stroke (the upper position of the lever)[observed in a motor domain-essential light chain complex (MDE) with either MgADP.AIF4- or MgADP-BeFx bound at the active site] and the end of the power stroke (the lower position of the lever) observed in the nucleotide free S1). In the figure the motor domain up to the hinge point (yellow) is shown only for the MDE since they superimpose in this representation. After this the two structures diverge at an angle of about 70o. The carboxyterminal of the heavy chain of skeletal S1 is magenta and that of the MDE structure is pink. In both structures the motor domain essential chain (ELC) is in blue. The RLC of the MDE is from a model and is greenish-blue (cyan). The RLC of S1 is red. From Dominguez et al. (1998) Cell 94:559-571, Copyright ©1998 by Cell Press. Reproduced by permission.
As summarized in Fig. 11 and 12, most current information on muscle contraction has suggested models in which movement is provided by the lever arm of the light chain domain of myosin, while the actinbinding motor domain has a single orientation (e.g., see Holmes, 1997). However, the possibility of a change in conformation also in the motor domain is supported by crystallographic data (e.g., see Bershitsky et al., 1997; Tsaturyan et al., 1999) and electron tomography (see Chapter 1) of quickly frozen insect flight muscle (Taylor et al., 1999). Details of the interaction of myosin components as the consequence of events following nucleotide binding are discussed in Section V. D. Attachment of the Thin Filaments: α-Actinin and Vinculin
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The contractile event, expressed in the shortening of the sarcomere, requires that the thin actin filament be attached to the Z disks. The latter are probably mainly made up of a rod-shaped protein of about 95 kDa, α-actinin. The location of the α-actinin in the Z disk has been demonstrated with immunofluorescence using anti-α-actinin antibodies (Lazarides and Burridge, 1975; Masaki et al., 1967). Since α-actinin cross-links in vitro with actin, it is presumed to constitute the attachment site for the thin filaments. In smooth muscle another protein, vinculin, of 130 kDa (Geiger, 1979) seems to play a similar role in connecting the actin filaments to the dense plaques (Geiger et al., 1980). E. Titin and Nebulin Titin and nebulin are huge molecules of myofibrils. Titin, also known as connectin, is as large as 3,000 kDa in skeletal muscle (for a review see Murayama, 1997) and nebulin is approximately 800 kDa. Their size precluded their early discovery because these molecules cannot enter most polyacrylamide gels that are generally used to study proteins. Titin Members of the titin family, also known as connectins, are giant proteins in the molecular weight range of 1,000 kDa or more (e.g. see Trinick and Tskhovrebova, 1999). Titins are modular proteins constituted in large part by tandems of immunoglobin- and fibronectin-like domains. Some of the titins are intracellular and are thought to provide scaffolding (e.g., Gregorio et al., 1999) that directs the correct assembly of contractile proteins. Extracellular titins are involved in adhesion and recognition. Titin is also suspected to be present in chromosomes (Machado et al., 1998). Vertebrate muscle titins (see Labeit and Komer, 1995a; Murayama, 1997) and non-muscle titins (Eilersten and Keller, 1992) are 3,000 kDa in size. The titin-like molecules of invertebrate smooth and striated muscle are smaller, about 800 kDa and go by various names such twichins (e.g., Benian et al., 1989), projectins (Daley et al., 1998) and mini-titins (Nave and Weber, 1990). The larger titins may also be present in some invertebrates (e.g., Vibert et al., 1996). All titins occur in various isoforms produced by differential splicing. In vertebrates, most of the diverging sequences are toward the amino-terminal of the protein producing molecules with different extensible properties responsible for the elasticity of the muscle. Cardiac muscle has a stiff titin. Other muscles have isoforms that are less stiff and therefore more easily stretched. In vertebrate striated muscle titin is about 1 µm long and about 4 nm wide. Each titin molecule extends for half a sarcomere with the amino terminal in the Z-disc and carboxy-terminal in the M-line (Labeit et al., 1992; Furst et al., 1988) (see Fig. 13). Most of its domains are immunoglobulin (Ig, I-set) and http://www.albany.edu/~abio304/text/24part1.html (20 of 23) [3/5/2003 8:27:18 PM]
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fibronectin (Fn, type III) repeats, each with about 100 amino acid residues folded in β-sheets sandwich. In the A-band, titin interacts with the thick filaments and M-line proteins and the amino terminal interacts with actin and Z-disc proteins. The Ig and Fn domains of the titin adjacent to the A-band are present in periodic patterns (Labeit et al., 1992; Labeit and Kolmerer, 1995a). The long-range repeat has been called a super-repeat and is 43-44 nm long. This distance (the myosin helix repeat of the thick filament) suggests that titin is bound to the thick filament. The observation that myosin binds to titin (Soteriou et al., 1993) or to constructs produced from cDNA encoding titin (Labeit et al., 1992), supports this view. The interaction appears to involve mostly the light meromycin part of the molecule, the backbone of the thick filament. Titin can therefore be considered an integral part of the thick filament. The I-band portion has mostly Ig domains as well as sequences rich in prolines, glutamates, valines and lysines (the PEVK region). Close to the carboxy-terminal (and the M-line) there is a protein kinase domain. The A-band portion of titin is thought to control the assembly of the thick filament (see Whiting et al., 1989). At the I-band, titin connects Z-disc and A-band and is thought to provide the muscle passive tension since it is elastic. The titin in the I-band maintains the distance between A-band and Z-disc. In the absence of titin, there would be imbalances between the opposing halves of the sarcomere (Horowits and Podolsky, 1987). In highly shortened muscles such as cardiac myocytes, it provides a sarcomere lengthening force (Helmes et al., 1996). When the elastic segment of titin in the I band was removed from the sarcomere by trypsin treatment, the restoring force of myocytes was found to be depressed. In Caenorhabditis elegans, twichin which also has a protein kinase domain, phosphorylates myosin (see Heierhorst et al., 1996). However, this is not the case for vertebrate titin. Its protein kinase domain phosphorylates a 22 kDa protein, telethonin (Mayans et al., 1998). Telethonin is a Z-disc protein, about 1 µm away from the kinase domain (Mues et al., 1998). How titin could phosphorylate telethonin was intitally a mystery. However, the phosphorylation is likely to occur in differentiating myocytes before the myofibrils are formed where the amino terminal and telethonin appear together (Mues et al., 1998) as shown by immunofluorescence (see Chapter 1) with antibodies against the amino-terminal region of titin and telethonin, which detect both proteins at the Z-disc of human myotubes. Therefore the kinase domain is thought to function in sarcomere assembly. The kinase activity has to be activated by Ca2+-calmodulin and phosphorylation of a tyrosine (Mayans et al., 1998). Titin is a major component of the Z-disc (see Gregorio et al., 1999). The titin amino-terminal contains several copies of a residue, the Z-repeat of 45 amino acid residues. These Z-repeats bind to the carboxy terminal of α-actinin. A domain in the central region of α-actinin binds to a region of titin close to the Zrepeats (Young et al., 1998). The titin from adjacent sarcomeres overlap for the full width of the Z-disc as shown by immunoelectron microcopy (Gregorio et al., 1998; Young et al., 1998). The configuration of titin is likely to vary when the muscle is stretched. When the sarcomere is relaxed, the I-band part of titin is likely to be coiled. When the sarcomere is stretched, the molecule is straightened out with a minimum of force (e.g., see Higuchi, 1996). After this however, extension http://www.albany.edu/~abio304/text/24part1.html (21 of 23) [3/5/2003 8:27:18 PM]
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requires unfolding and, therefore, high forces (50 pN or higher) as shown using atomic force microscopy (Rief et al., 1997).
Fig. 13 The sarcomere showing the extent and location of titin and the probable location of nebulin. Reproduced from Trends in Biochemical Science , vol. 19, Trinick, J., Titin and nebulin: protein rulers of muscle? pp.405-409. Copyright ©1994 with permission from Elsevier Science.
Nebulin Nebulin is a large actin binding protein of skeletal muscle, where a single polypeptide extends along the entire length of the thin filament with its carboxy terminal at the Z-line and the amino-terminal at the pointed filament end. Some of the probable features of nebulin in relation to the structure of the myofibril are also displayed in the model of Fig. 13 (Trinick, 1994). In different skeletal muscles the size of the nebulin differs and correlates with the length of the free portion of the thin filament. Consequently, it ranges from a molecular mass of 600 to 800 kDa (e.g., Wang and Wright, 1988; Kruger et al., 1991). A portion close to the carboxy terminal is located in the periphery of the Z-disc (Wright et al., 1993) as shown by immunoelectron microscopic localization (see Chapter 1) of epitopes of a number of sitespecific monoclonal antibodies. cDNA studies of nebulin revealed repeat modules (of 35 amino acid residues) and sets of modules (185 or more) (Labeit and Komerer, 1995b). These modules can bind actin and stabilize the filaments. The modules are present in approximately 20 tandem super-repeats, each containing 7 different modules. Near the amino and carboxy terminals these super-repeats are flanked by single-repeat regions containing 8 modules (Labeit and Kolmerer, 1995; Wang et al., 1996b; Zhang et al., 1996). Each repeat is predicted to have an α-helical configuration and contains an SDXXYK sequence (Labeit et al., 1991). The sevenfold periodicity in the super-repeat reflects that of the actin filament which contains a 385 Å unit composed of seven actin molecules and one complex of troponin/tropomyosin. Single repeats with the central SDXXYK motif are the smallest unit that can bind to actin (Chen et al., 1993; Pfuhl et al., 1994). The binding involves both charges as well as hydrophobic interactions. Nebulin has been proposed to form a stable extended configuration along the actin filament by a zipper-like actin binding mechanism,
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(Chen et al., 1993; Pfuhl et al, 1996). The binding of nebulin to actin suggests a role as a "ruler" to specify the length of the actin filament (Kruger et al., 1991; Labeit et al., 1991) (i.e. a progressive polymerization by addition of individual G-actin molecules binding to nebulin modules). Chemical crosslinking (Shih et al., 1997) was used to define the molecular contacts between actin and ND8, a twomodule nebulin fragment that promotes actin polymerization and inhibits depolymerization by binding to both G- and F-actin. A complex with a stoichiometry of 1:1 complex between ND8 and G-actin was demonstrated. Cross-linking of ND8 to F-actin occurred at the amino terminal of actin. The binding of nebulin to the amino terminal of actin is likely to have a role in actin polymerization. A much smaller protein (100 kDa), nebulette, has been found whose carboxy-terminal portion resembles nebulin. Nebulin and nebulette are coded by separate gene. A subgroup of repeats at the carboxy terminal of both nebulin and nebulette molecules, depends on the tissue and stage of development so that a variety of isoforms of both molecules are produced (Millevoi et al., 1998). Nebulette is likely to correspond to a nebulin-like protein of cardiac muscle (which lacks nebulin).
Go to Part 2 REFERENCES Search the textbook
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II. MECHANISMS IN OTHER MUSCLES We have seen that much of the information available for striated muscle points to contraction occurring as a consequence of the sliding of actin filaments in relation to myosin filaments. Some clues are presented as to how this might occur at the molecular level. As previously discussed, the striations are the consequence of the presence of fibers of myosin in the thick filaments of the denser anisotropic bands and the presence of actin in the thin filaments spanning the less dense isotropic bands. The thin filaments interdigitate with the thick filaments of the A bands. The sarcomeres are in register, so that the A or I bands of one sarcomere are adjacent to those of a sarcomere located next to it. Striated muscle is found in vertebrates and a number of arthropods. Presumably, the same contractile mechanism operates in all these striated muscles, although certain details may differ. Other muscles are arranged differently from striated muscle. Some, such as the body wall muscle of the earthworm, are obliquely striated (Fig. 14) (Heumann and Zebe, 1967); others, such as smooth muscles, have no apparent striations at all. Do these muscles contract in a different manner? The answer is not entirely clear. Smooth or obliquely striated muscles generally have some properties that distinguish them from striated muscle. For example, although striated muscle generally contracts to about 80% of its resting length, some smooth muscles can contract to 30% of their resting length (Hasselbach and Ledermair, 1958; Winton, 1926). Are these manifestations of fundamental differences or do they represent minor modifications of the same basic mechanism? Close examination of at least some of the cases indicates that usually it is not necessary to propose a different mechanism. Where shortening is extreme, the change in length could take place only if the filaments did not abut against a barrier, such as a Z disk. In this extreme, the thick filaments would have to slide relative to other thick filaments, as has been observed. An example of extreme contraction, that of the body wall muscle of the earthworm, is shown in Fig. 14. Different types of atypically striated or nonstriated muscle are listed in Table 1 (Rüegg, 1968). The table summarizes the type of muscle, the Z line structure, the proteins in the thin and thick filaments, and where these muscles are present.
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Fig. 14 Model of the sliding-filament mechanism in the obliquely striated body wall muscle of the earthworm. (a) Relaxed state; (b) contracted state. Note that the thick filaments slide not only relative to thin filaments but also relative to each other. From H. G. Heumann and E. Zebe, Zeitschrift feur Zellforschung und Mikroskopische Anatomie, 78:131-150, with permission. Copyright ©1967 Springer-Verlag, Heidelberg.
All the systems that have been studied seem to have both actin and myosin and may well work by a similar mechanism, although differences must occur. Not surprisingly, the properties of the component molecules are different. This is particularly true of myosin, which differs depending on the system from which it has been isolated. Evidence as to whether sliding actually occurs or whether the thick and thin filaments actually correspond to actin and myosin is not always readily available. In addition, the thick filaments have been difficult to demonstrate in vertebrate smooth muscle by means of electron microscopic techniques. Smooth muscle contraction has been recently reviewed (Horowitz et al., 1996). Table 1 The Diversity of Smooth Muscle
Proteins of myofilaments Type of muscle
Z-line structure
Thin filaments
Thick filaments
Helical smooth or obliquely striated
Z-column or dense bodies
Actin
Myosin and tropomyosin A (paramyosin)
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Examples
Earthworm body wall; oyster yellow adductor
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Invertebrate smooth type I (paramyosin muscle)
Dense bodies
Actin
Myosin, very much paramyosin
Muscle anterior byssal retractor, oyster opaque adductor
Invertebrate smooth type II (classical smooth)
Dense bodies
Actin
Myosin
Pharynx retractor, snail
Vertebrate smooth muscle
Dense bodies
Actin
Thick fibers difficult to see, no paramyosin
Uterus, taenia coli, chicken gizzard
From J.C. Rüegg, Symposia of the Society for Experimental Biology, XXII: Aspects of Cell Motiity, with permission. Copyright ©1968 Academic Press.
As already discussed, in striated muscle the arrangement of cross-bridges is bipolar. The cross-bridges have the same polarity in one half of the filament and the opposite polarity in the other half. They are arranged helically with an axial spacing of 14.5 nm between one level of cross-bridges and the other. This allows for contractions consistent with the structure of the sarcomeres. Smooth muscle could be arranged in the same way as striated muscle. Another possible alternative is an arrangement in a non-helical side polar structure. Fig. 15 (Xu et al., 1996a) presents these two alternatives in diagrammatic form. EM studies of a variety of smooth muscles indicate that the myosin filaments of smooth muscle are side-polar (Xu et al., 1996). This arrangement allows for a much more extensive contraction because myosin can move along actin filaments in a much longer path.
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Xu et al., ©1996. Reproduced from The Journal of Cell Biology by copyright permission of the Rockefeller University Press.
III. MOVEMENT IN CILIA AND FLAGELLA In Chapter 23 we saw that the bending of cilia or flagella may occur through a mechanism analogous to the sliding filament of striated muscles. Nevertheless, the details of the mechanism are likely to differ significantly. As discussed in that chapter, the MTs of cilia or flagella are made up of tubulin α and β, as are the MTs present in the cytoplasm. Dynein, responsible for the ATPase activity, corresponds to the cross-bridges in the tubular doublets (see Chapter 23). Tubulin seems to be analogous to actin, and dynein to myosin. However, tubulin differs from actin, and dynein does not correspond to myosin. The details of the interactions between the two proteins making up the microtubular system of cilia or flagella are still not well understood. Dynein is made up of several polypeptides (Johnson, 1985). In Tetrahymena cilia and in Chlamydomonas flagella the dynein is three-headed, whereas in sea urchin sperm flagella the dynein is double-headed. A comparison of myosin and dynein is shown in Fig. 16 (Johnson, 1985). The base of the dynein is anchored to one microtubule, the so-called A subfiber, by ionic interactions. The head, which contains the ATP binding site, is free to interact with the B subfiber of the adjacent doublet, as shown in Chapter 23. Although this arrangement is entirely analogous to the one in actomyosin, the sliding is between microtubules and the dynein heads are much larger.
Fig. 16 Structures of dynein and myosin. The structure of Tetrahymena 22S dynein is schematically compared to that of myosin, which is drawn to the same scale. The dynein structure combines information obtained from the three dynein sources (see text). IC, Dynein intermediate chains; LC, myosin light chains. From Johnson (1985). Reproduced, with permission, from the Annual Review of Biophysics and Biophysical Chemistry, Vol. 14, copyright ©1985 by Annual Reviews Inc.
Dynein forms a so-called rigor bond with the B subfiber and is released by the addition of ATP. The rate of this dissociation suggests that the microtubule-dynein complex may operate in essentially the same http://www.albany.edu/~abio304/text/24part2.html (4 of 22) [3/5/2003 8:27:28 PM]
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way as the actomyosin complex, with the same cycle of dissociation, movement of bridge, ATP hydrolysis, formation of bond, and generation of force as outlined for actomyosin. IV. MOVEMENT IN THE CYTOPLASM In myofibrils, the structure of the contractile elements is obvious. Many other systems lack apparent contractile structures. Perhaps the proper molecular arrangement could be assembled when needed and then disassembled (e.g., mitotic apparatus) or could actually be labile and present only for a short while. In slime molds, the flow of the cytoplasm reverses direction continuously (Chapter 23). Fibers could produce contraction of the kind observed in slime molds if arranged across the flowing cytoplasmic channel at one location, and could then be disassembled while another contractile structure is formed elsewhere. The idea that the underlying mechanisms of motility are similar is reinforced by the fact that microtubules or fibers have been found in several contractile systems and in some cases actomyosin and actin molecules have been isolated. A. Microfilaments and Microfilament Bundles As mentioned, filaments and tubules are present in the cytoplasm. Not only tubules but also filaments are prominent in neurons (neurofilaments). These are 8-10 nm in diameter (Davidson and Taylor, 1960) and appear to be formed of globular subunits. Filaments also appear in many other cell systems. Generally the filaments fall into two groups. Some of the fibers, the microfilaments range from 5-7 nm in diameter and correspond in size to actin filaments. Others, such as the neurofilaments, are in the range of 8-10 nm. These intermediate filaments, thought to have a structural role, are discussed below in Section IVD. There is extensive evidence that the neurofilaments form a network with microtubules. They form crossbridges, possibly through the so-called microtubule-associated proteins (MAPs) (Leterrier et al., 1982). This section examines the possible role of the actomyosin system of cells other than muscle and then the role of actin-binding proteins. The actomyosin system In Nitella and Chara, as we saw in Chapter 23, the pattern of flow of the cytoplasm suggests that the motile force is produced in the boundary between the stationary cortex and the outer edge of the cytoplasm. Fibers in this zone have been recognized with the light microscope (Ishikawa et al., 1969; Kamitsubo, 1966, 1972) and the electron microscope (Nagai and Rebhun, 1966). The fibrils are 5-6 nm thick, which suggests that they are actin filaments. Immunofluorescence techniques (Williamson and Toh, 1979) and HMM decoration (Korn and Hammer, 1988) confirm that actin is in fact involved. In Nitella, myosin has been isolated (Kato and Tonomura, 1977) suggesting that an actomyosin contractile system is present. http://www.albany.edu/~abio304/text/24part2.html (5 of 22) [3/5/2003 8:27:28 PM]
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The mechanism of cytoplasmic flow implied by the boundary model described above for Nitella and Chara, together with the location of the filaments in the cortex, suggests that whereas actin is in the cortex, myosin is most likely to be present in the moving endoplasm. This model is supported by experiments in which cortex and endoplasm are separated out by centrifugation of these very large cells. Centrifugation at low speed collects the endoplasm in the centrifugal end of the cell, whereas the cortex remains in place. The two components are differentially treated and then reassembled by centrifugation in the reverse direction (Chen and Kamiya, 1975; Nagai and Kamiya, 1977). N-Ethylmaleimide (NEM) is known to interfere with the F-actin-activated ATPase activity of myosin. In the separation-reassembly experiments, treatment the cortex with NEM did not interfere with streaming after reconstitution. However, treatment of the endoplasm blocked the streaming. Similarly, cytochalasin B, which reacts with actin, blocked streaming when the cortex alone was treated. These observations seem to be in agreement with the model previously proposed for motility in this system, discussed in Chapter 23 Another experiment supports this interpretation. The ectoplasmic fibers were isolated by cutting open the cell and washing away the cytoplasm. The oriented fibers and the chloroplasts stayed behind. Fluorescent beads 0.7 µm in diameter were coated with HMM and then placed on these fibers, on which they proceeded to move unidirectionally along the array of filaments. This motion required the presence of ATP and did not take place when the HMM was inactivated (Sheetz and Spudich, 1983a). Therefore, all indicators support the models that were proposed. Actins The actins are ubiquitous in cells. Their polymerization is thought to play a significant role not only in cytoplasmic movement, but also in the structure and mechanical properties of the cytoplasm. G-actins, the monomeric forms, correspond to a family of globular protein. Like the skeletal muscle counterpart, they are of approximately 42 kDa and are composed of 374 to 375 amino acids, depending on the variant. Various isoforms are present in mammalian tissues. The actins have been conserved to a remarkable extent. Animal and fungal actins are very similar. These, however, differ significantly from the plant actins. Most actins have been modified by post-transcriptional acylation of the amino-terminal and methylation of a histidine residue. The structure of actin has been studied with electron microscopy and X-ray diffraction. For X-ray diffraction of G-actin, it was necessary to study the structure of the actin-DNase I complex (Kabsch et al., 1990). This complex, by blocking actin polymerization, allows the formation of crystals necessary for the X-ray diffraction. The reconstruction of the structure of actin is shown in Fig. 17 (Bremer and Aebi, 1992). The molecule has two domains, one slightly larger than the other. Actin contains centrally located bound ATP and a divalent cation. G-actin polymerizes to form actin filaments (F-actin). The structure of these filaments, was studied with electronmicroscopy obtained with frozen-hydrated or negatively stained fibers and also studied by X-ray diffraction of the phalloidin stabilized filaments (Holmes et al, 1990). A model is shown in Fig. 18 http://www.albany.edu/~abio304/text/24part2.html (6 of 22) [3/5/2003 8:27:28 PM]
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(Bremer and Aebi, 1992). Polymerization of G-actin first requires the formation of a nucleating seed, then monomers assemble at either end. One end generally grows faster (the plus-end) than the other (the minus-end). The two can be distinguished by decorating with HMM or the S1 fragment of myosin. As discussed in Chapter 23, the barbed end (B-end) is the plus-end and the pointed end (P-end) is the minus end. The polymerization is much faster in the presence of ATP. Under appropriate conditions, while Gactin is added at one end, it is removed from the other, eventually reaching a steady state. Although the length and appearance of the filament does not change, the actin molecules are continuously exchanged with the medium, with a migration of each individual molecular from the plus to the minus end. This is known as treadmilling. The polymerization of actin can be blocked using drugs such as the cytochalasins (alkaloids of fungal origin), which block the ends of the actin filaments, or the latrunculins (derived from the red sea sponge), which bind to G-actin monomers. Phalloidin (a compound present in the deadly mushroom Amanita phalloides) complexes to filaments and prevents them from depolymerizing. Combined with rhodamine it can be used as a fluorescent stain for actin filaments. Apart from its binding to myosin, actin binds to many other proteins.
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Fig. 17 The actin molecule. (a) A schematic view of G-actin. The arrow defines the orientation of the molecule relative to the myosin subfragments-1 decoration pattern of the F-actin filament. (b) Folding of the actin molecule represented by ribbon tracing of the α-carbon atoms. An ATP molecule with its associated Ca2+ atom (bottom) are shown in a Van-der-Waals-radius representation. The cleft separating the two domains, the DNase I-binding loop, the hydrophobic loop, α-helix 222-223, the amino terminus, and the two carboxyl-terminal α-helices 358-372 are marked. The orientation is the same as in (a). (c) Different views of the atomic structure of the actin molecule reduced to 1.0 nm resolution. From Bremer and Aebi, 1992. Reproduced by permission. (Available in the BioMedNet library at: http://biomednet.com/cbiology/cel)
Fig. 18 The F-actin filament. Low resolution representation of (a) the atomic model of the F-actin filament and three-dimensional reconstruction of negatively stained (b) and of frozen-hydrated (c: corrected for the contrast transfer function) F-actin filaments. The main panel shows a filament stretch of about 1.4 crossovers long. The top of the figure depicts an end-on view of the filament stretch shown in the main panel. To the left is a comparison of the three-dimensional filament model (bottom half) with a computed projection perpendicular to the filament axis (top half). (d) Myosin subfragment-1-decorated filament stretch, negatively stained with uranyl formate. Scale bars are 10nm ( for a-c; main panel and top inset) and 50nm (for d). Reproduced from Bremer and Aebi, 1992 by permission. (Available in the BioMedNet library at: http://biomednet.com/cbiology/cel)
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The proteins involved in actin-polymerization in relation to the lamellopodia and pseudopodia such as the Arp2/3 complex and α-actinin are also discussed in Chapter 23. Several different groups of proteins bind actin (see Ayscough, 1998; Pollard and Cooper, 1986). The capping proteins generally bind to the barbed end of the filaments and thereby interfere with the binding of monomers at that end. They can then grow with their barbed ends attached to particles or surfaces coated with capping proteins such as villin or severin. The capping proteins in the presence of Ca2+ favor the polymerization of actin monomers by facilitating nucleation to form short filaments, possibly by severing longer segments. Capping may stabilize filament length by stopping treadmilling, that takes place in vitro and in the intact cell. By facilitating the formation of short segments, they favor the sol state and not the gel state and hence presumably favor the flow of cytoplasm. Some low molecular weight proteins, the severing proteins, bind to actin monomers and sever longer actin filaments to produce shorter ones. Various bundling proteins, which are present as dimers, cross-link actin filaments to form bundles. Generally, three to five actin molecules are cross-linked by one bundling protein molecule. Bundling proteins play a role in intact cells; in the brush border of the intestinal microvilli, for example, fimbrin and villin cross-link actins to form bundles. In addition, cross-linking favors the formation of a gel. The role of these actin-binding proteins is summarized in Fig. 19 (Craig and Pollard, 1982). Much of the recent information on the actin-based interactions has been derived by primary amino acid sequence data of the binding proteins, determined by using recombinant DNA techniques. The search for common sequences has allowed the recognition of actin-binding domains. Many of these actin-binding proteins mediate interactions by binding to yet other proteins. Many of the actin-binding proteins can bind directly to polyphosphoinosides (i.e., a phospholipid component of the membrane); others to integral membrane proteins, possibly implicating the actin system in the regulation of both structure and metabolism. For example, profilin (see Chapter 23) inhibits the hydrolysis of PIP by some phospholipases C and, therefore, has a potential regulatory role in the production of second messenger of the inositol system (Chapter 7).
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Fig. 19 Regulation of actin assembly by three classes of actin-binding proteins. Class I cross-links filaments into networks or bundles. Class II caps an end of the filament and may sever preformed filaments. Class III inhibits polymerization by binding to actin monomers. From S. W. Craig and T. D. Pollard, Trends in Biochemical Sciences, with permission. Copyright ©1982 Elsevier Science Publishers, England.
The known actin-binding proteins share a 270 amino acid region that is presumed to be the actin-binding domain (see Hartwig and Kwiatkowski, 1991). Subfragments of these domains bind actin (e.g., a 17 kDa fragment of Dictyostelium ABP-120, Bresnick et al., 1990). These molecules generally are rod-shaped, and apart from at least one actin-binding domain, the rest of the rod contains tandems of structural repeats of variable lengths, presumably to distance the actin from specific binding sites to other proteins that are also present. For example, spectrin has binding sites in the rod region for calmodulin, ankyrin, and band 4.1. Many of these proteins also contain domains that have homology to sequences of the Ca2+binding proteins (e.g., Vandekerkhove, 1990), suggesting a regulatory role of Ca2+. Such a region is at the carboxy-terminal of α-actinin (Noegel et al., 1987) and of α-spectrin (Wasenius et al., 1990), and at the amino-terminal region of fimbrin (de Arruda et al., 1990). Except for fimbrin/plastin, the actin-binding proteins are thought to be in the form of dimers in antiparallel subunit chains, so that the actin-binding domains, generally present at the amino-terminal, are at both ends of the composite rods (Fig. 20, Hartwig and Kwiatkowski, 1991). The subunits are either side by side (e.g., spectrin, α-actinin, and ABP-120), or end-to-end (e.g., filamin). Spectrin is formed by α and β chains that self-associate in overlapped antiparallel alignment to form heterodimers 100 nm in length; these associate end to end to form tetramers (see Fig. 20). The rate of treadmilling of F-actin in intact lamellipodia is much greater than that in vitro (e.g., see Wang, 1985). This is because the in situ system has accessory proteins that are missing in vitro. Actin binding proteins such as capping proteins and ADF/cofilin (see Theriot, 1997) regulate the rate of turnover. The details of the various interactions between accessory proteins, ATP, ADP, G-actin and Factin have acquired sufficient complexity to require detailed kinetic analysis facilitated by the use of computers (e.g., Dufort and Lumsden, 1996, see Carlier and Pantaloni, 1997). Profilin binding to an actin monomers blocks the pointed but not the barbed end and facilitates the exchange of ATP for the bound ADP (Dufort and Lumsden, 1996). ATP-bound actin is the form that is added at the barbed end (Pantaloni and Carlier, 1993). Capping proteins also decrease the total number of growing barbed ends "funneling" polymerization to the remaining free barbed ends. In contrast the cofilin/actindepolymerizing factors (ADFs) increase the depolymerization at the pointed end (Carlier et al., 1997) without affecting the barbed ends. In this process the increase in ATP-bound G-actin speeds up the association at the barbed ends. The balance of the various actions together produce a continuous movement of actin molecules through the actin thread. The proteins of the gelsolin family sever and cap the barbed ends of actin filaments (see Ayscough, 1998). Gelsolins are required for rapid motile responses of cells. Ca2+ favors the binding of gelsolin to Fhttp://www.albany.edu/~abio304/text/24part2.html (10 of 22) [3/5/2003 8:27:28 PM]
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actin. In contrast PIP2 (phospatidylinositol 4,5 bisphosphate) dissociates gelsolin from actin. Tropomyosin have been found in many kinds of cells and it is thought to have a role in stabilizing actin filaments. Several isoforms have been found suggesting that they may have different roles. Coronin was first found in an actin-myosin complex (de Hostos, 1991). Unlike mutants of genes coding for ABP-120, α-actinin or severin that only have slightly altered phenotypes, mutants of the gene coding for coronin of the slime mold Dictyostelium, were found to be impaired in cell locomotion and cytokinesis (de Hostos et al., 1993; Fukui et al., 1999) phagocytosis (Maniak et al., 1995) and macropinocytosis (Hacker et al., 1997). Members of the coronin family were found in a variety of organisms including mammals (see de Hostos et al., 1999). Coronin, a protein of 55 kDa, binds to actin in vitro (de Hostos et al., 1991). Immunofluorescence localizes the protein in actin-rich crown-like extension of the cytoplasm. Coronin has five WD (trp-Asp) repeats similar to the β subunit of the GTP-heterotrimeric binding protein (or G-protein) (see Chapter 7, Section II). These are flanked by other domains. WD proteins have been found only in eukaryotes where they function in cell division, cell-fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion (Neer, 1994). Close to the amino-terminal, the molecule forms a β propeller structure similar to that of the β-subunit of G-proteins (see de Hostos, 1999). Generally the last 25 to 40 amino acids at the carboxy-terminal are likely to form a coiled-coil configuration. Between the WD-containing regions and the coiled-coil domain there is a unique region (Goode et al., 1999) of variable length (22 to 50 amino acids) which differs in the various coronins. The coronin present in Saccharomyces cerevisiae (Crn1p) (Goode et al., 1999) is a component of the cortical actin cytoskeleton and binds to F-actin with high affinity. Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them. Filament cross-linking depends on the coiled-coil domain suggesting that dimerization is required. Assembly-promoting activity is independent of cross-linking. Crn1p also binds to microtubules in vitro in the unique region not found in other coronins and which is much longer than in other organisms. This region is homologous to the microtubule binding region of MAP1B. The binding of microtubules to Crn1p is enhanced by the presence of actin filaments. The possibility that actin polymerization alone may have a role in some movements inside cells has been suggested. The pathogenic bacteria Listeria monocytogenes move in the absence of motor molecules (e.g., Loisel et al., 1999). The bacteria propel themselves in the cytoplasm of the host cells by nucleating actin filaments at surface of their outer membrane. The actin filaments rearrange into a tail that varies in length and trails behind the moving bacterium (the so-called "comet tail"; see Cossart, 1995). The actin filaments cross-link and the actin polymers are rapidly disassembled by depolymerizing factors. The actin-based propulsion is linked to actin polymerization dependent on ATP hydrolysis, but not requiring myosin. An activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein
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(see above) are also required for motility. These maintain a high level of G-actin required for the unidirectional growth of actin filaments at the surface of the bacterium. Similar mechanisms of motion have been shown for Shigella, as well as Rickettsia and vaccinia virus (Higley and Way, 1997). Is it possible that a similar mechanism operates in cells? Although the role of this "comet tail" mechanism in movements of vesicles in the cytoplasm is probably not common, recent evidence suggests that it does occur. The possible involvement of the actin-assembly mechanism in the movement of some cytoplasmic organelles was examined in Xenopus eggs (Taunton et al., 2000). Dynamic actin "comet tails" were found on a portion of cytoplasmic vesicles which exhibited movement and contained protein kinase C (PKC). The process was enhanced by the activator of PKC, phorbol myristate acetate (PMA). The phenomenon was also found in a cell free system. In vitro, N-WASP (N-Wiscott-Aldrich syndrome proteins) was found to be recruited to every vesicle with a comet tail. WASPs are thought to have a role as adaptors needed for coupling the GTPases to the Arp2/3 complex (see Chapter 23) and N-WASP, a protein homologous to WASP, is thought to transmit signals that lead to rearrangements of cortical actin filaments (see Miki et al., 1996). These findings suggest, that in these experiments, N-WASP recruits Arp2/3 which, in turn, nucleates actin. The vesicles exhibiting this behavior were found to be probably endosomes and lysosomes, because they accumulate acridine orange (indicating an internal acid pH). In addition., endosomes and lysosomes isolated from mammalian cells also nucleated actin and moved in the Xenopus extracts. The formation of comets has been implicated in endocytosis involving caveolae (see Chapter 9) Other experiments using mouse fibroblasts reveal more facets to the role of actin, implicating membrane components in actin nucleation and the formation of comets. Phosphatidylinositol 4,5-bisphosphate (PIP2) (see Chapters 4 and 7) stimulates actin polymerization by activating the pathway of the WASP and the actin-related protein complex Arp2/3 (see Chapter 23 and Higgs and Pollard, 1999). The binding of phosphoinositides to actin has been discussed in Chapter 23. In addition, actin polymerization is initiated at cholesterol-sphingolipid-rich membrane sites, the 'rafts' (see Chapter 4), in a process requiring tyrosine phosphorylation. Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP2, initiates actin polymerization from vesicles to form motile actin comets (Rozelle et al., 2000). The vesicles attached to comets, were enriched in PIP5KI and tyrosine phosphoproteins. WASP-Arp2/3 were involved as shown with WASP-null mutants. Extraction of cholesterol reduced comet formation, suggesting an involvement of the rafts. The formation of comets may be an exaggeration of a normal process where actin assembles around transport vesicles or may represent an actual movement of vesicles under certain conditions.
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Fig. 20 Comparison of predicted structure of actin-binding proteins sharing a common actin-binding domain. Reproduced from J. H. Hartwig and D. J. Kwiatkowski, Current Opinion in Cell Biology, 3:8797. Copyright ©1991 Current Biology Ltd., by permission. (available in the BioMedNet library at: http://biomednet.com/cbiology/cel)
The assembly of cytoskeletal elements containing actin has been examined in detail in the red blood cells (see Bennett, 1990; Anderson and Marchesi, 1985) and in intestinal microvilli. In the red blood cell, a meshwork of spectrin tetramers interacting with F-actin is attached to the plasma membrane through two high affinity associations: a binding to ankyrin, which attaches to band 3 (anion exchanger) protein, and a binding to band 4.1 attached to glycophorin, an association favored by polyphosphoinositides. In the brush border of microvilli, a myosin I (see next section) complex containing calmodulin subunits attaches to actin, while at the carboxy-terminal it binds to acidic phospholipids (Hayden et al., 1990). The complex also contains the bundling proteins villin and fibrin. These four proteins can assemble in vitro to form the microvillar core (Coluccio and Bretscher, 1989). A model of this association is shown in Fig. 21 (Bretscher, 1991).
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Fig. 21 Model of the molecular organization of the microvillus cytoskeleton. The microvilli are approximately 100 nm in diameter. The 100 kDa-calmodulin-17 kDa is a myosin I (BB myosin I). Reproduced from the Annual Review of Cell Biology, copyright ©1991 by Annual Reviews Inc.
B. Myosins As previously discussed, skeletal muscle myosin is double-headed and composed of two approximately 200 kDa heavy chains with an amino-terminal globular domain. A single unit of the latter (S1), which corresponds to one head, is responsible for the ATPase activity and, furthermore, has all the machinery needed to generate force (Toyoshima et al., 1987; Kishino and Yanagida, 1988). However, myosin molecules represent a family of proteins of which striated muscle myosin, myosin II, is just one. Other myosins have been referred to as unconventional myosins (e.g. see Titus, 1997). Generally the various myosins move toward the plus (barbed) end of F-actin (e.g., see Sellers and Goodson, 1995). The exception, however, is a myosin VI which moves in the opposite direction (Wells et al., 1999). Myosin VI is supposedly involved in the cytoplasmic transport of vesicles (Mermall et al., 1994) and is probably involved in endocytosis (see Chapter 9). A diagrammatic summary of the myosins is shown in Fig. 22 (Cheney and Moosker, 1992).
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Fig. 22 The myosin motor superfamily. Schematic diagrams of the several classes of myosins are shown with the myosin head domains indicated by large open ovals and the various myosin light chains by small filled ovals. From Cheney and Mooseker, 1992, reproduced by permission.
Unconventional myosins became the focus of interest with the demonstration that Dictyostelium cells lacking myosin II retain many actin based functions such as phagocytosis, pseudopod extension and cell http://www.albany.edu/~abio304/text/24part2.html (15 of 22) [3/5/2003 8:27:28 PM]
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movement (see Pollard et al., 1991). The possibility of a role in these functions for myosin I was suggested by the localization of this protein in phagocytotic cups and the leading margins of locomoting cells (Fukui et al, 1989, 1993a,b). Mooseker and Cheney (1995) have grouped the currently known myosins into 13 classes, with the understanding that this classification may have to be revised as our knowledge expands (see Mooseker and Cheney, 1995, Cope et al., 1996). There is evidence for 25 myosin genes in vertebrates. At least half of these are thought to be distinct from myosin II and considered "unconventional". A single vertebrate cell has been reported to express mRNA or protein for at least 11 distinct myosins, 9 of them unconventional (Bement et al., 1994a). Unconventional myosins are thought not to form filaments in contrast to myosin II. The myosins have a head domain at the amino-terminal, a neck domain which is the site of light-chain binding, and a class specific tail domain. A current working hypothesis proposes that the tail domains dictate subcellular localization and function because these sectors are the most variable from myosin to myosin. However, there is evidence for a role of the tail region for only one myosin (class III, NINAC, Porter et al., 1992). The head domains have some specific features and have been shown to have a role in localization (Porter and Montell, 1993; Ruppert et al., 1995; Durrbach et al., 1996). Some unconventional myosins have large insertions which would alter the motor activity considerably. The neck regions have repeat motifs referred as IQ motifs, which are thought to bind the light chains of myosin II and calmodulin. The neck domain might also act as a lever (e.g. Spudich, 1994). The tail domains of some unconventional myosins are thought to be able to interact with membranes. Some also have SH3, kinase, PH and GTPase-activating domains associated with signal transduction. Phosphorylation of the myosin light chains has a regulatory function in striated muscle, increasing isometric tension (Persechini et al., 1985). Smooth muscle and nonmuscle myosin assembly depends on the phosphorylation of the light regulatory subunits for their ability to assemble in filaments; in addition, the phosphorylated form has a high ATPase activity (see Trybus, 1991). In at least some cases, phosphorylation has been shown to be in response to physiological stimuli (Devreotes et al., 1987). A regulation of myosin and actin filaments assembly by phosphorylation-dephosphorylation of the myosin light chains has been demonstrated by injection of a protein phosphatase into living human fibroblasts (Fernandez et al., 1990). The microinjection resulted in a disassembly of the actin network, as seen by immunofluorescence. After long incubations, the cells' actin returned to the original distribution. Neutralization of the phosphatase by microinjection of the corresponding antibody prevented the disruption. In contrast to this function in vertebrate cells, in lower eukaryotes (such as the cellular slime mold Dictyostelium discoideum), phosphorylation of the tail inhibits assembly and the assembly of myosin II of Acanthamoeba castellanii is not affected by phosphorylation (see Trybus, 1991). Unconventional myosins and vesicle movement We saw that the movement of vesicles inside the cell is often on microtubular-based motors (Chapter 10, 11 and 23, see also below, Section IVB). The role of actin-based motors are beginning to be studied (see http://www.albany.edu/~abio304/text/24part2.html (16 of 22) [3/5/2003 8:27:28 PM]
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Langford, 1995; Hasson and Mooseker, 1995). Information has been obtained in a variety of systems. In squid axoplasm (Kuznetsov, et al. 1992, 1994; Bearer et al., 1993), where the role of microtubules is well established, a given organelle can move on both types of filaments (Kuznetov et al., 1992). At this time, it would seem likely that the microtubular system provides movement over long distances, whereas the actin-myosin system provides movement to local sites (Atkinson et al., 1992; Langford, 1995). This idea is supported by the organization of the two kinds of filaments in axons. Microtubules are long (25 µm or more) and are oriented longitudinally in parallel to each other. In contrast, actin filaments are short (less than 1 µm) and form a cross-linked filamentous network (Fath and Lasak, 1988). Furthermore, the kinetic properties of myosin I suggest that it is suited only as a short duty motor (Ostap and Pollard, 1996) and may function in locations where actin is clustered. However, there is considerable evidence as well for the involvement of unconventional myosins in vesicular transport. An association between the microtubular and the actin-based system is suggested by the demonstration of interactions between actin and microtubules (e.g. Griffith and Pollard, 1982) and the finding of a protein, p150 glued which is a component of the dynactin complex. The dynactin complex is an activator of dynein-mediated vesicle movement. p150 glued is attached to an actin-like filament (Schafer et al., 1994). In yeast, the malfunction of MYO2 (a myosin-V) can be overcome by the overexpression of kinesin (Lillie and Brown, 1992) suggesting some interaction between the microtubular and the actin-based systems that is still not understood. In addition, there is evidence that myosin V and microtubular systems interact in the transport of vesicles (e.g., see Rogers and Gelfand, 1998; Huang et al. 1999 and discussion below). Unlike the microtubular-based movements that have been shown to be bidirectional, actin-based movements have been shown to be only unidirectional, toward the barbed end (e.g. Bearer et al., 1993) with the exception of myosin VI (see above). The idea that unconventional myosins are involved in vesicular transport is supported by the inhibition of the movement of endogenous vesicles on actin filaments by antimyosin I and not antimyosin II in Acanthamoeba cell extracts (Adams and Pollard, 1986). When absorbed to lipid surfaces, myosin I can also move actin filaments along the surface (Zot et al., 1992). Furthermore, the localization of myosin I, as well as the phenotype of myosin I mutants of Aspergillus, Dictyostelium and Saccharomyces cerevisiae, have implicated this class of motors in the control of the activity of the cell cortex, rich in actin. Myosin I is present on the plasma membrane and the actin-rich cortex (McGoldrick et al., 1995; Goodson et al., 1996). Loss of myosin I function produces defects in pseudopod formation, actin rearrangement, endocytosis and secretion (McGoldrick et al, 1995; Novak et al., 1995; Geli and Riezman, 1996; Goodson et al., 1996; Jung et al., 1996; Wessels et al., 1996). A question that remains unresolved is whether a single kind of myosin is involved in all vesicular or http://www.albany.edu/~abio304/text/24part2.html (17 of 22) [3/5/2003 8:27:28 PM]
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organelle transport or each vesicle has a different motor. Multiple motors for different vesicles is possible, considering that the same cell hosts several distinct myosins. This idea is supported in a study of Dictyostelium discoideum (Titus et al., 1989). The genome of Dictyostelium was probed with DNA fragments containing parts of the conserved region of conventional myosin. These experiments identified several genes coding for other actin-based motors. The role of myosin I is still unclear. Ameboid-type myosin-I is generally associated with motile cells. However, one of these is present in motile and non-motile cells as well (Bement et al., 1994b). The function of another characterized subclass that includes brush border myosin I (BBMI) is unknown. BBMI itself cross-links the microvillar core bundle to the plasma membrane. A myosin I, similar or identical to BB-myosin I, has been isolated in rat kidney brush border (Coluccio, 1991) and a similar but slightly different protein has been isolated from brain and the adrenal cortex (Barylko et al., 1992). An interaction of myosin I with the microtubular system in Golgi vesicle transport is suspected. BBMI is present in the cytoplasmic face of Golgi derived vesicles (Fath and Burgess, 1993, Fath et al., 1994). Dynein, a microtubular motor, is present in a portion of these vesicles. As discussed above microtubules are likely to be involved in the transport of the vesicles over long distances. Therefore this observation suggests that microtubules deliver the vesicles to actin arrays. Following this, fusion with the apical plasma membrane may then involve BB-myosin I moving on actin fibers. Myosin IC is associated with the contraction of the contractile vacuole of Acanthamoeba (Doberstein et al., 1993). Microinjection of antibodies to myosin IC have been shown to interfere with vacuole contraction. Myosin V has been found in yeast and vertebrate cells. In vitro, myosin V acts as a molecular motor (Cheney, et al., 1993a). In mammals myosin V is most abundant in secretory and nervous tissue. It is also found in microvilli. The indications that myosin V is involved in vesicular traffic are many. In brain myosin V is associated with vesicles and can be activated to function on actin filaments (Evans et al., 1998) and ER vesicles have been found to be transported in neurons by myosin V (Tabb et al., 1998). Saccharomyces cerevisiae lacking one of the two genes for MYO2 (coding for myosin V), accumulate small vesicles in the mother cells and vacuolar inheritance is lost (Johnston et al., 1991, Govindan, 1995, Hill et al., 1996). Myo2p is needed for polarized secretion (e.g., Santos and Snyder, 1997; Catlett and Weisman, 1998; Schott et al., 1999). It attaches to secretory vesicles and transports them on actin filaments to the site of secretion (Schott et al., 1999) (see Chapter 11). Cultured melanocytes from the mice mutant dilute, alleles at the dilute unconventional myosin heavy chain locus (corresponding to the gene for myosin V), accumulate melanosomes around the nucleus (Provance et al., 1996). Myosin V localizes with melanosome markers
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(Provance et al., 1996) and with tubovesicular organelles (Tabb et al., 1996) in extracts of squid axons. In addition, dilute-lethal mice and dilute-opisthonus rats have an abnormal distribution of endoplasmic reticulum in Pukinje cells of the brain (Dekker-Ohno et al., 1996; Takagishi et al., 1996). Melanosomes purified from Xenopus melanophores move on microtubules. They have also been shown to move along actin filaments as well (Rogers and Gelfand, 1998). Immunological techniques identify myosin V as the motor associated with the organelles. Isolated ER vesicles (Tabb et al., 1998) were found to be capable of moving on actin filaments adsorbed to coverslips. Immunogold EM techniques (see Chapter 1) using an antibody to squid myosin V showed that this myosin localized to the vesicles. In addition, dual labeling with a squid myosin V antibody and a kinesin heavy chain antibody showed that the two motors colocalized on the same vesicles. A more direct interaction between the myosin based systems and the microtubular system is indicated by the finding that myosin VA can interact directly with a kinesin (KhcU) to form a complex (Huang et al. 1999). This observation suggests that the two motors may move together and switch from microtubules to actin tracks without any complicated transition. Drosophila 95F myosin of the myosin VI class has been shown to be associated with vesicle movement in cells of Drosophila embryos. The injection of antibody to this protein was found to block particle movement (Mermall et al., 1994). Unconventional myosins and cell movement Besides a role in vesicle and organelle transport, the association of myosin I with membranes and actin suggests that myosin I may be able to support pseudopod extension and membrane ruffling. The tailregion actin-binding site allows cross-linking with actin to form gels that condense when phosphorylated by special kinases (Fujisaki et al., 1985). This phenomenon suggests a mechanochemical function that could play a role in phagocytosis and the extension of pseudopods. For example, some myosins I from Acanthamoeba (IA, IB and IC) or Dictyostelium (IB and IC) have a tail region that binds the acidic phospholipids or membranes and a second actin-binding domain independent of ATP binding (see Pollard et al., 1991). The myosin, therefore, can not only be involved in movement but also interact with membranes and cross-link with the actin structural framework of the cytoplasm. Apart from the well recognized actin-binding domain of the motor, myosin of Dictyostelium has three other domains. An approximately 200 amino acid domain is rich in basic residues (tail homology domain 1, TH-1) and binds membranes. An adjacent region (tail homology domain 2, TH-2 or GPA domain) is rich in glycine, proline and alanine. A third domain (TH3) of approximately 50 amino acids, homologous to src protein, is present either at the tail or within TH-2 and constitutes a second actin-binding domain. src is a membrane associated protein kinase that phosphorylates tyrosine and is coded by an oncogene. Immunofluorescence microscopy shows that Acanthamoeba myosin I is associated with membranes and is likely to play a role in vesicle transport, phagocytosis and cell movement. Phosphorylation of ameboid myosin I greatly increases the activity of this protein. The phosphorylation may correspond to autophosphorylation or a response to the presence of acidic phospholipids such as phosphatidylinositol (which also has a role as a second messenger). Dictostylium myosin IA and IE are similar to other myosins I but lack the ATP-independent actin binding site. http://www.albany.edu/~abio304/text/24part2.html (19 of 22) [3/5/2003 8:27:28 PM]
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Insights to the relative roles of myosin I and II in vivo are provided by experiments in which the expression or the function of one of these myosins is blocked. In prectice, it has been easiest to block myosin II (e.g., in Dictyostelium, there is a single gene corresponding to this myosin, whereas there are five for myosin I). Dictyostelium cells lacking myosin II can be produced by genetic manipulation (e.g., DeLozanne and Spudich, 1987) or by using antisense RNA (which by hybridizing to the mRNA blocks its translation) (e.g., Knecht and Loomis, 1987). These myosin II-deficient cells are unable to carry out cytokinesis and receptor capping and to maintain cortical tension (as determined by deformability) (Pasternak et al., 1989). Capping of certain receptors is a sign of mobility of the receptor, shown by an accumulation due to cross-linking to multivalent ligands (e.g., concanavalin A). However, cell locomotion, formation of pseudopods, and phagocytosis remain unaffected. A similar strategy was used by injecting an excess of antimyosin II (Sinard and Pollard, 1989) which slows down but does not stop motility. Therefore, locomotion is believed to be driven by myosin I. Other properties, such as cytokinesis and receptor capping, are likely to depend on myosin II. In yeast, myosin I has been found to associate with WASP-like adapter proteins and components of the Arp2/3 actin nucleation complex (Evangelista et al., 2000; Lechler et al., 2000). These findings suggests that these molecules bridge interactions between actin and myosin and play a role in actin polymerization. The Arp2/3 complex is also discussed in relation to formation of lamellopodia and pseudopodia in Chapter 23. The binding of myosin I was shown by the two-hybrid system and immunoprecipitation (Evangelista et al., 2000), as well as affinity chromatography and mass spectroscopic analysis (Lechler et al., 2000). The carboxy-terminal tail of the yeast myosin-I (Myo3p and Myo5p) contains an Src homology domain 3 (SH3) followed by an acidic domain. The SH3 domain binds to the yeast homologues of human WiskottAldrich syndrome protein (WASP) and the Wiskott-Aldrich syndrome protein-interacting protein (WIP). These are adapters linking actin assembly and signaling molecules. In contrast, the acidic domain binds to Arp2/3 complex subunits which are associated with Arp2/3-mediated actin nucleation. In addition, assays with permeabilized yeast cells showed that the WASP-like components and phosphorylation of the myosin [by p21-activated kinases (PAKs)] are required for actin polymerization which also requires the action of the small GPPase, Cdc42p (Lechler et al., 2000). A role of myosin V in the formation of filapodia is demonstrated in experiments using chromophoreassisted laser inactivation (CALI) with myosin V-specific antibodies. These antibody-targeted laser inactivations implicate myosin V in the formation of filopodial extensions (Wang et al., 1996a). Three distinct monoclonal antibodies to chicken brush border myosin were injected into chicken fibroblasts (Höner et al., 1988). The antibodies were to three distinct epitopes of the tail region of the molecule. The microinjection resulted in the loss of stress fibers, change in shape, and increased locomotory activity. Other changes were consistent with the interpretation that there was an increase in fluidity in the cells. http://www.albany.edu/~abio304/text/24part2.html (20 of 22) [3/5/2003 8:27:28 PM]
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Sensory role of myosins Movement appears to be only one of the functions of the myosins. Myosin-Iβ is associated with the sensory hair cells of the frog sacculus (Gillespie et al., 1993). This myosin is thought to have a role in an actin based motor regulating mechanochemically Ca 2+ channels. These channels present within the hair bundle respond to mechanical stimulation (Assad and Corey, 1992; Gillespie et al, 1993). Myosin VII is also thought to have a role in sensory transduction. It is found in inner and outer hair cells of the inner ear and in the pigmented epithelium of the retina (Hasson et al., 1995). Mutations can produce deafness and blindness (e.g. Gibson et al., 1995). Class III myosin ninaCs of Drosophila melanogaster are involved in phototransduction. The ninaC gene codes for two proteins, p132 and p174, by alternative splicing. Each has a protein serine/threonine domain fused to a myosin head domain (Montell and Rubin, 1988). The tail domains of the two proteins differ and are thought to be responsible for the localization of p174 in the microvillar rhabdomeres, whereas p132 are in the sub-rhabdomeral cell bodies (Porter et al., 1992). In the case of p174 the head domain is thought to have a role in its localization. In p174 (Porter and Montell, 1993; Porter et al., 1993, 1995), the kinase domain is required for normal electrophysiology. The head domain has a role in phototransduction and rhabodomere maintenance. The neck region binds calmodulin at two sites. Both domains are required in vivo to terminate phototransduction. Myosin and GTP-binding proteins Class IX myosins have been found to have a domain homologous to GTPase activating proteins (see Lamarche and Hall, 1994; Hall, 1994). They are presently thought to be a link between rearrangements of the actin-based cytoskeleton which are modulated by the rho family of GTPases, such as membrane ruffling and stress fiber formation. Obviously, the interactions between the myosins and actin are very complex and many questions still remain unanswered. The structural versatility and the dynamics of all cells are in large part the result of dynamic interactions of the components of the cytoskeleton. As we have seen some interactions require actin and actin associated proteins, and these are addressed in this section. Others are based on the system of microtubules and associated proteins discussed in the next section.
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C. Microtubular Motors Like actin filaments, microtubules are ubiquitous in cells and are thought to play a role in both structure and movement. The general role of microtubules in movement was briefly described in Chapter 23. Aside from their role in chromosome movement, microtubules are likely to be associated with Golgi stacks (Allan and Kreis, 1987), endoplasmic reticulum (Franke, 1971), mitochondria (Lindén et al., 1989), and lysosomes (Swanson et al., 1987). Chapter 10 examined intracellular transport and discussed evidence for an involvement of microtubules. Chapter 23 examined the role of microtubules in axonal movement and in cell division, and the dependence of microtubular transport on motors was noted. This section will take a closer look at the currently known motors and their possible modes of action. Current studies have suggested two major kinds of microtubule-associated motors, the dynein and the kinesin classes. Conventional kinesin drives the transport of organelles from the minus to the plus end (see Fig. 23). In neurons and undifferentiated cells, this corresponds to the movement from the cell nucleus to the periphery (axon in the case of neurons). Kinesin can also translocate microtubules linearly in a minus direction when attached to a surface. Dynein drives movement in the opposite direction, translocating particles toward the minus end and moving microtubules in the plus direction (Vale et al., 1986). The experimental design where the motor molecules are attached and the microtubules are free to move has been referred to as the microtubular-gliding assay. The role of these motors is summarized in Fig. 23 (Vallee and Bloom, 1991). The two motors are also distinguishable by their different sensitivities to inhibitors or ATP-analogs as well as direction and rate of movement along microtubules. Categorizing motors according to directionality of movements can be too simplistic. Some kinesins move particles toward the minus end (see below). However, it is not yet clear whether it is the microtubules in the lysed cell model that are moving relative to one another. This is an important consideration because dynein can effect bidirectional microtubule sliding in the ciliary axoneme. In the protist Reticulomyxa (related to foraminifera) the bidirectional traffic of organelles (Schliwa et al, 1991, Orokos and Travis, 1997) and surface transport of latex beads in the pseudopodia (Orokos et al., 1997; Orokos and Travis, 1997) are powered by the motor protein dynein on MT-tracks (see Chapter 23). These findings introduce an apparent paradox. In these pseudopodia the microtubules are oriented uniformly with the plus ends toward the distal ends of the pseudopodia (Euteneuer et al., 1989) and dynein is involved only with the unidirectional movement of organelles toward the minus end. This paradox can be resolved as indicated above by the observation that microtubules slide along each other (Chen and Schliwa, 1990). The microtubules with their cargo attached would then slide in both directions (see Orokos and Travis, 1997) as they do in reactivated demembranated sperm axonemes (Brokaw, 1991).
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Fig. 23 Axonal transport. Organelles are depicted using either kinesin for anterograde transport or cytoplasmic dynein for retrograde transport. Vallee and Bloom (1991). Reproduced from the Annual Review of NeuroSciences, Volume 14, copyright ©1991 by Annual Reviews Inc.
Apart from directional motion some cytoplasmic dynein and kinesin can produce torque, causing rotation of microtubules coincident to linear translocation. This is the case for the nonclaret disfunctional protein, Ncd (Walker et al., 1990) and ciliary dynein from Tetrahymena (Vale and Toyoshima 1988). Cytoplasmic dynein Dynein represents a family of motors (see King, 2000) related to the AAA (ATPases Associated with a variety of cellular Activities) family of of chaperone-like ATPases (e.g., Mocz and Gibbons, 2001). Cytoplasmic dynein is required for (a) the steady state localization of the Golgi cisternae and the endosomal vesicles toward the center of cells (Burkhardt et al., 1997; Harada et al., 1998), (b) the movement of cargo from ER to Golgi (Presley et al., 1997) and (c) microtubular organization at interphase (Quintyne et al., 1999) and mitosis (see Compton, 1998). The structure and mechanism of movement of dynein has lagged behind that of other motors probably because of its complexity (see Vallee and Sheetz, 1996). Axonemal and cytoplasmic dynein are very similar. Cytoplasmic dynein was isolated from microtubules and found to bind microtubules in an ATPdependent manner (Paschal et al., 1987). It was originally named microtubular associated protein 1C http://www.albany.edu/~abio304/text/24part3.html (2 of 46) [3/5/2003 8:27:44 PM]
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(MAP1C), because its homology to the dynein of cilia and flagella was not immediately recognized. The protein had ATPase activity, markedly stimulated by binding to microtubules. When tested with the microtubule-gliding assay (Paschal and Valee, 1987), it was found to be a motor. When tested on isolated axonemes of Chlamydomonas reinhardi, a biflagellate single celled alga, the axonemes were found to move toward the plus end (so that the force exerted by the motor was toward the minus end). As indicated earlier, this polarity is opposite that of kinesin. Dynein has been found in a variety of cells and tissues. MAP1C was identified as cytoplasmic dynein (DHC1) from its biochemical and physical properties. Perhaps most significantly, scanning transmission electron microscopy (STEM) (Vallee et al., 1988) revealed the same morphology and molecular mass as the two-headed forms of ciliary and flagellar dynein. Dyneins typically have two heads and in some cases three for a total molecular weight of 1.2 to 2 x 103 kDa. The native protein has two 410 kDa heavy chains, three 74 kDa subunits, and one subunit each of low molecular weight subunits ranging from 53 to 59 kDa (Vallee et al., 1988). The head domain of dynein (350 to 400 kDa) is much larger than that of kinesin (40 kDa) or myosin (95 kDa). Each head contains four putative ATP-binding domains (P-loop motifs) separated by approximately 300 amino acids (Holzbauer and Vallee, 1994). The heavy chains are responsible for ATPase activity and force production. A very large domain at the carboxy terminal is needed for ATP binding and the microtubule-binding domain is far removed from the ATP-binding domain (Gee et al., 1997). These properties suggest that dynein might function very differently from other motors. The structure of isolated dynein also argues for a distinct mechanism. Electron microscopy has shown that the heavy chain fold in such a way that it forms globular head with two elongated processes, the stalk and the stem (Goodenough et al., 1984, 1987; Goodenough and Heuser, 1989). Supposedly, the stalk binds the microtubules whereas the stem binds to the cargo as well as the intermediate and light chains. In the head region six AAA modules form a ring, 125 Å in diameter. Negatively stained dynein from Dictyostelium has been studied using transmission EM and image reconstruction techniques (Samsó et al., 1998; Burgess et al., 2003). Fig. 24A shows an EM view of purified cytoplasmic dynein and 24B a model showing attachment to the microtubule and a membrane bound vesicle (Gee et al., 1997) and possible power strokes.
Fig. 24 Structure of cytoplasmic dynein. A. Cytoplasmic dynein seen with the EM after freeze-drying and
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platinum replication. B. Models of cytoplasmic dynein showing attachment to microtubule through the stalk and to a vesicle. The arrows indicate possible direction of power stroke. Reproduced with permission from Nature , from Gee et al. 1997, Copyright ©1997 MacMillan Magazines Ltd.
In addition to the structural similarity between axonemal and cytoplasmic dynein, microtubular gliding in vitro is supported by either kind of dynein. However, the rates of movement are different. For cytoplasmic dynein they are 1-2 µm/s (Paschal et al., 1987, Lye et al. 1987). The movement is much faster for outer arm dyneins (5-10 µm/s; Vale and Toyoshima, 1988) or inner arm dyneins (2-12 µm/s; Kagami and Kamiya, 1992). In contrast to kinesin, single headed dynein does not follow a protofilament but moves throughout a microtubular lattice (Wang et al., 1995) (see below). The conformational changes that accompany cross-bridge cycles that must accompany movement have been studied in axonemes (Goodenough and Heuser, 1984; Burgess, 1995). In the study of Burgess (1995), after removal of the plasma membrane, cockerel sperm flagella were observed with freeze etching in three different nucleotide states: no added ATP (rigor); relaxed (1 mM ATP plus vanadate); and active (i.e. 1 mM ATP). Each state produced a statistically significant morphology of outer dynein arms. The relaxed and active morphologies differed only in the angulation of their heads. The dynein in the relaxed state were in a more tilted position. The rigor morphology showed a conformational change of 12 nm shift in the position of the dynein head in relation to the base, suggesting an ability to develop tension. Active flagella showed all three morphologies. In this case, no unattached stalks were observed suggesting a very prolonged duty cycle. In a study of the dynein from the flagella of of the alga Chlamydomonas reinhardtii using the EM and an image averaging technique (Burgess et al., 2003), the stalk was found to be bent. The conformation in the presence of ADP and vanadate, supposedly being equivalent to ADP and phosphate right after the hydrolysis of ATP, was compared to that without ATP or ADP, supposedly the condition after the power stroke. The conformation changes so that in the latter state the angle between the stalk and stem shifts, a movement that would be reflected in a 15 nm movement at the tip of the stalk. In addition, the stalk becomes straighter. It is unclear how these changes are related to movement. The mechanism is thought to involve the AAA ring head structure changing its orientation in relation to the stem and of moving the stalk toward the plus end of the microtubules. To produce force two possible mechanisms have been proposed (see Gee et al., 1997; Vallee and Gee, 1998). The stalk could act as a rigid lever arm amplifying the conformational changes in the rest of the dynein head. Alternatively the stalk could act as a passive tether where conformational changes in the head region could pull at the base of the stalk dragging the microtubule along. Two dynein heavy chains that differ from the heavy chains of DHC1 have been identified. Both are present in mammalian cells that lack cilia or flagella. DHC3 is localized in a unidentified structures that may be transport intermediates. Microinjection of antibodies to DHC2 disrupts the Golgi apparatus, suggesting that this protein has a role in maintaining the organization of the Golgi (Vaisberg et al., 1996).
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Dyneins bind to different partners where the heavy chain combines with various accessory subunits. These in turn bind to dynactins forming large complexes. These complexes distinguish different cargos and have a broad spectrum of functions. These include the endocytotic pathway, retrograde membrane transport and other functions during cell division (see Hirokawa et al., 1998). The dynein/dynactin complex is also involved in anchoring microtubules to centrosomes (Quintyne et al., 1999) (see Chapter 23). Dynactin is part of the cytoplasmic dynein motor and is needed for dynein-based movement (e.g., see Holleran et al., 1998). Dynactin is believed to function as an adapter, mediating dynein attachment to cargo. It contains two domains. A side arm (p150glued) interacts with dynein and binds to MTs. An actin like filament is thought to bind to cargo. Several subunits of dynactin have been identified and characterized (Eckley et al., 1999). Dynactin, acting as a cytoplasmic dynein activator, has been shown to bind microtubules and increases the average length of dynein movements while the velocity or ATPase kinetics remains the same (King and Schroer, 2000). Both the increase in microtubule binding and motility by dynactin are blocked by an antibody to dynactin's microtubule-binding domain. The complexes are regulated by phosphorylation of the dynein light intermediate chain which reduces the level of membrane associated dynactin decreasing the rate of retrograde transport (Niclas et al., 1996). In addition, isoforms of p150glued bind to dynein without binding to microtubules, presumably downregulating the motor by competing with the functioning dynein (Tokito et al., 1996). A study using a chimera of the green fluorescent protein (GFP) and the p150Glued subunit of dynactin showed that dynactin interacts with the growing microtubule plus end (Vaughan et al., 2002). The binding is regulated by the phosphorylation of p150Glued. Effectors of protein kinase A affected the binding suggesting a role for p150Glued phosphorylation in plus-end binding specificity. During the dyneindependent transport of Golgi membranes the Golgi membranes temporarily interacted with GFPp150(Glued)-labeled microtubules before transport toward the minus end was seen. Kinesins Movement of particles on single microtubules can be followed using DIC and enhanced contrast video technology (Chapter 23). Not surprisingly, giant axons were used in the early studies. The microtubules of extruded axoplasm from giant squid axons supported the fast movement of particles on microtubules in either direction and, the transport was found to require ATP (Brady et al., 1982, 1985). Kinesin was revealed when the ATP analog 5'adenylimidodiphosphate (AMP-PNP), that cannot be hydrolyzed, was used to block fast transport of vesicles in the axoplasm (Brady et al., 1985). The effect of the analog differed from that observed with either dynein or myosin. AMP-PNP weakens the association between motor and fibrous elements in the myosin-actin or dynein-microtubule systems. In contrast, it favors binding of kinesin (and therefore vesicles) to microtubules. This led to the realization that a new motor was involved and soon after to its isolation by extracting microtubules isolated in the presence of AMPPNP. This protein, which was found later to be an ATPase, could be tested for motor activity readily, for example, by the microtubular gliding assay. In a different kind of assay, synthetic microspheres can be http://www.albany.edu/~abio304/text/24part3.html (5 of 46) [3/5/2003 8:27:44 PM]
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coated with the protein and tested on oriented microtubules. Because of its involvement in movement this protein was called kinesin (e.g., Vale et al., 1985a). When tested on isolated microtubules assembled on a centrosome (see Chapter 23) (with the plus end out) using the microbead assay, the beads were found to move away from the centrosome (i.e., in the anterograde axonal direction, toward the plus end, as represented in Fig. 23) (Vale et al., 1985b). The involvement of kinesin is in vesicular transport is shown also by the block of the movement along microtubules provided by kinesin antibodies (Ingold et al., 1988; Brady et al., 1990). A similar assay system that permits dissecting the various molecular components needed for transport has been developed. In essence, components of extracts to be tested are mixed with organelles and assembled microtubules (Pollock et al., 1999) and the movement is observed with video enhanced differential interference microscopy (see Chapter 1). This system has recognized two new kinesin motors involved in organelle movement in Dictyostelium (Pollock et al., 1999). Green fluorescent protein (GFP) has been used to observe movement of motors such as kinesin-GFP constructs (see Chapter 1) moving along a microtubule (e.g., Romberg et al., 1998). This technique has allowed examining the role of components of the neck region in the directionality of the movement (see below). The kinesin family of motors has over 140 proteins in organisms that include plants, protists, fungi and animals (see Hirokawa et al., 1998); www.blocks.fhcrc.org/~kinesin/). The 380 kDa protein is tetrameric, consisting of two copies of a 110-130 kDa (the kinesin heavy chains, KHCs) and two copies of a 60-65 kDa complex (the kinesin light chains, KLC) (Kuznetsov et al., 1988; Bloom et al., 1988). The heavy chains contain the motor domain. However, the kinesin light chains are essential for the functioning of the heavy chain motor domain (see below). In conventional kinesin, the two KHCs subunits, (referred to as KIF5A and KIF5B or KIF5C) are parallel and form a coil-coil domain, so that the two amino terminals and the carboxy-terminal are adjacent (see Vale and Fletterick, 1997). In contrast, the two KLCs have an α-helical coiled-coil domain which associates with the carboxy-terminal of the KHC (e.g., Verhey et al., 1998; Diefenbach et al., 1998). A domain of about 590 amino acids at the amino terminal differs between different species and the sequence of the extreme carboxy-terminal differs between species and between splice variants (Cyr et al., 1991). In mammals, the polypeptide components of the KHC are encoded by three genes, kif5A, kif5B and kif5c (e.g., Xia et al., 1998) and the KLC genes by klc1, klc2 and klc3 (e.g., Rahman et al., 1999). The structure of the motor domain of of conventional kinesin (Kull et al., 1996; Kozielski et al., 1997; Sack et al., 1997) and Ncd (Sablin et al., 1996; Sablin et al., 1998; Kozielski et al., 1999) has been determined by X-ray crystallography. Like other motors, conventional kinesin was found to be rod shaped with a pair of globular heads at one end (e.g., Hirokawa et al., 1989). cDNA corresponding to Drosophila kinesin has been isolated. Like myosin II or dynein from cilia and flagella, the globular portions of kinesin were found at the amino-terminal (however, note that the location of the globular motor domain differs in http://www.albany.edu/~abio304/text/24part3.html (6 of 46) [3/5/2003 8:27:44 PM]
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different kinesins, see below). Kinesin binds to microtubules and hydrolyses ATP at a head site (see Goldstein, 1993). Like myosin, the kinesin molecule was found to have three distinct domains (e.g., Yang et al., 1989). The head domain alone was found to have the motor activity (Yang et al., 1990). A model of a kinesin molecule is shown in Fig. 25.
Fig. 25 Model of a kinesin molecule. The head domain binds MTs and ATP and generates the motor activity. Attached to the head in an α-helical coiled-coil stalk and a tail composed of heavy and light chains (solid elipses). From Goldstein, 1993. With permission from the Annu. Rev. Gen., Vol. 27, copyright ©1993, by Annual Reviews Inc.
Proteins of the kinesin family have been identified by determining the amino acid sequence through the techniques involving the isolation of cDNA and PCR amplification (Endow, 1991) (Chapter 1). Once the sequence was known, database searches for amino acid homologies were then carried out and many proteins belonging to this family were identified. The head portions are very similar for all kinesins. In contrast, the sequences of the tail portion have little in common. Hybridization of PCR-amplified cDNA produced from degenerate primers to the Drosophila polytene chromosomes (where the genome is amplified) suggests the existence of as many as 25 members of the kinesin family (Endow and Hatsumi, 1991). Phosphorylation is the only well documented post-transcriptional modification both in the heavy and light chains (Hollenbeck, 1993). Various members of the kinesin family are represented in Fig. 26.
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Fig. 26 Various members of the kinesin superfamily represented diagrammatically. The motor region (represented by the solid circle) is similar for all of the molecules. Proteins that are predicted to be α-helical coiled-coils in the tail domain are represented as dimers. The α-helical coiled-coil is represented as a chain of ovals. Other proteins are arbitrarily represented as monomers. From Goldstein, 1993. With permission from the Annu. Rev. Gen., Vol. 27, copyright ©1993, by Annual Reviews Inc.
Based on the position of their motor domain containing the ATP and the microtubules binding domains, the kinesin family proteins (KIF) are also referred to as amino-terminal type, middle-type and the carboxyterminal-type. In all plus-directed kinesins the motor domain is attached to the amino-terminal (conventional kinesins) and all minus-directed kinesins have the motor attached to the carboxy-terminal (e.g., Ncd). The kinesin KIF1A is an amino-terminal type of kinesin present in neurons. It moves microtubules at 1.5 µm/s . Mutants of a kinesin of the nematode Caenorhabditis elegans, similar to KIF1A retain vesicles in the cell bodies, and have few synapses suggesting the major role in the transport of synaptic vesicles
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(Otsuka et al., 1991). KIF1B is a smaller amino-terminal type kinesin similar to KIF1A which occurs in many tissues and is involved in the transport of mitochondria (Nangaku et al., 1994). Alternative splicing of the Kif1b gene (which codes for KIF1B) produces eight kinesin isoforms (Gong et al., 1999). One of these isoforms, KIF1Bβ, has a different cargo binding domain ( Zhao et al., 2001) and has been shown to bind to synaptic vesicle proteins and to be essential for proper development of neurons. KIF3, also an amino-terminal kinesin, occurs as a heterodimer and associates with an associated protein (KAP3). KAP3 is approximately 11 nm in diameter and it is possibly involved in membrane binding of the KIF3 heterodimer (Yamazaki et al., 1996). Another member of the KIF3 family is associated with melanosomes in Xenopus laevis (Rogers et al., 1997). The middle-type KIFs have a motor domain in the middle of the molecule. KIF2 forms homodimers that move at approximately 0.4 µm/s. KIF2 occurs in several cell types but primarily in neurons where its expression is controlled developmentally and appears to be present mostly in juvenile neurons (Noda et al., 1995) where it carries a receptor for the β subunit of the insulin-like growth factor 1 receptor (Morfini et al., 1997). Carboxy-terminal-type KIFs have varied functions including the transport of organelles (Saito et al., 1996; Harlon et al., 1997) as well as functioning in meiosis, mitosis and nuclear division. KIFC2 seems to function in transporting a multivesicular organelle functioning between early and late endosomes (Saito et al., 1996). At least some of these kinesins propel cargos toward the minus-end rather than the plus-end, notably Ncd, a carboxy-terminal type kinesin from Drosophila (see below). The profusion of kinesin isoforms suggests that they might be targeted to different structures. In agreement with this notion, the carboxy-terminal of the light chain domains are similar to targeting domains (Cyr et al., 1991) (e.g., a mitochondrial import sequence). Furthermore, in axons, vesicles are transported at different rates. Vesicles propelled by KHC-A moved much faster than those using KHC-B (Elluru et al., 1995). In addition to this complex picture of kinesins, there are many genes related to that of kinesin which code for the kinesin-related proteins (KRPs) (see Goldstein, 1993). The attachment of kinesin to vesicles may require a receptor attached to the membranes (see Section VI). The polypeptide kinectin has been reported to bind to both kinesin and cytoplasmic dynein (Toyoshima et al., 1992) and might serve as a receptor attached to membranes. What happens in the absence of cargo? In this case, the movement of the motors must be inhibited or the unneeded motion would be bioenergetically very expensive. It has been estimated to correspond to 100 ATP molecules/s per molecule of kinesin (Coy et al., 1999) (extrapolated for an individual to be comparable to the human basic metabolic rate!). In the case of kinesin the inhibition results from the kinesin tail binding to the motor domain (Coy et al., 1999; Friedman and Vale, 1999). The tail does not interfere with the attachment of the kinesin to the microtubule. The experiments of Coy et al.(1999) were carried out with KHC constructs derived from the DNA coding for heavy chain of the Drosophila kinesin. http://www.albany.edu/~abio304/text/24part3.html (9 of 46) [3/5/2003 8:27:44 PM]
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The study of Friedman and Vale (1999) used constructs of the human KHC. A sequence in the globular tail domain which has been termed the IAK sequence is responsible for blocking the ATPase activity of the motor domain inactivating kinesin (see Hackney and Stock, 2000). Although the mechanism of inactivation of kinesin is clear, the mechanism or mechanisms responsible for activation of inactive kinesin is less well defined (see Verhey and Rapoport, 2001) and may be multiple. Activation may correspond simply to the binding of cargo to the tail of kinesin. Head-to-tail interactions have also been found to regulate several myosins (see Reilein et al., 2001). A variety of arguments suggest that the KLC component of kinesin imparts specificity of binding to different cargoes (see Manning and Snyder, 2000). This is attested by the multiplicity of the KLC isoforms and in some cases the demonstration of specific binding to certain organelles or structural components. For example, five different KLC isoforms are present in a mammalian culture line, one of which binds only to mitochondria (Khodjakov et al., 1998). In addition, kinesin complexed to vimentin (see below) contains a specific KLC isoform (Liao and Gundersen, 1998). As discussed below, the binding of kinesin to cargo is mediated by scaffolding proteins. Another possible role of the KLC is the regulation of the kinesin motor or the binding to microtubules. KLC has been proposed to inhibit the motor activity. The kinesin conformation change during movement brings the KHC tail domain in close contact with KLC (e.g., Hackney et al., 1992). Furthermore, removing KLC from the KHC increases the ATPase activity of the motor (Hackney et al., 1991). When KHC and KLC are cotransfected into COS cells (transformed kidney cells from the African green monkey), the binding to microtubules is inhibited (Verhey et al., 1998). These observations suggest that KLCs are involved in cargo recognition and the microtubule binding capacity. As discussed above, without a cargo, kinesin is probably in the folded conformation so that the KLC domain may bind to the motor domain blocking the ATPase. Although the role of KLCs is uncertain, they are essential for function. In Drosophila disruption of a single klc gene is lethal (Gindhart et al., 1998) because of a failure axonal transport. In mice, knockout mutation of the klc1 gene causes a defect in the activation and targeting of KIF5A and KIF5B (Rahman et al., 1999). Motors and mitosis Some of the details of mitosis were discussed in Chapter 23. The role of various motors will be discussed in this section. Since the motors act in concert with microtubules (MTs), these will have to be discussed as well. As we saw in Chapter 23, the organization of MTs is dynamic and plays a role in many functions of the mitotic apparatus. The spindle MTs are oriented so that their minus ends are toward the centrosomes and their plus ends toward the equator (see Chapter 23, Fig. 26). The astral MTs have a similar orientation, however, since they point in the opposite direction, their plus ends are toward the cytoplasmic cortex rather than the spindle. As we saw, some of the interpolar MTs overlap. Since they originate from different poles they are antiparallel. As discussed below, these MTs in conjunction with the appropriate motor(s) move the spindle poles away from each other. The MTs attached to the kinetochores (K-MT in the figure) and their motors move the condensed chromosomes toward the equator (prophase and prometaphase) or http://www.albany.edu/~abio304/text/24part3.html (10 of 46) [3/5/2003 8:27:44 PM]
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the poles (anaphase). On the other hand, the astral MTs, attached to the cell's cortex and the spindle poles also function in pulling the poles apart and maintain the orientation of the spindle in relation to the cortex. In addition, a set of MTs connects the centrosomes to the arms of chromosomes and should also function in chromosomes movement. At least eight distinct motors have been found to be involved in mitosis (see Sharp et al., 2000). It is generally agreed that movements related to the mitotic spindle involve the coordinated interaction of several motors that can operate antagonistically or in a complementary manner. These motors are thought to function in three different ways. When they form cross-bridges between MTs, they slide an MT in relation to adjacent MTs. Some may bridge MTs to other structure structures and also provide movement. MTs transport cargos along the surface of the spindle. In addition, they regulate the assembly and disassembly of the MTs. Some members of the kinesin family form bipolar kinesin-like homotetramers with two motor domains at opposite ends of the kinesin molecule ( Kashina et al., 1996; Gordon and Roof, 1996). The motors can attach to adjacent MTs and are likely to slide antiparallel spindle MTs in opposite directions. They are thought to have a role in the elongation of the spindle. For example, interference with these bipolar kinesins produce spindles with abnormally close poles (see Sharp et al., 1999). As seen with the EM, the Saccharomyces cerevisiae bipolar motor (Kip1p) has two globular domains 14 nm in diameter connected by a 73 nm long stalk (Gordon and Roof, 1999). Monopolar motor molecules can also produce a sliding. In this case, a domain at one end of the molecule attaches to an MT and the motor end is attached to an adjacent MT. Other motors (dynein/dynactin) act on MTs attached to the cell's cortex and are thought maintain the centrosomes in their appropriate position and play a role in spindle elongation. The positioning of the chromosomes could be driven to the metaphase plate by plus-directed motors. In contrast, the minus-directed motors would drive the chromosomes toward the poles. The kinetochores are involved in these movements. The kinetochores can move bidirectionally on the surface of MTs (e.g., Hyman and Mitchison, 1991). In fact, a plus-end (CENP-E) ( Yen et al., 1992) and a minus-cytoplasmic dynein ( Steuer et al. 1990; Pfarr et al., 1990) have been shown to be located in the kinetochore. For example, immunological microscopic techniques (see Chapter 1) showed the presence of cytoplasmic dynein near or at kinetochores, centrosomes and spindle fibers during mitosis but found it distributed throughout the cytoplasm in interphase ( Pfarr et al., 1990). CENP-E was found to be in the kinetochores ( Yen et al., 1992) during the formation of the metaphase plate, in the spindle midzone at anaphase. Eventually, it is degraded at the end of mitosis. In agreement with a role of these motors in chromosome movement, inhibition of CENP-E produces defects in chromosome alignment ( Wood et al., 1997) and mutations of genes coding for components of the dynein complex produce defective chromosome segregation (Bowman et al., 1999; Lee et al., 1999). Several plus-end motors contain chromatin-binding motifs and are likely to bind to the chromosome arms rather than kinetochores. They are thought to be involved in the transport of the chromosomes in the formation of the metaphase plate (e.g., Molina et al., 1997).
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There are indications that some motors regulate the assembly and disassembly of MTs. Chromosomes can move while attached to MTs that are shortening by depolymerization. In lysed Tetrahymena cells, antibodies to the dynein CENP-E were shown to block the movement of chromosome fragments toward the MTs minus-ends during MTs depolymerization ( Lombillo et al., 1995). These observations suggest that CENP-E helps couple chromosomes to depolymerizing MTs. In a similar coupling activity, the MTs of the spindle may remain attached to kinetochores while their lengths change during both prometaphase and anaphase A. D. Intermediate Filaments Intermediate filaments (IFs) are cytoskeletal fibers generally 8-10 nm in diameter. They are intermediate in thickness between the thinner F-actin filaments (the microfilaments) and the thicker microtubules. In most cells the IFs form a basketlike array around the nucleus but also reach the periphery of the cell. They are present at specialized junctions, and they are prominent throughout the length of axons. However, they are not evenly distributed. Neurofilaments (IFs of neurons, or NFs) are six times more numerous at distal levels of the mouse optic axon (Nixon et al., 1994) or in the squid giant axon (Martin, 1996). 50 different IF genes are differentially expressed in various cells depending on tissue and developmental stage. In many cells IFs constitute only 1% of the total protein. However, in certain cells such as epidermal keratinocytes and neurons, IFs account for as much as 85% of the total protein of mature cells. Genetically characterized keratin disorders involve fourteen of the known keratins (see Fuchs and Cleveland, 1998). The complexity and variety of IF proteins and tissue-specific expression suggest that they have an important role. They are most likely to play a role in specialized cell functions and in maintaining cell structure. However, a general housekeeping role seems unlikely because at least some cultured cells function well and even divide in the absence of IFs (although in these cases, lamins, the IFs of the nucleus are present) (see Steinert and Roop, 1988). Strictly speaking, IFs are not part of the machinery responsible for movement. However, a discussion of IF is necessary in any consideration of movement. We have seen how difficult it is to separate a discussion of movement from that of structure (e.g., for the case of microvilli whose scaffolding is in part composed of actin and myosin). The neurofilaments are thought to function in the maintenance of the caliber of large myelinated axons, a factor with obvious implications in intra-axonal transport and in the structural and dynamic environment needed for other functions. It has been suggested that IFs are also implicated in signal transduction (Baribault et al., 1989; Skalli et al., 1992). As is the case for MT and actin, IF filaments are also in a dynamic state. IF incorporate microinjected unpolymerized subunits. Fluorescence recovery studies (FRAP) (see Chapter 4) demonstrate that the polymerized IF and unpolymerized subunits are at a steady state (Vikstrom et al., 1992 and Okabe et al., 1992). Hyperphosphorylation of IF is frequently accompanied by disassembly (see Skalli et al., 1992). As discussed below, the organization of the IF network is thought to be the result of interactions between http://www.albany.edu/~abio304/text/24part3.html (12 of 46) [3/5/2003 8:27:44 PM]
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IF, IF-associated proteins (IFAPs), microtubules and actin filaments. IF networks have been studied in living cells transfected with DNA coded for a green fluorescent protein-vimentin construct (e.g., Yoon et al., 1998; Prahlad et al., 1998). Vimentin is one of the subunits of IF (see below). GFP-vimentin is incorporated into the IF network and accurately reports the movement of IFs. Interphase arrays of vimentin containing fibrils were found to constantly change in configuration even when the cells do not change in shape. Fluorescence recovery after photobleaching (FRAP) shows a rapid recovery of bleached spots indicating that the IFs are in continuous motion. Areas that are still bleached frequently move, indicating whole fibrils are moving. In view of the association between IF and microtubules and actin filaments the movements are thought to reflect to motion mediated by these components. The movement of small fragments containing vimentin were found to be inhibited by kinesin antibodies, indicating that this is the motor responsible for the movement of these short fibers (Prahlad et al., 1998). The role of IFs has to be evaluated from contradictory data, probably because defects in IFs may be rather subtle and difficult to observe. Microinjection into cultured fibroblasts of peptides of the helix region of the IFs disrupts the IF, MT and microfilament network (Goldman et al., 1996). These so called mimetic peptides disassemble IF assemblies into small oligomeric complexes and monomers within 30 min at room temperature in vitro. These observations argue for a very important role of IFs in cell organization. In agreement with this interpretation disruption of proteins that cross-link IFs to other filamentous elements (including IFs) does have serious consequences (see below). However, earlier work in which IF organization was disrupted by anti-IF antibodies had no effect on other components (Quinlan et al., 1994). In addition, cell lines lacking IFs were found not to have a major disarray in actin filaments or MTs (Sarria et al., 1994; Colucci-Guyton et al., 1994). Neurofilaments form bundles by combining with other neurofilaments by cross bridges in the axon as they do in vitro (Hirokawa, 1991) as also shown by the persistence of the spacing of 50-55 nm between neurofilaments corresponding to the side arm of the heavy chain of the filaments (NF-H) (Xu et al., 1996b). NFs are heteropolymers of NF-L, NF-M, and NF-H. Their carboxy-terminal tail form sidearms along the length of the fibers ( Mulligan et al., 1991). All indications are that the elements of the cytoskeleton form a cross-linked network. Generally, ultrastructural studies support this concept. They show that IFs are cross-linked to microtubules and Factin (e.g., Hirokawa 1982; Yang et al., 1999). Furthermore, when microtubules depolymerize, the IF network collapses, a process which depends on actin (see Gard and Klymkowsky , 1998). In addition, disruption of microtubules and microfilaments produces a reorganization of keratin-type IFs (Gard and Klymkowsky , 1998). The three-dimensional structuring of the cytoplasm requires the interaction of the various components of the cytoskeleton (see Chou et al., 1997). A variety of intermediate-filament-associated proteins (IFAP) cross-link the IFs to other IFs and to F-actin and MTs and cell membranes (see below). IFs are unique among the cytoskeletal structure in that their protein subunits are fibrous (for a review on http://www.albany.edu/~abio304/text/24part3.html (13 of 46) [3/5/2003 8:27:44 PM]
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IF, see Steinert and Roop, 1988). As previously discussed, F-actin and microtubules are formed from the polymerization of globular subunits. The IF proteins form long filaments of high-tensile strength. Five types of IF are recognized, spanning a size range between 40 to 130 kDa, and they have been classified from their amino acid sequences and assembly properties (see Parry, 1999, Herrmann and Aebi, 2000). The central portions of all IF proteins (the so-called rod portions) of approximately 310 amino acids are highly homologous and in an α-helical configuration (see Fig. 27 for a diagrammatic representation; Steinert and Roop, 1988). Antiparallel association of the central region of two fibrous subunits produces a double-stranded coil (i.e., a dimer), a 3 nm protofilament (Fig. 28b). In the case of keratins, the two chains constituting the dimers are different (one of type I and the other of type II). Two of the dimers align in parallel to form a tetramer with two globular domains at each end, the 4.5 nm protofibril (Fig. 28c). Associations of these protofibrils produce the ~10 nm IF fiber. These further arranged in a staggered manner (Fig. 28d) produce a striated appearance when viewed with the EM. Type I and II IFs (so-called assembly group I, see Herrmann and Aebi, 2000), the keratins (acid, basic, or neutral), are heteropolymers formed from an equal number of subunits from each subgroup. They are found predominantly in epithelial cells (besides hair and nails). There are at least 19 different types of keratins in human epithelia. The keratin network links the plasma membrane, the nucleus and other cytokeletal components. Keratin-null mutants in humans and mice show that it is an essential component neeed for the integrity of epithelial tissues (see Albers, 1996). Type III (assembly group 2) proteins include vimentin (mesenchyme), desmin (striated and smooth muscle), and glial fibrillar acidic proteins (astrocytes and Schwann cells). These IF proteins assemble spontaneously in vitro to form homopolymers and heteropolymers. Desmin-null animals have severe muscle deficiencies such as misaligned muscle fibers (Li et al., 1996; Milner et al., 1996). Desmin is thought to be associated with the Z discs of muscles (see Sections I C and I D). Type IV proteins are neurofilament proteins of neurons. In vertebrates the type IV IF proteins represent three different polypeptides. Type V are nuclear IF proteins, the lamins, that form a highly organized network, the nuclear laminae.
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Fig. 27 Subdomains of IF chains. All central rod domains are flanked by end domains. For details see Steinert and Roop (1988). Reproduced from the Annual Review of Biochemistry, Volume 57, copyright ©1988 by Annual Reviews Inc.
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Fig. 28 IF chains and formation of oligomers. (a) IF chain, (b) two coiled-coil molecules in parallel and in register, (c) Proposed models for four chain complexes, (d) possible 8-chain complex and (e) lattice of an entire IF containing 32 chains 147 nm. The axial banding at 22 and 47 nm is that observed with the EM and X-ray diffraction. Steinert and Roop (1988). Reproduced from the Annual Review of Biochemistry, Volume 57, copyright ©1988 by Annual Reviews Inc.
The view that is emerging with more studies suggests the IFs form a network that has a dynamic role in the arrangement of the cytoskeleton and attachment of the plasma membrane to the cytoskeleton (see Houseweart and Cleveland, 1998). IFs can contribute to rapid changes in the cell's cytoskeleton because they are present in readily available pools of subunits. The interactions involving IFs are thought to determine cell shape and resistance to mechanical stress. Phosphorylation of specific domains in the IF proteins controls assembly. For example, vimentin (Inagaki et al., 1987) is disassembled by phosphorylation, whereas phosphorylation of desmin inhibits assembly (Geisler and Weber, 1988). In fact, a dynamic remodeling of the cytoskeleton during neuronal growth and the establishment of directionality is thought to depend on the phosphorylation of specific domains of neurofilament proteins (Nixon and Sihag, 1991).
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E. The Plakin Family The interdependence of the cytoskeletal components has been firmly established by the finding of cytoskeletal linkers, proteins of the plakin family (see Ruhrberg and Watt, 1997; Leung et al., 2002). Plakins crosslink fibrous elements of the cytoskeleton and junctional complexes ( see (Ruhrberg and Watt, 1997; Leung et al., 2002). Plakins are expressed mostly in tissues subject to mechanical stress (e.g., epithelia, muscle, etc.). They contain a globular amino-and carboxy-domains separated by a central coiledcoil rod. All plakins possess one or more plakin domains (PRD) usually flanking the rod domain. Some members also have other domains such as the actin-binding, microtubule binding and spectrin-repeat domains. Desmoplakins are to be found in junctional plaques (see Green and Bornslaeger, 1999) such as desmosomal plaques and they anchor IFs to the plasma membrane to form connections between cells. The desmoplakin gene produces two alternatively spliced forms DPI and DPII of 322 and 259 kDa respectively (Green et al., 1990; Virata et al., 1992). The plectins are found in most tissues (except neurons). They are more than 500 kDa in size and they have been found associated with IFs (e.g., Clubb et al., 2000) and in stress fibers . They also connect IFs to microtubules ( Svitkina et al., 1996). Homozygous mutants of the plectin gene are responsible for epidermolysis bullosa simplex which produces blistering of the epidermal basal layer and muscular dystrophy. Bullous pemphigoid antigen 1 (BPAG-1) is present as two isoforms BP230 (BBPAG-1) and BP180 (BPAG-2). BP230 is cytoplasmic and associates with hemidesmosomal plaques, BP180 is a transmembrane glycoprotein. The BPAG-1 gene gives rise to several distinct proteins with a distinct tissue distribution by alternative splicing. They are important for the maintenance of cell structure of neurons, muscle and epithelia. One of the plakin isoforms contains an actin-binding domain and some isoforms have been shown to link IFs and the actin cytoskeleton (Yang et al., 1996; Andra et al., 1998a). Immunoelectron microscopy has shown plectin cross-bridges between IFs and microtubules, IFs and actin filaments and IFs and myosin filaments (Svitkina et al., 1996). In BPGA1-null mice IFs fail to tether neurofilaments to the actin cytoskeleton (Yang et al., 1996) and the microtubules are disorganized. The mice exhibit dystonia musculorum, characterized by rapid degeneration of sensory neurons, and display a mild skin blistering, a reflection of disorganization of the IFs. In humans the homologous condition is epidermolysis bullosa simplex (EBS)-MD, a hereditary skin blistering disease with muscular dystrophy, caused by defects in the BPFGA-1 gene. A form of plakin ( Yang et al. 1999) lacks the domain that binds to actin but is capable of binding to microtubules and appears to stabilize them. Plectin has been shown to bind to vimentin, keratins, Lamin B, MAPs, α-spectrin and neurofilament proteins (Foisner et al. 1988,1991) as well as actin (Yang et al., 1996). An involvement of plectin with the cell membrane and the actin network is shown by its ability to interact with G-actin in vitro in a phosphatidylinositol-4,5biphosphate dependent manner and its association with actin stress fibers in living cells (Andra et al., 1998a). Microtubule-actin cross linking factor (MACF) is a 600 kDa protein is thought to functionin providing http://www.albany.edu/~abio304/text/24part3.html (17 of 46) [3/5/2003 8:27:44 PM]
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interations between actin and microtubules (e.g., Leung et al., 1999). V. MECHANISMS OF MOVEMENT AND COMPARISON OF MOTORS Myosin and kinesin have been studied much more intensely than cytoplasmic dynein. The mechanism of movement of myosin has been discussed in Section IA. For these reasons much of the discussion that follows is directed toward kinesin. However, the intimate mechanism linking the conformational changes to the nucleotide binding site are discussed in some detail for both myosin and kinesin. A. The Movement As in the case of myosin, the kinesin heads have been shown to change in conformation during the ATPase cycle involved in motion (Hirose et al, 1995). Kinesin molecules were found to move very slowly without a neck domain. However, they gained very rapid movement when joined to a very short (about 11 amino acids) flexible random chain (Inoue et al., 1997). This latter finding suggests that the mechanism of movement differs significantly from that of myosin, where a rigid lever arm is likely to play a role in magnifying the conformational changes. Kinesin is a processive motor: it remains bound to the tubulin while undergoing multiple rounds of activity. This is probably because kinesin is double headed and moves hand-over-hand and at any one time one of the two heads is attached to the MTs. The hand-over-hand model proposes that the heads of double headed kinesin attach alternatively, with the head that is not bound moving to face the direction of the movement. However, simultaneous transient binding of both heads are required for processivity, otherwise the motor would detach entirely. For kinesin, processivity is shown by a variety of experiments. Single molecules typically travel over distance as long as 1 µm representing a 1% probability of dissociation per molecular step or about 100 enzymatic turnovers. It has been proposed that myosin V, which is two headed, is also a processive motor (e.g., Nascimento et al., 1996). Studies using an optical trapping system (see Chapter 1) have concluded that myosin V is indeed processive (see Mehta et al., 1999) and moves in large steps of about 36 nm. Myosin II and dynein are are not processive. In the case of dynein, Wang et al.(1995) studied the movement on MTs of single or few cytoplasmic dynein molecules bound to each bead. In contrast to kinesin, dynein moved on more than one protofilament indicating that it frequently detaches from its original track. Similarly, the results obtained with subfragment 1 (a single head) and heavy meromyosin (double-headed) are approximately the same (Molloy et al., 1995b). The hand-over-hand mechanism of kinesin movement is supported by a several experiments. In the absence of ATP one head is attached to the microtubule, the other is attached and bound to ADP. Addition of ATP releases the bound head and the ADP from the detached head (Hackney, 1994). Furthermore, the results of other studies of Ma and Taylor (1997) suggest that ATP binding to one head is required for the ADP release by the other head so that the two heads act cooperatively and alternate in their function.
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Structural information is in harmony with the hand-over-hand model. Cryoelectron microsope images of kinesin attached to a MT show one head attached with the other detached and oriented at right angle to the filament (Arnal et al., 1996; Hirose et al., 1996). If the hand-over-hand model were correct a single headed kinesin should not be processive, in contrast to the double headed variety. Furthermore, it is difficult to see how it could be directional (Inoue et al., 1997). The dimeric motor works at extremely low concentrations down to the single molecule level (Howard et al., 1989) and follows the track of protofilaments closely (Ray et al., 1993). In contrast, in the same assays, single headed monomeric constructs of Drosophila kinesin move slowly, do not follow straight tracks and require high concentration of the motor (Gelles et al., 1995). This simple picture is complicated by the observation that a supposedly single headed kinesin is processive, being able to move along a microtubule for as much as 1 µm (Okada and Hirokawa, 2000). This kinesin was a chimeric construct of conventional kinesin with the motor of KIF1A, a monomeric kinesin, supposedly unable to dimerize (Jiang et al., 1997). Apparently, the explanation for this behavior rests on the structure of the molecule. This protein possesses a lysine rich region in the motor domain referred to as a K-loop. The K-loop is thought to keep the motor in close contact with the negatively charged microtubule (Okada and Hirokawa, 2000). Other experiments have also been directed toward this question. The properties of single headed kinesin containing the whole "rod" segment were compared to those of wild-type kinesin (Hancock and Howard, 1998). The microtubule-gliding assay was used, where the motor is attached to glass and the microtubules are free to move. The microtubules were labelled with a visible marker, in these experiments at the front end of the microtubule. The movement could then be recorded and analyzed. The procedure was carried out with various densities of the motor molecules. The Hill coefficient derived from a Hill plot provides information on cooperativity, i.e., how many kinesin molecules produce motion. The maximum value of n corresponds to the number of subunits or binding sites. Unity indicates independent action of each molecules. In the case of the double headed kinesin the Hill coefficient was unity indicating that only one was needed for motion. When the single headed kinesins were used, however, the Hill coefficient ranged from 4 to 6 indicating that motion required the participation of that many kinesin molecules. Furthermore, single heads remained attached for as long as 3 s. This indicates that the two heads act in concert in agreement with the hand-over-hand model. The role of the neck region in kinesin motion has lead to a good deal of speculation. The neck region is needed to allow the two heads to interact during the cycle. In addition, the geometry of of MTs and kinesin in its most frequent conformation is such that both heads cannot be bound simultaneously at the required 8 nm distance between binding sites, whereas the two heads are 6 nm apart. Transient binding is needed for processivity (see Kozielski et al, 1997). Therefore, the neck region must undergo some conformational change to allow for the two heads to attach simultaneously. In contrast to conventional dimeric kinesin, Ncd (also dimeric) has low processivity (Case et al., 1997; Block, 1998). However, the two heads must move in relation to each other to allow for the conformation seen in the three dimensional reconstructions from cryoelectronmicroscopy (Hirose et al., 1996; Arnal et al., 1996; Hirose et al., 1998; Hirose et al., 1999). This conformation could correspond to a melting of the coiled-coil neck region to allow for the
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separation of the two heads (Endow and Fletterick, 1998). In most kinesins, the neck consists of two parts. At the amino-terminal approximately 10 amino acids form a β sheet (Case et al., 1997; Henningsen and Schliwa, 1997). The domain that follows forms heptad repeats of about 30 amino acids that form a coiled-coil structure (Huang et al., 1994; Morii et al., 1997; Tripet et al., 1997). The carboxy-terminal of kinesin has a coiled-coil stalk domain followed by a globular tail. In contrast, the Ncd neck is a coiled-coil continuous with the stalk (Sablin et al., 1998). The difference in structure of the two molecules leads to a different orientation of the heads in relation to the stalks. The structural differences suggest that two distinct mechanisms operate in the neck region. Recent models of conventional kinesin suggest a cycle in which the neck serves to integrate the function of the heads (Hackney, 1994; Hirose et al., 1996, Tripet et al., 1997). At the beginning of the cycle the neck holds the two heads close together and so tightly that only one can bind to the microtubule. The binding of the nucleotide to the second head causes a segment of the coiled-coil to unwind. This unwinding alters the spacing between the two heads and allows the second head to reach a new binding site in the microtubule. Then the coiled-coil is reformed at the end of the cycle, a process that pulls the first head off the microtubule. There is substantive evidence for a rearrangement of the two heads. Microtubules decorated with constructs of kinesin molecules (Hoenger et al., 1998) were examined with cryoelectron microscopy using image reconstruction techniques and X-ray crystallography. A monomeric construct with a short neck (insufficient for a coiled-coil formation) decorates microtubules with a stoichiometry of one kinesin/αβ tubulin dimer. A longer kinesin construct with a longer neck forms a helix coiled-coil is correspondingly dimeric. A we already saw, the stoichiometry was found to be unchanged corresponding to one kinesin head per tubulin heterodimer. In order for both head to bind simultaneoulsy they must be able to separate to a distance of 8 nm, beyond the 6 nm normal distance between the two. The conformational change may not involve a rearrangement of the coiled-coil region. Romberg et al. (1998) tested the role of the neck region by deleting or altering it and then examining the effect on single molecule motility. Eliminating the coiled-coil region of the neck entirely, decreases processivity ten-fold but does not abolish it. Stabilizing the neck by replacing the neck region with a peptide sequence that forms a very stable coiled-coil, only reduced processivity 45% suggesting that unwinding of the coiled coil region does not play a significant role. Increasing the flexibility between the two heads by introducing a three residue glycine linker at the beginning of the neck reduced processivity by 60%. When the first heptad was duplicated processivity was enhanced ten-fold. These results suggest that unwinding of coiledcoil region is not involved in the motion but that the neck structure is still very important. Romberg et al. (1998) propose that the β sheet region is disrupted during the hydrolysis cycle. There is some structural support for this model. The β sheet of the neck have been found in a highly ordered (Kozielski et al., 1997) and in a disordered form (Kull et al., 1996) suggesting that it may be able to alternate between two different conformations.
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An interesting approach (see Howard, 1997; note however that several aspects are still under discussion, Huxley, 1998 and Howard, 1998) that has considerable predictive value and may help understand the functioning of motors, is that of duty ratio, r, defined as fraction of the time a motor domain spends in contact with the corresponding filament (originally called duty cycle) (Eq.(1).
A two headed molecule that stays attached most of the time will have an r that is high (e.g., 0.5), otherwise it will diffuse away. On the other hand, when many cross-bridges are involved as in the case of muscle myosin, the r can be quite small (approaching 0.01 or 0.1). A low value of r (0.1-0.2) was deduced by paramagnetic resonance spectroscopy which showed that only 20% of the heads were ordered as expected if bound to actin (Cooke et al., 1982) (the reciprocal of the number of cross bridges needed for continuous motion). This simple relationship has several implications. If during a contractile event each head moves over a distance δ then v =δ/τon. where v is the velocity of the motor. However, we expect the total cycle time to be τtot= 1/V, where V is the rate at which each head hydrolyzes ATP. Substituting these values in the original equation, assuming that 1 ATP is hydrolyzed per mechanical cycle, the ratio becomes:
This approach has promise because of its predictive value. For example, (see Howard, 1997) unloaded fast skeletal muscle glides twenty times faster than conventional kinesin per ATP hydrolyzed. In addition, the movement is for approximately 400 nm. This is because myosin has a low value of r, about 0.01 and an ATPase rate of 20 per head per second. From Eq. (2), δ = r.v/V or 0.01 x 8000 nm s-1= 4 nm. 4nm is well within the size of the displacement of one myosin head. Basically this means that of one hundred myosin heads, each contributes only 4 nm of displacement. While the motor is detached, the other heads displace the filament for 400 nm. This will not be the case for kinesin where a distance of 8 nm and an ATPase rate of 50 s-1 permits a speed of only 800 nm s-1 because r = 0.5. The values of r are, in actual fact, limited because the displacement must conform to the presence of a binding site on the filament. Another way of looking at r is in terms of distance (Eq. 3). If we consider d the path distance as the minimum distance between consecutive binding sites of the same motor, the r ought to equal the fraction of the distance to the next binding site. Where it is smaller than d, each individual crossbridge must spend a significant amount of time detached while other crossbridges move the filament. In this case, the duty ratio must be less than 1; it should be equal to the fraction of the distance to the next binding site. n can be
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greater than 1 if a binding site is skipped.
Does the definition formalized by Eq. (3) agree with the previous definition? In the case of kinesin there is one dimer binding site per kinesin molecule and the kinesin proceeds straight on the protofilament. Recent tracking studies have shown that the steps are probably 8 nm. Since n = 2 (one kinesin head has to skip one site occupied by the other head), r would be 0.5 consistent with previous considerations. Myosin on actin filaments also proceed on a straight or near straight line. If the myosin head binds to correctly oriented actin subunits, the path distance should be 36 nm corresponding to the half pitch of the actin helix. When an actin filament is suspended between to beads held in optical traps, binding is observed in multiples of 40 nm (Molloy et al., 1995a). B. Inside the Motors Movement is linked to ATP hydrolysis. The intimate details of the mechanism of motion must involve the catalytic site of the motors. Therefore, it would seem necessary to examine the details of nucleotide binding and hydrolysis and their structural correlates in order to understand the mechanisms. Although a good deal has been found in recent years, the emerging picture is not always consistent (e.g., see Volkmann and Hanein, 2000) Myosin, kinesin and G-protein (the heterotrimeric GTPase) (see Chapters 7 and 11) have several common structural features (see Smith and Rayment, 1996b; Kull et al., 1996, 1998) and many differences. The present discussion will center on myosin and kinesin. Members of the kinesin family have catalytic core of about 330 amino acids. This core differs little from one kind of kinesin to another (see Vale and Fletterick, 1997) and is very similar to the myosin core (Kull et al., 1996; Kull et al., 1998). Despite the similarities between the kinesin and the myosin motor domains, there are significant differences in the mechanism of motion (for animation models for both myosin and kinesin see Vale and Milligan, 2000 www.sciencemag. org/feature/data/1049155.shl). As discussed above (Section IC), muscle myosin undergoes a large angular rotation of a long and relatively rigid element corresponding to an α-helix and associated light chains. Each ATP-hydrolyzed corresponds to a 10 nm displacement (note however that this may be controversial point; 6 nm is indicated by single molecule analysis, see above, however, in these experiments two steps are seen with a total of about 12 nm). ATP causes myosin to dissociate from actin and to undergo its recovery stroke. The release of Pi following nucleotide hydrolysis, produces a tight binding to actin and the forward swing of the lever arm. (Rayment et al., 1993a; Dominguez et al., 1998; Corrie et al., 1999; Suzuki et al., 1998; Houdusse et al., 1999). In contrast, in conventional kinesin, a conformational change occurs in the neck linker peptide of http://www.albany.edu/~abio304/text/24part3.html (22 of 46) [3/5/2003 8:27:44 PM]
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approximately 15 amino acids. The neck linker is rigid only in the ATP/ADP.Pi-bound state. In the case of kinesin, ATP-binding is responsible for the power stroke in the form of a forward motion of the neck linker. ATP hydrolysis releases the binding of kinesin to the microtubule and detaches the head linker. The small conformational change of the head linker is amplified to produce an 8 nm movement by the swinging of the partner head to the next binding site. Three segments of myosin have been traditionally referred to as 15, 50 and 20 kDa segments (in that order from the amino terminal) corresponding to the segments separated by tryptic digestion (see Rayment, 1996 and Fig. 10). The ATPase site is located in the cleft that splits the central 50 kDa segment into lower an upper domains. The bound nucleotide is surrounded by the P-loop and two loops in the 50 kDa segment (referred to as switch II and switch I loops). The switch I loop runs along the ATPase pocket and some ot its side chains are arranged toward the pocket to form non-covalent bonds with nucleotide and Mg2+. One of the side chains of switch II points toward the ATPase pocket and is hydrogen bonded to the water surrounding the Mg. Other chains outside of the ATPase pocket connect the ATPase site and the long αhelix (the switch II helix). In the myosin-MgADP/VO4 complex (considered equivalent to myosin-ADP.Pi, the metastable state after hydrolysis; see Fig. 9), a residue of the switch I loop forms an ionic bond with with one of the switch II loop residues so that switch II is held close to the nucleotide. In contrast, when myosin is complexed to MgADP/BeFx (considered equivalent to ATP), these residues are separated and the conformation is open with the loops away from each other. In this process, switch II moves by almost 6 Å relative to switch I. The movement is propagated to the long adjacent helix and rigid loop (the relay), to the lever arm (Dominguez et al., 1998). A domain referred to as the converter domain which is connected to the lever arm has been shown to rotate about 70o, calculated to correspond to a 10 nm displacement of the lever arm. Vale and Milligan (2000) view the mechanical factors to be in response to the γ-phosphate of ATP. A sensor of the γ-phosphate produces a change conformation when undergoing the transition from the ATP and ADP bound states. Other components link the catalytic site, the mechanical element and the filament binding sites. In myosin, the sensor would correspond to Switch I and Switch II which form hydrogen bonds with the γ-phosphate. In addition, they position a catalytic water and important side chains for cleavage of the β to γ-phosphate bond. Switch II swings in to interact with the γ-phosphate and swings out when the γ-phosphate is removed. This has been shown when the myosin structures were compared with and without ATP analogs (Fisher et al., 1995b; Smith and Rayment, 1996a). The switch mechanism in kinesin (see Kull et al., 1996) is almost identical and it also resembles that of GTPase proteins (see Vale and Fletterick, 1997; Kull et al., 1998). The movements of the γ-phosphate sensor are transmitted via a long helix connected to the switch II loop at the amino terminal and to the filament binding sites at the carboxy-terminal. This helix is the switch II helix of kinesin and the relay helix of myosin. In myosin, the helix undergoes a conformational change similar to that of a piston. With ATP-ADP.Pi analogs, the motion of the switch II loop toward the γhttp://www.albany.edu/~abio304/text/24part3.html (23 of 46) [3/5/2003 8:27:44 PM]
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phosphate tilts and translates the relay helix along its axis toward the upstroke position. In the absence of nucleotide, switch II swings away and the relay helix goes to the downstroke position (see Vale and Milligan, 2000). Similar conclusions can be reached for kinesin. The movement of kinesin is produced by a conformational change of the 'neck linker" (Rice et al., 1999) which consists of a 15 amino acid region. In the absence of nucleotide or when it binds ADP, the neck linker is mobile when kinesin is attached to microtubules. When the attached kinesin binds an ATP analog, the neck linker becomes immobilized by attaching to the catalytic core with its carboxy-terminal pointing toward the plus-end of the microtubule. In this way, the binding of ATP drives the forward motion of the head linker and whatever is attached to the carboxy-terminal. The motion of one head is conveyed to the other via the coiled-coil dimerization domain, so that, the trailing head detaches and from its binding site and is moved forward to the next binding site. A major advance has been the determination that 1 ATP is hydrolyzed for each step in the movement of kinesin on a microtubular track (Schnitzer and Block, 1997; Hua et al., 1997). In principle, without corrections for Brownian motion or statistical analysis, the ATP hydrolyzed per step can be computed directly. For a single dynein dimer, the velocity of the movement (nm/s) (measured experimentally) divided by the size of the steps (8 nm), will give us the number of steps per second (n s-1). The rate of ATP hydrolysis per g of purified dynein (measured independently in the presence of tubulin) x the molecular weight of the dynein dimer will give us the rate of hydrolysis per molecule of dynein (ATP s-1). Then the ATP/step = (ATP s-1)/ (n s-1). In one set of experiments, beads attached to a single molecule of kinesin were tracked with interferometry (Svoboda et al., 1993) (see Chapter 1 for the principles involved in interference microscopy). Statistical analysis of the interval between steps, together with measurement of motor speed as a function of ATP concentration allows the calculation that 1 ATP is hydrolyzed per 8 nm step. In another study carried out at the same time, a very low ATP concentration (150 nM) was used allowing for corrections of Brownian motion. The study also arrived at the same conclusion (Hua et al., 1997). A study of motion of single kinesin molecules at various loads and ATP concentrations used an optical trap (see Chapter 1) which mantains the load constant, (Visscher et al., 1999). A kinesin molecule attached to a silica bead moves along an immobilized microtubule while an optical trap follows the movement. The steps were found to be tightly coupled to the hydrolysis of ATP with a single hydrolysis for each 8 nm step regardless of load. The force required to stall the kinesin depends on the ATP concentration. Increasing load not only reduces the velocity of the movement but also the Km (in relation to ATP). The in vitro properties of the motors need not reflect how they actually operate in vivo. A case in point is that of the bidirectional movement of lipid droplets in early Drosophila embryos. Each droplet is moved by multiple motor molecules (Welte et al. 1998). The minus motor is cytoplasmic dynein (shown by mutations that alter both the dynein molecules and the movement) and the positive motor is presumably, kinesin. Video-enhanced DIC microscopy and optical tweezers were used to track single droplets and measure the forces generated (Gross et al., 2000). The droplets travel shorter distances from what we http://www.albany.edu/~abio304/text/24part3.html (24 of 46) [3/5/2003 8:27:44 PM]
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would expect from in vitro estimates of cytoplasmic dynein processivity. Usually, the interruption of movement results in an immediate switch in travel direction. The switching appears to occur similarly in either direction, suggesting the presence of a switch mechanism for both motors. The quick switch in direction suggests that both motors are present in the droplet and both are in contact with the switch. C. Directionality of the Movement In addition to a mechanism providing movement, the kinesin molecules must have the means to move on the microtubules in a specific direction. All plus-directed kinesins have their motor domain attached to the amino-terminal (e.g., conventional kinesins) and all minus-directed kinesins have the motor domain attached to the carboxy-end (e.g., Ncd). The Ncd neck is a coiled-coil, whereas this region in conventional kinesin corresponds to an interrupted β-strand (Sablin et al., 1998; Kozielski et al., 1999). At one time the direction of the microtubule-based movement was thought to be a property of the motor domain (e.g., Stewart, 1993) because replacing the other regions of the molecule did not alter the direction of the movement. However, the answer is more complex. A clue to the mechanism responsible for the directionality of the movement is provided by the observation that Ncd and Nkin at any moment in time bind with only one of their two heads. As already mentioned, the head that does not bind faces the direction of the movement (Hirose et al., 1996; Arnal et al., 1996). This suggests a role of the neck region. The involvement of the neck region was studied by producing hybrid proteins containing the Ncd motor domain fused to the kinesin heavy chain, α-helical coiled-coil stalk and containing the region just adjacent to the motor domain (Henningsen and Schliwa, 1997; Case et al., 1997). The Ncd stalk-neck region fused to a kinesin motor core reversed the directionality of kinesin. This reversed (minus) motor could go back to its original polarity (plus, however with slow motility) by the mutation of certain residues in the neckmotor junction (Endow and Waligora, 1998). These mutations are thought to inactivate the minus-end determinants and suggest the presence of plus-end determinants in the motor. In another study of the Ncd molecule, site directed mutations of 13 class-specific residues in the Ncd neck, next to the motor domain (Sablin et al., 1998), resulted in a reversal from a minus to a slow plus-end determinant indicating that the Ncd motor domain also contains directionality determinants. These findings identify residues in the Ncd neck that confer minus-directionality and show plus-end determinants in both the kinesin and Ncd motors. Although residues needed for minus-end motility have been identified in the Ncd neck (Sablin et al., 1998; Endow and Waligora, 1998), this is not the case for kinesin. It might be possible that the neck region of kinesin only amplifies the plus-directed motility of the motor. This hypothesis would explain the slow plusend movement of the Ncd-kinesin-heavy chain and Ncd neck mutants (Sablin et al., 1998; Endow and Waligora, 1998). Interestingly, a mutations in a single amino acid in Ncd neck produce a motor that will move in either direction. In experiments using the gliding assay (see above), the microtubules were found to move either
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in one direction or the other and at times reverse the direction (Endow and Higuchi, 2000). Half of the MTs will initially move in one direction and the other half in the other direction. In addition, single motor molecules attached to beads in contact with microtubules in a single beam optical trap indicated the same properties. These observations are not easy to explain. However, they emphasize the theme that the directionality depends on a neck-motor core interaction. A similar situation occurs in the case of the myosins. The myosins generally move toward the plus end of actin (the barbed end). Recently, myosin VI has been found to move in the opposite direction (Wells et al., 1999). This property is associated with a large insertion in the converter domain (between head an tail) (see above). How the different structure can determine polarity of movement is not known, although simple mechanical models can be designed to account for the observations (e.g., see Schliwa, 1999). VI. ASSOCIATION OF MOTORS WITH CARGO The melanophore system of amphibians and fish (see Halmo and Thaler, 1994) has been one of the most useful in examining the role of the motor systems responsible for organelle movement. This system has revealed a role for microtubules as well as an intimate interaction between microtubules and actin. Melanophores can change in appearance by rearranging their pigment granules, the melanosomes. The distribution of melanosomes is hormonally regulated and is mediated by cytoplasmic cAMP. When the melanophores are dispersed, the cells appear dark. When aggregated, the cells appear light. In part the movement, either anterograde or retrograde, involves the microtubular motors dynein and kinesin (e.g., see Rogers, 1997) on uniformly polarized microtubules (Euteneur and McIntosh, 1981). Although the anterograde transport toward the periphery depends on microtubules. The dispersion in the cytoplasm is independent of microtubules. This distribution requires movement on actin filaments driven by myosin V (Radionov et al., 1998). Motors can move not only a variety of vesicular elements but also protein and RNP complexes (see Karcher et al., 2002). The ability of the motors to carry such disparate cargoes is due to the divergence of their tail domains and their ability to bind cargo or proteins which can serve as mediators by binding to both cargo and motor. The interactions between motor proteins and their binding partners have been studied by standard biochemical methods which have identified such molecules as kinectin, dynactin and pericentrin. The yeast two-hybrid strategy (see Chapter 1) has revealed many other binding partners. The motors have multiple domains that can bind to various receptors. For example, kinesin, can bind to several proteins through its tricopeptide (TPR) motifs of the light chains. TPRs are known to mediate protein-protein interactions (Gindhart and Goldstein, 1996).However, kinesin can also bind through its heavy chain (e.g. see Ong et al., 2000). Similarly, in myosin V from yeast, the tail contains subdomains so that random mutations of this region have different effect on the transport of various vesicles (e.g, Catlett et al., 2000). When associated with vesicles, the motors were found to be located on the organelle surfaces (Langford et
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al., 1987). In the native system, binding of the motor to the organelle is likely to require an integral membrane protein, as phospholipid vesicles or trypsinized organelles (i.e., with the surface proteins cleaved) do not move on microtubules (Gilbert et al., 1985; Vale et al. l985b). Specific binding to such motor receptors could determine the sorting out of vesicles accompanying intracellular transport (Chapter 10 and 11). A role of microtubules in this transport is supported by the observations that microtubules are associated with the endoplasmic reticulum (Franke, 1971), Golgi vesicles (Allan and Kreis, 1987), and lysosomes (Swanson et al., 1987). The receptor for kinesin, kinectin, has been isolated only in the ER (Toyoshima et al., 1992). Kinesin also binds to c-Jun N-terminal kinase (JNK)-interacting proteins (JIPs) (Bowman et al., 2000; Verhey et al., 2001). JIPs are so named because they were first found to be scaffolding proteins for JNK. Mammalian Sundaydriver (SYD), a JIP, binds to the TPRs motifs of the kinesin light chain. Using the TPR motif and the two-hybrid procedure (see Chapter 1), two other JIP proteins were found to bind to kinesin (Verhey et al., 2001). In agreement with the notion that the TPRs have a role in cargo transport, monoclonal antibodies (see Chapter 1) against the third tandem repeat, blocked axonal transport and released kinesin from vesicles (Stenoien and Brady, 1997). The complex of kinesin and JIP was found to bind to dual leucine zipper-bearing kinase (DLK), part of the JNK pathway (see Chapter 7). In addition, it was attached to the ApoER2 (LR7/8B) complex, a receptor of the LDL receptor family highly expressed in neurons. These associations suggest a role of JIP and kinesin in signaling. Other proteins have been found to be possible adaptors for kinsine based transport (see Karcher et al., 2002). An involvement of JIP-3 in vesicle transport is shown by the accumulation of organelles in axons in JIP-3 mutants, similar to what takes place in the kinesin heavy chain null mutants (Bowman et al., 2000). JIPs 1, 2 and 3 were also shown to bind to the TPR motif (Verhey et al., 2001) and kinesin-JIP combinations were found to coprecipitate using immunological techniques. Presumably, the motor proteins are linked to their membranous vesicles or organelles via these scaffolding proteins. Accordingly, in neurons, mutations in JIP-1 that abolish the interaction with kinesin light chains or the expression of dominant negative constructs of JIP-1, eliminate its localization at the terminals ( Verhey et al., 2001). In addition to serving as links between vesicles and kinesin, the scaffolding proteins carry with them transmembrane signaling molecules (e.g., see Chapter 6). This finding indicates that signal transduction pathways may regulate motor activity or even distribute signaling molecules at definite locations in the cell (see Verhey and Rapoport, 2001). Some experiments implicate the protein fodrin in providing a link between kinesin motors and vesicles. Fodrin is an actin/calmodulin-binding protein with similarities to spectrin (see Chapter 4, Fig. 7 and Fig. 8) and a protein of the intestinal epithelium brush border (see Cheney et al., 1983). Fodrin is composed of two polypeptide subunits of 235 and 240 kDa (Glenney and Weber, 1985). Fodrins are highly concentrated in the cortical cytoplasm of neurons and other tissues (e.g., skeletal muscle, uterus, intestinal epithelium) (Levine and Willard, 1981). The movement of fodrins has been demonstrated in neurons and in some other tissues. The redistribution of fodrin is accompanied by concomitant redistributions of actin, myosin, and http://www.albany.edu/~abio304/text/24part3.html (27 of 46) [3/5/2003 8:27:44 PM]
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calmodulin, and the movements proceed at similar velocities (Cheney et al., 1983), suggesting that fodrin serves to link various organelles or proteins. Other kinesins [so called kinesin superfamily proteins (KIFs)] play a role in vesicle transport and mitosis (see Goldstein, 2001). There is direct evidence of the involvement of fodrin in tethering the kinesin KIF3 to vesicles. KIF3, located in the nervous system, corresponds to a heterotrimer of KIF3A (e.g., Kondo et al., 1994) and either KIF3B (Yamazaki et al., 1995) or KIF3C (Muresan et al., 1998; Yang and Goldstein, 1998) and an associated protein, KAP3 (Yamazaki et al., 1995). Microinjection of antibodies against KIF3 in neurons was found to block fast axonal transport and to block neuronal processes extension (Takeda et al., 2000) implicating this kinesin as the primary microtubular based motor in this system. The yeast twohybrid system (see Chapter 1) showed that KIF3 is attached to fodrin with KAP3 bridging the association (Takeda et al., 2000). Fodrin and KIF3 colocalized in the same vesicle as shown by immunoprecipitation and immunoelectron microcopy. The evidence supports a role of fodrin and KAP3 in tethering KIF3 to vesicles for transport in neurons. In some cases, KIFs attach directly to the cargo proteins (e.g., an NMDA receptor subunit which is in vesicle membranes; Setou et al., 2000) or to the coat of vesicles, for example thorugh the β1 adaptin subunit of AP1 in the trans-Golgi. ( Nakagawa et al., 2000). Some KIFs are linked to cargo through Rab proteins (see Echard et al., 1998). KIF1A (Unc104), involved in vesicle transport, has been found to attach to membranes directly through its PH domain (see Chapter 4; Chapter 6 and linked discussion). This motor can transport artificial vesicles containing phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) at high concentrations . Much lower concentrations are required when cholesterol and sphingomyelin are added to the vesicles suggesting a role of rafts in the transport (Klopfenstein et al., 2002). Like kinesin, dynein often acts through other proteins. The dynein intermediate, light intermediate and light chains associate with the synactin complex, a dimer of 150-kDa required for most functions of dynein such as the transport of vesicles (Gill et al., 1991). Three isoforms of dynactin are coded by a single gene, probably equivalent to the Drosophila Glued. Synactin action is through spectrin, generally present in the membranes (Fath et al., 1997; Muresan et al., 2001). In addition, dynein binds directly to cargo proteins such as an isoform of visual rhodopsin and pericentrin, a centrosomal protein. We have seen that myosin V is involved in the transport of vesicles (see Wilson et al., 2000) . The attachment of myosin V to membranes is mediated by Rab proteins (e.g., melanosome movement, e.g. Bahadoran et al., 2001). However, Myosin Va binds to synaptic vesicles through the protein VAMP/synaptobrevin ( see Chapter 11), a member of the SNARE family of proteins (Prekeris and Teriian, 199). Another myosin V (myo4) transports ASH1 RNA (a yeast mRNA that localizes to the bud tip in Saccharomyces cerevisiae), possibly through an adaptor protein such as She3p (e.g., Bohl et al., 2000). Interestingly, conventional kinesin has been found to bind to myosin V (Huang et al., 1999), although an http://www.albany.edu/~abio304/text/24part3.html (28 of 46) [3/5/2003 8:27:44 PM]
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actual role in vesicle transport has not been shown. Myosin VI colocalizes with clathrin and AP-2 structures and binds to clathrin coated vesicles (Buss et al., 2001) The binding of motors to cargo is regulated, thereby controlling its transport. For example, the dephosphorylated form of dynein binds dynactin and phosphorylation abolishes the binding (Vaughan et al., 2001). Similarly, the kinesin Eg5, involved in spindle formation during mitosis, is phosphorylated when it binds to microtubules through the mediation of dynactin (Blangy et al., 1997). In addition, the phosphorylation of myosin V in the globular part of the tail by Ca2+-calmodulin dependent protein kinase II dissociates the myosin from the transport of pigment organelles in Xenopus (Karcher et al., 2001). Calculations of the energy required for movement on microtubules (Sheetz and Spudich, l983b) show that only a few of these motor molecules need to be attached to the organelles, and electron microscopic observations suggest only five cross-bridges between organelles and microtubules (Langford et. al., 1987). VII. TRIGGERING OF CONTRACTION The events that trigger contraction are reasonably well understood for vertebrate skeletal muscle. Several steps are interposed between the excitation of the nerve and contraction. The action potential of the nerve is transmitted to the muscle. The nerve terminals release acetylcholine. A specialized structure on the muscle fiber, the end plate responds to the arrival of acetylcholine by depolarizing, giving rise to the end plate potential (Chapter 22). The end-plate potential in turn triggers an action potential in the muscle, which results in contraction by processes discussed in this section. The sequence of events in other motile systems is less well understood. The coupling between stimulus events and motility in the various motor systems is the topic of this section. Calcium is necessary to couple excitation to contraction; it has been implicated in muscle contraction from the earliest experiments. Heart muscle, for example, exhibits an action potential but fails to contract when Ca2+ is absent from its external medium (Mines, 1913). The situation is somewhat more complex in the striated muscle of the frog, which fails to conduct or contract when stimulated in a medium lacking Ca2+ (Ishiko and Sato, 1957). Apparently, the two independent mechanisms, conduction and contraction, are affected by the lack of Ca2+. When depolarization is induced by increasing the K+ concentration in the medium, contraction still cannot be elicited without Ca2+ (Franck, 1960). Although Ca2+ is necessary for contraction, muscle can contract in the absence of the action potential. Microinjection of Ca2+ into the fiber (Heilbrunn and Wiercinski, 1947; Portzehl et al., 1964) in concentrations as low as 0.3 to 1.5 µM (Portzehl et al., 1964) induces a contraction. In contrast, injections of Mg2+, Na+, K+, ATP, AMP, arginine, and inorganic phosphate are ineffective (Caldwell and Walster, 1963; Heilbrunn and Wiercinski, 1947). The Ca2+ could then serve as a trigger for initiating contraction. This view is supported by the contraction elicited by delivering small amounts of Ca2+ with a micropipette on muscle fibers stripped of their surface membrane (skinned preparations) (Constantin et al., 1965). The http://www.albany.edu/~abio304/text/24part3.html (29 of 46) [3/5/2003 8:27:44 PM]
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contraction is limited to a small area on which the Ca2+ has been delivered, and it is followed soon by relaxation. The requirement of Ca2+ for contraction, either in intact muscle or in stripped fibers, suggests that the release of Ca2+ into the fiber's interior induces contraction. Its subsequent removal by some special mechanism induces the contractile elements to relax. The Ca2+ required for contraction does not originate from the medium external to the fiber. The influx of Ca2+ during activation is insufficient to provide enough for contraction (Franck, 1961; Winegrad, 1961). On the other hand, in vitro vesicles derived from the muscle's endoplasmic reticulum (the sarcoplasmic reticulum, the SR) can remove enough Ca2+ from the medium to induce isolated fibrils to relax after having been made to contract by the addition of ATP (Weber et al., 1963). Therefore, the SR could provide the system for removal and release of the Ca2+ necessary for the relaxation-contraction cycles of functional striated muscle. The structural arrangement of the internal membranes is well suited for a role in triggering contraction. It is composed of a system of transverse tubules continuous with the surface membrane (Fig. 29a and b) and a longitudinal system, the lateral sacs, that reach each sarcomere (Fig. 30) (Peachey, 1965; Porter, 1961). The T tubules and the two associated cisternae at the sarcomere level constitute the triad. The continuity of the transverse system with the surface membrane has been demonstrated by electron microscopy (Fig. 29a) (Franzini-Armstrong, 1964). Similarly, the longitudinal and transverse systems are closely associated (Fahrenbach, 1965; Franzini-Armstrong 1964, 1973). The continuity of the transverse tubules (T tubules) with the surface membrane is supported by observation of the passage of large molecules, such as ferritin (Huxley, 1969; Page, 1964) or albumin (Hill, 1964), into the transverse system from the medium. Threedimensional reconstructions are shown in Fig. 30 (Peachey, 1965).
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Fig. 29 (a) Tail myotome of guppy. The A and I bands and the Z line are labeled in the left corner at the bottom of the figure. At each Z line a triad [the central tubule (part of the T system) and the lateral sacs], is indicated by the triple arrows. The rest of the SR (single, large arrows) is probably continuous with the lateral sacs. The central tubule is convoluted and was originally present in and out of the section. It appears in this section as vesicles. Bar corresponds to 786 nm. From Franzini-Armstrong (1964), with permission. (b) Opening of the T-system to the outside showing continuity with the cell membrane (the plasmalemma or sarcolemma). White twitch fiber from the black mollie. Bar corresponds to 300 nm. From FranziniArmstrong (1973), with permission. (c) Sarcoplasmic reticulum of frog sartorius muscle. From Peachey,©1965. Reproduced from Journal of Cell Biology by copyright permission of the Rockefeller University Press.
A reconstruction of the triad is shown in Fig. 31 (Franzini-Armstrong et al., 1987, Block et al., 1988). The T tubule (in the center of the image, toward the viewer) is shown with two terminal cisternae (TC). The lower one is shown occupied by calsequestrin, an acidic Ca2+-binding protein of skeletal, cardiac, and smooth muscle (MacLennan et al., 1983). Calsequestrin has a molecular mass of 42 kDa and can bind http://www.albany.edu/~abio304/text/24part3.html (33 of 46) [3/5/2003 8:27:44 PM]
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approximately 50 Ca2+ per molecule. The surface of the TC shows projection of the Ca2+-ATPase, visible also as intramembranous particles. The projections are lacking in the junctional portion of the triad. The large structures of four subunits in the junctional portion are the so-called junctional feet (JF) identified in other studies as ryanodine receptors. Calsequestrin binds Ca2+ with low affinity (Kd = 1 mM), so that the bound Ca2+ is readily avalable for release. The formation of T-tubules is induced by amphiphysin 2 (Amph 2), a molecule previously shown to induce invaginations at endocytotic sites (see Chapter 9). When expressed in non-muscle cells, an isoform of Amph2 induced tubular structure resembling T-tubules and where it was found in high concentrations (Lee et al., 2002). The induction of tubular structures was found to depend on the presence of a phosphoinositide-binding module in the amphiphysin molecule (binding to 4,5 bisphosphate, PIP2). Caveolin, thought to have a role in myogenesis is also recruited to the tubules (along with dynamin 2 and Amph 2). That the SR is involved in triggering a contraction is supported by experiments in which small portions of the muscle fibers are given electrical subthreshold stimuli (Huxley and Taylor, 1958; Huxley, 1969) at the location of the T tubules and in experiments in which direct electrical stimulation is applied to fibers stripped of surface membranes (Constantin and Podolski, 1966; Csapo, 1959). In contrast to the effect of normal stimulation, the responses are local, not propagated, and graded depending on the strength of the stimulus. The effectiveness of the stimulation varies dramatically with the position of the electrode. In frog muscle, stimulation at the Z line is effective (Huxley, 1959; Huxley and Taylor, 1958), and in the crab and lizard, stimulation between the A and I bands (Huxley, 1959) is effective. These susceptible spots correspond to the location of the openings of the transverse tubules in these animals. The portions of the sarcomeres that contract correspond in distribution to the longitudinal vesicles. These experiments support a role of the SR in the control of muscle contraction and also implicate the T tubules in conducting depolarization. In the proposed mechanism, Ca2+ would be released from the SR to produce a contraction, whereas it would be transported into the vesicles during relaxation. Tests of this premise have been carried out with several approaches. The Ca2+ can be detected in a variety of ways. It can be traced by using a radioactive isotope such as 45[Ca2+]. Alternatively, its concentration can be estimated using Ca2+ indicators. These can be dyes such as murexide, which complexes with Ca2+ and whose light absorption varies with the degree of binding. Ca2+ sensitive dyes were discussed previously in connection with a study of the interaction between Ca2+ and Ca2+-transporting enzyme, the Ca2+-ATPase. A convenient indicator is the protein aequorin, extracted from the luminous jellyfish, which fluoresces when complexed with Ca2+ so that the light emitted is proportional to the concentration of Ca2+ (see Chapter 1) . The results obtained with these three approaches essentially agree. Muscle can be incubated in the presence of 45[Ca2+] and then fixed in either the contracted or in the relaxed state (Winegrad, 1965a,
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1965b). The position of the radioactivity viewed with autoradiography then serves to locate the Ca2+. At rest, the 45[Ca2+] was found in a position corresponding to the lateral sacs close to the I-band. During contraction it was found to shift to the A-band, where fiber overlap would be expected. The Ca2+ can also be followed using Ca2+ indicators. The experiments with murexide showed that the amount of free Ca2+ inside the muscle fiber increases with contraction (Jobis and O'Connor, 1966). In other experiments in which aequorin was injected into barnacle muscle fibers, the time course of the fluorescent emission preceded the contraction, and the decrease in fluorescence preceded the relaxation. The results are shown in Fig. 32 (Ashley and Ridgeway, 1970). Curve 1 indicates the changes in membrane potential that precede the release of Ca2+, shown as light emission in trace 2. The isometric tension of the muscle, shown in trace 3 (the muscle is held at constant length), follows the release of Ca2+ and decreases after the decrease in internal Ca2+. These experiments suggest that Ca2+ is indeed the trigger for muscle contraction.
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Fig. 30 Three-dimensional reconstruction of the sarcoplasmic reticulum of the frog. From Peachey (©1965). Reproduced from The Journal of Cell Biology, by copyright permission of the Rockefeller University Press.
Fig. 31 Three-dimensional reconstruction of a triad. From Franzini-Armstrong et al., (1987) and Block et al., (©1988). Reproduced from The Journal of Cell Biology, by copyright permission of the Rockefeller University Press.
The portion of the SR that intimately controls the Ca2+ release-sequestration cycles must correspond to the triad. Physiologically, excitation-contraction must depend on three processes that correspond to the structures. There must be a mechanism of coupling the depolarization of the cell membrane and the release of Ca2+. The release of Ca2+ needed for contraction of the myofibril is most likely to involve Ca2+ channels of the junctional SR. Removal of the Ca2+ by the terminal cisternae involves the operation of Ca2+-ATPase, which we discussed in part in Chapters 20 and 24. In addition, calsequestrin helps retain the Ca2+ in the SR and serves as a Ca2+ store. http://www.albany.edu/~abio304/text/24part3.html (36 of 46) [3/5/2003 8:27:44 PM]
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Fig. 32 Results of applying a single depolarizing pulse to a single barnacle muscle fiber. Curve 1, membrane potential; curve 2, light emission; curve 3, isometric tension; curve 4, stimulation marker. Calibration: ordinate, 20 mV (curve 1) or 5 g (curve 2); abscissa, 100 ms. From C. C. Ashley and E. B. Ridgeway, Journal of Physiology, 209:105-130, with permission. Copyright ©1970 The Physiological Society, Oxford, England.
Electrical currents in the T tubules in every way analogous to action potentials are involved in excitationcontraction coupling. Voltage clamp experiments on isolated portions of muscle fibers have demonstrated an inward delayed Na+ current that follows the current of the action potential as shown in Fig. 33, curve A (Mandrino, 1977). When the Na+ in the medium is reduced (in this case to half the normal amount), the delayed Na+ current is also reduced, as shown in Fig. 33, curve B. In agreement with this observation, tetrodotoxin, which blocks Na+ channels, also delays the current. The delayed current disappears when the T tubules are removed by treatment with glycerol. The presence of Na+ channels in the T tubules is also supported by the localization of monoclonal antibodies to the Na+ channels in the T tubules (Haimovich et al., 1987).
Fig. 33 Effects of decreasing the external sodium concentration on the inward currents. A, Record in normal
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Ringer's solution; B, Record in a solution with 50% of the normal sodium concentration. The peak current is approximately 1 x 10-7 A. From M. Mandrino, Journal of Physiology, 269:605-625, with permission. Copyright ©1977 The Physiological Society, Oxford, England.
The mechanism of coupling between the T tubules and the terminal cisternae has been studied in detail. The closeness of the association between the terminal cisternae and the T tubules suggests the possibility of a direct mechanical connection between the two. A molecular voltage sensor in the T tubules in contact with the Ca2+-release channels could induce a conformational change in the latter, initiating the Ca2+ release. As we shall see, this is probably the main mechanism for the activation. The voltage-dependent Ca2+ channel (VDCC) (or dihydropyridine receptor, DHPR) composed of five subunits, is a voltage sensor (see Catterall, 1991) that undergoes a conformational change responsible for activating the Ca2+release channels. The "foot structures" of the terminal cisternae of the SR (Franzini-Armstrong and Nunzi, 1983) are in contact with the transverse tubules. They function as Ca2+-release channels (Hymel et al., 1988) that are blocked by the drug ryanodine (a neutral alkaloid derived from the plant Ryana speciosa and generally used as an insecticide) (see Fleischer and Inui, 1989). Binding of ryanodine provides a convenient way to recognize the channel protein during fractionation procedures. The channels have been referred to as ryanodine receptors (RyRs). In contrast, in other cells the inositol 1,4,5-tris phosphate receptors (IP3Rs) act as Ca2+-release channels of the SR and the endoplasmic reticulum (see Chapter 7, section IA or section IF). The IP3Rs are tetramers very similar to the RyRs, (see Chadwick et al., 1990). Most cells have a prevalence of one of the two kinds of SR-channels. Smooth muscle cells, cardiomyocytes and Purkinje cerebellar neurons have both at high levels but they have a different spacial distribution in each type of cell (Kijima et al., 1993; Walton et al., 1991). In cardiomyocytes, immunological methods showed the RyR to be at the transverse bands throughout the length of the cells, coincident with the triad junctions at the Iband. Immunogold particles localized the IP3Rs at the intercalated discs (the structures containing gap junctions and connecting two adjacent cardiomyocytes) of rat ventricular and atrial cardiomyocytes (Kijima et al, 1993 Three different RyRs isoforms have been isolated and reconstituted in bilayers. RyR1 is the release channel of skeletal muscle, RyR2 is predominant in cardiac muscle and the brain, whereas RyR3 is present in other tissues including the brain. Smooth muscle has both RyR2 and RyR3 in minor amounts. The three RyRs have been cloned. They are arranged similarly in bilayers and have similar properties (see Marks, 1997). The native Ca2+-release channels of striated muscle are constituted by four RyR1 subunits of 565 kDa and four 12 kDa proteins (FKBP12) (Jayamaran et al., 1992). The combination of the two kinds of proteins allow the four subunits, each functioning as a channel, to open in a coordinate fashion to be fully open (Brillantes et al., 1994). Removing FKBP12 from RyR1 produces multiple subconductance levels, http://www.albany.edu/~abio304/text/24part3.html (38 of 46) [3/5/2003 8:27:44 PM]
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indicating that each subunit is acting independently. FKB12 is also associated with the (IP3R) that has many similarities to RyR (Cameron et al., 1995). In striated muscle, Ca2+ in the micromolar range and adenine nucleotides in the millimolar range activate RyRs channels. Calmodulin (CaM), present in the muscles, increases the open probability of RyRs at Ca2+ concentrations corresponding to relaxed muscle. At higher concentrations of Ca2+, it has the opposite effect (see Schneider, 1994). The cytoplasmic domain of RyR1 projects into the space between the transverse tubule and the SR. Activation of RyR during excitation-contraction coupling, requires the cytoplasmic domain of the α1 subunits of the VDCCs (Tanabe et al., 1990). The excitation-contraction coupling probably involves the physical interaction between these two kinds of channels because pieces of the α1 subunits of VDCCs (the cytoplasmic loop) can activate or inactivate the RyR1 channels (e.g., Lu et al., 1994, 1995; el-Hayek, 1995). In the arrangement of VDCC and RyR1, four VDCC overlie only every other RyR1 channel (FranziniArmstrong and Kish, 1995), so that the cytoplasmic loop of VDCC can only be in contact with half of the RyR1 channels. However, studies with RyR1 channels reconstituted in planar bilayers show that two channels are coupled by the presence of FKBP12 so that they open and close simultaneously (Marx et al., 1998). The cardiac SR has three distinct continuous regions: the network SR, the interior and peripheral junctional SR (jSR) and the corbular SR (cSR) (e.g., Segretain et al., 1981). The network SR consists of vesicular tubules forming a network of connected compartments along the length of the sarcomere. The junctional and corbular SR represent specialized domains that contain electron dense material. The junctional SR is physically connected to either the T tubules or the sarcolemma via feet structures that correspond to the RyRs. The corbular SR is not connected in this way although it might have feet structures protruding into the cytoplasm (Sommer and Johnson, 1979). In agreement with this view the RyR, thought to correspond to the feet structures can be shown by immunofluorescence and immuno-gold techniques to be present in both (Jorgensen et al., 1993). In the cardiac cells, the area of surface occupied by VDCC in the sarcolemma is equal to that occupied by feet structures in the SR (Protasi et al., 1996). However, they are not positioned in relation to the feet structures; the two sets of channels are close to each other but not intimately connected (Sham et al., 1995). As we shall see the VDCC in cardiac muscle allow entry of Ca2+ which in turn triggers the release from the SR. Depolarization of the sarcolemma and T-tubes of cardiac muscle cells is not enough to produce the Ca2+ release from the sarcoplasmic reticulum. Depolarization triggers Ca2+-release from cardiac SR only via activation of the VDCCs in the sarcolemma (e.g., Grantham et al., 1996). This influx of Ca2+, directly or
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indirectly induces the release of Ca2+ from the sarcoplasmic reticulum (SR) (e.g., Reuter, 1984). In smooth muscle Ca2+ is also released from RyR receptors (see Marks, 1992; Nelson et al., 1995). The end result may be very indirect. For example, increases in local Ca2+ concentration can result in vasodilation of the vessels surrounded by the smooth muscle by activation of Ca2+-dependent K+ channels, an effect that leads to hyperpolarization. In the mammalian intestine, smooth muscle contractions move from cell to cell in waves controlled by the local nervous system (Furness and Costa, 1987) and the interstitial cells of Cajal (Sanders, 1996). A key element in the control of the contractility resides in intracellular concentration of Ca2+ which is discharged in waves as shown using a fluorescent Ca2+ indicator (introduced as an ester which enters intact cell and is hydrolyzed by esterases, generating and trapping inside the intracellular calcium indicator) (Stevens et al., 1999). In summary, in cardiac muscle and other tissues the activation of RyR are thought to be mediated not by a conformational change but by intracellular Ca2+ entering via the VDCCs. In certain cerebellar neurons, Ca2+-channels activated by glutamate receptors are thought to play a similar role to the VDCCs (Chavis et al., 1996). Fig. 34 (Wagenknecht et al., 1989) shows an image reconstruction of the isolated RyR1 protein from electron micrographs (see Chapter 1). Table 2 Possible Role of Ca2+ in the Motility of Various Cells
Organisms or cell
Evidence
References
Vorticella
Ca2+ activation of contraction
a
Xenopus laevis
Cleavage of eggs requires Ca2+
b
Spirostomum
Release of Ca2+ at contraction followed with aequorin
c
Fibroblasts
Ca2+ required for contraction, vesicular fraction inhibits contraction
d
Stentor
Contraction requires Ca2+
e
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Leukocytes
Demonstration of a contractile protein which requires Ca2+ for contraction
f
Amoeba
Ca2+ required for contraction of cytoplasm in ruptured cells
g
Physarum polycephalum
Ca2+- requiring actinomyosin for contraction Ca2+-requiring vacuoles Release of Ca2+ with electrical charge
hij
a
Amos, W.B., Nature 229: 127 (1971). bBaker, P.F. and Warner, A. E., J. Cell Biol.. 53: 579 (1972). cEttienne, E.M., J. Gen. Physiol. 56: 168 (1970). dKinoshita S. et al., Biochim. Biophys. Acta 79: 88 (1964). eHuang, B. and Pitelka, D.R., J. Cell Biol. 57: 704 (1973). fShibata, N. et al., Biochim. Biophys. Acta 256: 565 (1972). gTaylor, D.L. et al., J. Cell Biol. 59: 378 (1973). hKato, T. and Tonomura, Y. J. Biochem. 81: 207 (1977). iRdigway, E.B. and Durham, A.C.H., J. Cell Biol.. 69: 223 (1976).
Many studies have suggested that Ca2+ activation also occurs in primitive motile systems. The freshwater protozoan Spirostomum ambiguum, for example, contracts when stimulated electrically. Release of Ca2+, detected by the aequorin assay, coincides with the contraction. Removal of Ca2+ coincides with relaxation (Ettienne, 1970). A role of Ca2+ is also indicated in the ciliary motion of Paramecium (Eckert, 1972) and in other systems as well, but the evidence is indirect. Some examples of the indirect evidence for Ca2+ involvement are shown in Table 2. The initiation of chromosomal movement in the mitotic spindle also seems to be triggered by a release of Ca2+ from vesicles distributed in the mitotic spindle (Hepler, 1980; Hepler and Callahan, 1987).
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Fig. 34 Computer-generated surface representations of the three-dimensionally reconstructed junctional channel complex. (a-c) Stereo pairs of the reconstruction in various orientations related by rotation about a vertical axis. (d and e) The two complementary halves of the reconstruction after slicing it in half to reveal internal structural features. In (e) the view is from the interior of the channel toward the surface adsorbed to the grid (the "platform" side) and in (d) it is toward the T-tubule surface. Abbreviations: BP, base platform; PL, peripheral lobes; PV, peripheral vestibules; CC, central channel; RC, radial channels. Scale bar, 10 nm. Reproduced with permission from Nature , Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Inui, M. and Fleischer, S. (1989) Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum, Nature 338:167-170. Copyright ©1989 MacMillan Magazines Ltd.
Relaxation follows the removal of Ca2+. Ca2+ is removed by the Ca2+-transport system of the SR, a process likely to be facilitated by the presence of parvalbumin in the sarcoplasm.
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Parvalbumins constitute a family of soluble intracellular proteins of approximately 11 kDa, present in the cytoplasm of certain cells in high concentrations. In striated muscle of fast fish the parvalbumin concentration is in the range of 0.3-0.5 mM. One molecule of parvalbumin binds 2 Ca2+ with high affinity (108 M-1) and therefore this protein can serve as a calcium buffer. Parvalbumin has a role in relaxation, at least in frog muscle, where Ca2+ replaces Mg2+ and acts in parallel with the sarcoplasmic Ca2+sequestering system (Rall, 1996). SUGGESTED READING General Amos, L. A. and Amos, W. B. (1991) Molecules of the Cytoskeleton, The Garland, The Guilford Press, New York. Adams, R. and Pollard, T. D. (1989) Prediction of common properties of particle translocation motors through comparison of myosin I, cytoplasmic dynein, and kinesin, In Cell Movement, Vol. 2. pp 3-10. Liss, New York. Bárány,M. and Bárány, K. graduate course lectures, Biochemistry of muscle contraction. Updated: March 2000. http://www.uic.edu/classes/phyb/phyb516/ Block, S.M. (1995) One small step for myosin, Nature 378:132-133. (Medline) Bray, D. (1992) Cell Movements, Garland Publishing Inc., New York and London, Chapters 6, 8, 11 and 16. Vale, R.D. (1996) Switches, latches and amplifiers: common themes of G proteins and molecular motors, J. Cell Biol. 135:291-302. (Medline) Vallee, R.B. and Sheetz, M.P. (1996) Targeting of motor proteins, Science 271:1539-1544. (Medline) Actins and actin-binding proteins Bretscher, A. (1991) Molecular aspects of microfilament structure and assembly. Curr. Opin. Struct. Biol. 1:281-287. Hartwig. J. H. and Kwiatkowski, D. J. (1991) Actin-binding proteins. Curr. Opin. Cell Biol. 3:87-97. (Medline) Myosins
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Baker, J.P. and Titus, M.A. (1998) Myosins: matching function with motors, Curr. Opin. Cell Biol. 10:8086. (Medline) Irving, M. and Piazzesi, G. (1997) Motions of myosin heads that drive muscle contraction, News in Physiol Scie. 12:249-254. Mermall V., Post P.L. and Mooseker M.S. (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction, Science 279:527-533. (Medline) Microtubule-based motors Endow, S.A. (1999) Determinants of molecular motor directionality, Nature Cell Biol. 1:E163-E167. (Medline) Endow, S.A. and Fletterick, R.J. (1998) Reversing a 'backwards' motor, BioEssays 20:108-112. Gee, M. and Vallee, R. (1998) The role of the dynein stalk in cytoplasm and flagellar motility, Eur. Biophys. J. 27:466-473. (Medline) Hirokawa, N., Noda, Y. and Okada, Y. (1998) Kinesin and dynein superfamily proteins in organelle transport and cell division, Curr. Opin. Cell Biol. 10:60-73. (Medline) Lane, J. and Allan, V. (1998) Microtubule-based membrane movement, Biochim. Biophys. Acta 1376:2755. (Medline) Excitation-contraction coupling Caswell, A. H. and Brandt, N. R. (1989) Does muscle activation occur by direct mechanical coupling of tranverse tubules to the sarcoplasmic reticulum? Trends Biochem. Sci. 14:161-165. (Medline) Franzini-Armstrong, C., Protasi, F. (1997) Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions, Physiol. Rev. 77:699-729. (Medline) Martonosi, A. N. (1983) The regulation of cytoplasmic Ca2+ concentration in muscle and non-muscle cells. In Muscle and Nonmuscle Motility. Vol. 1, pp. 233-357 (Stracher, A., ed.). Academic Press, New York. Rüegg, J. C. (1986) Vertebrate smooth muscle. In Calcium in Muscle Activation, Chapter 8, pp. 201-238. Springer-Verlag, Berlin. Somlyo, A. P. (1984) Cellular site of calcium regulation. Nature 309:516-517. (Medline) http://www.albany.edu/~abio304/text/24part3.html (44 of 46) [3/5/2003 8:27:44 PM]
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Somlyo, A. P. and Himpens, B. (1989) Cell calcium and its regulation in smooth muscle. FASEB J. 3:22662276. (Medline) Molecular mechanisms of motion Brokaw, C. J. and Johnson, K. A. (1989) Perspectives, dynein induced microtubule sliding and force generation. In Cell Movement, Vol. I, pp. 191-198. Liss, New York. Huxley, H. E. (1990) Minireview: Sliding filaments and molecular motile system, J. Biol. Chem. 265:83478350. (Medline) Huxley, A.F. (1998) Biological motors: Energy storage in myosin molecules, Curr. Biol. 8:R485-R488. (Medline) Rayment I. (1996) The structural basis of the myosin ATPase activity, J. Biol. Chem. 271:15850-15853. (MedLine) Rüegg, C., Veigel, C., Molloy, J.E., Schmitz, S., Sparrow, J.C. and Fink, R.H. (2002) Molecular motors: force and movement generated by single myosin II molecules, News Physiol. Sci. 17:213-218. (MedLine) Satir, P. (1989) Mechanism of ciliary movement - what's new. News Physiol. Sci. 4:153-157. Smith, C.A. and Rayment, I. (1996) Active site comparisons highlight structural similarities between myosin and other P-loop proteins, Biophys. J. 70:1590-1602. (MedLine) Vale, R.D. and Milligan, R.A. (2000) The way things move: looking under the hood of molecular motor proteins, Science 288:88-95. (MedLine) Online animated models of myosin and kinesin motors: www.sciencemag.org/feature/data/1049155.shl Volkmann, N. and Hanein, D. (2000) Actomyosin: law and order in motility, Curr. Opin. Cell Biol. 12:2634. (MedLine) Intermediate filaments Albers, K. and Fuchs, E. (1992) The molecular biology of intermediate filament proteins, Int. Rev. Cytol. 134:243-279. (Medline) Fuchs, E. and Cleveland. D.W. (1998) A structural scaffolding of intermediate filaments in health and disease, Science 279:514-519. (MedLine) http://www.albany.edu/~abio304/text/24part3.html (45 of 46) [3/5/2003 8:27:44 PM]
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Houseweart, M.K. and Cleveland, D.W. (1998) Intermediate filaments and their associated proteins: multiple dynamic personalities Curr. Opin. Cell Biol. 10:93-101. (Medline) Nixon, R.A. (1998) The slow axonal transport of cytoskeletal proteins, Curr. Opin. Cell Biol. 10:87-92. (Medline) WEB RESOURCES Barth, A. and de Hostos, E.L Filaments on the move: cells expressing GFP-actin or tubulin. http://wwwbioc.rice.edu/~hostos/gfptubMDCK.html Doyle, T. Yeast actin cytoskeleton, GFP fusions to the yeast actin gene http://genomewww.stanford.edu/group/botlab/people/doyle.html Duke University Medical Center: Movies created from data analysis of a fusion of the Ncd microtubule motor protein to the green fluorescent protein of jellyfish http://abascus.mc.duke.edu/moviepage.html Greene, L. and Henikoff, S. Kinesin home page. www.blocks.fhcrc.org/~kinesin/. Kaech, S., Ludin, B. and Matus, A. Cytoskeletal plasticity in cells expressing neuronal microtubule associated proteins. http://www.fmi.ch/groups/AndrewMatus/Video.html Section of the Cell Biology & Cytoskeleton Group Division of Hematology, Brigham & Women's Hospital, Harvard Medical School, Research Overview : information and movies in relation to actin crosslinking, actin dynamics, actin severing and capping, cell motility and mechanics, cytoskeletal polymer physics and rheology, genetics of motility, interactions between cytoskeletal systems (e.g., neurofilaments-microtubules interactions and signaling to the actin cytoskeleton. Vale, R.D. and Milligan, R.A. (2000) Science Online. Animated models of myosin and kinesin motors. http://www.sciencemag.org/feature/data/1049155.shl Waddle et al. Movement of Cortical Actin Patches in Yeast http://www.cooperlab.wustl.edu/Waddle_Ms:Waddle_Ms.html REFERENCES Search the textbook
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