International Review of Cytology: A Survey of Cell Biology, Volume 178

VOLUME 178

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander Jonathan Jarvik

1949-1988 1949-1984 19671984-1992 1993-1995

EDITORIAL ADVISORY BOARD Aimee Bakken Eve Ida Barak Rosa Beddington Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay P. Mark Hogarth Anthony P. Mahowald M. Melkonian

Keith E. Mostov Andreas Oksche Vladimir R. Pantic Thomas D. Pollard L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred D. Stein Ralph M. Steinman M.Tazawa Yoshio Watanabe Donald P. Weeks Robin Wright Alexander L. Yudin

Edited by

Kwang W. Jeon Department of Biochemistry University of Tennessee Knoxville, Tennessee

VOLUME 178

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Front cover photogruph: Demyelination within the cerebellum of a Biozzi ABH mouse infected with Semliki Forest virus. (See Chapter 4, Figure 10 for more details.)

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CONTENTS

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Karyotypic Evolution of Cells in Culture: A New Concept S. E. Mamaeva I. Introduction ............................................ II. Factors of Karyotypic Destabilization and Criteria for Purity and Stability ............... of Cell Lines ........................ 111. Cytogenetic Approach to Analysis of Cultured Cells ............... IV. Karyotypic Evolution of Cell Lines during Th V. Regularities of Karyotypic Variability of Cells in Culture ......................... VI. Cell Lines with Autosomal Monosomies and Compensation for the Loss of Genetic Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Concluding Remarks .................... ........... ....................... References . . . . . . . . . .

2 2 3 9 13

21 20 29

Sucrose Transport in Higher Plants John M. Ward, Christina Kuhn, Mechtild Tegeder, and Wolf B. Frommer I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. SugarTransport ........................................................ Ill. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 46 63 64

vi

C0NTEN TS

Interaction of Cytoskeletal Proteins with Membrane Lipids G. lsenberg and V. Niggli .................... I. Introduction . . . . Lipids and Posttranslational Lipid Modifications , . , . . . . , . . . , . .... .. ... .. .... ... . II. Ill. Methods of Choice to Study Cytoskeleton-Lipid Interactions . . . . . . . . . . . . . . . . . , . . . IV. Interactions of Actin and Associated Proteins with Membrane Lipids . . . . . . , . . . . . . . . V. Interactions of Microtubules and Associated Proteins with Membrane Lipids . . . . . , . . . VI . Interaction of Intermediate Filaments with Membrane Lipids . , . . . . . . . . . . . . . . . . , . . . .......................... . . . VII. Conclusions References ............................. . . . . . . . .

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75 78 84 99 107 111 112

Cell Biology of Autoimmune Diseases Johannes M. Van Noort and Sandra Amor I. 11. 111. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . , . , , . . , . . . . . . . . . . . . . . , . . . . Immune Mechanisms in Autoimmune Diseases . . . . . . . . . . . Factors That Contribute to the Development of Autoimmune Diseases Models of Autoimmune Diseases . . . . . . . . . . . . . . . . . ................ T Cell Recognition of Autoantigens . . . . .................... Intervention in Autoimmune Diseases Perspectives on Future Therapies in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . References . . . .....................................

127 133 149 167 179 185 195 198

Multiple Forms of Tubulin: Different Gene Products and Covalent Modifications Richard F. LudueAa I. II. 111. IV.

Introduction . , .. , . . . . . . . . . . , . , .. , , . . .. . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . Multiple Genes for a-and PTubulin . , . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . Posttranslational Modifications of Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . , . , . Concluding Remarks . . . . . , . , . . , . , . . . . . . . . . . . . . . . . . . . . . . , . . . . , . . . . . . . . . . . References . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207 208 246 252 255

Translocation of Proteins across the Endoplasmic Reticulum Membrane Jeffrey L. Brodsky I. Introduction . . . . , .. , .. , .. . . . . . . . . , .. . . . ... . . . . , .. , . . . . .. .. , . . . . . . . . .. ..

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........ 111. Preprotein Targeting to the ER . . , . , IV. ER Membrane Translocation Machine

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279 203

VI. Concluding Remarks . . . . . . . . References . . . . . . . . . . . . . . . . .

Index

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CONTRIBUTORS

Number in parenlheses indicate the pages on which the authors' contributions begin.

Sandra Amor (1 27),Department of lmmuno/ogy, The Rayne Institute, St. Thomas' Hospital, London SEI 7EH, United Kingdom Jeffrey L. Brodsky (277),Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Wolf 8.Frommer (41),lnstitut fur Botanik, Universitat Tubingen, D-72076 Tubingen, Germany G. lsenberg (73),Biophysics Department E-22, Technical University of Munich, D85747 Garching, Germany Christina Kuhn (41),lnstitut fir Botanik, Universitdt Tubingen, 0-72076 Tubingen, Germany Richard F. Luduena (207),Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

S. E. Mamaeva (l),Laboratory of Cell Morpho/ogy, Institute of Cflology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersberg, Russia (correspondence should be sent to T. R. Sukhikh at this address) V. Niggli (73),Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland Mechthild Tegeder (41), lnstitut fur Botanik, Universitat Tubingen, 0-72076 Tubingen, Germany Johannes M. Van Noort (127),Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, The Netherlands John M. Ward (41), lnstitut fur Botanik, Universitdt Tubingen, 0-72076 Tubingen, Germany ix

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Karyotypic Evolution of Cells in Culture: A New Concept S.E. Mamaeva' Laboratory of Cell Morphology, Institute of Cytology, Russian Academy of Sciences, 4 Tikhoretsky Avenue, 194064 St. Petersburg, Russia

The Chapter summarizes peculiarities of karyotypic variability during establishment and long-term cultivation of permanent cell lines. A new concept on pathways of karyotypic evolution of cells in culture is put forward. A detailed description is presented of the author's original approach of cytogenetic analysis of cell lines provided for a principally new characteristic of the cell line: its generalized reconstructed karyotype (GRK). Its use as a criterion to evaluate authenticity, purity, and stability of cell lines is discussed. Based on analysis of the GRK, two stages of karyotype evolution of cell lines are revealed: establishment and stabilization, different in karyotypic variability of the cell population and in peculiarities of clone selection. Comparison of peculiarities of karyotypic variability of leukemic and tumor cells both in vitro and in vivo was made, and general regularities of their karyotypic evolution have been established, such as nonrandom changes in the number and structure of chromosomes and deletion of one of the sex chromosomes, as well as regularities characteristic only of cells in culture in most human and animal cell lines (at least 85%) of disomy on all autosomes. The rest of the cell lines, 15%, are characterized by either partial or total monosomies on certain autosomes during long-term cultivation. Three main compensatory mechanisms of maintaining viability of cell lines that have lost genetic material are discussed: polyploidizationof the initial cell clone, amplification of oncogenes (predominantly of mys family), and extracopying of whole autosomes or of their fragments. KEY WORDS: Tumor cell lines, Karyotype variability regularities, Monosomic autosomes, Compensatory mechanisms, Poliploidization, Myc oncogene amplification, Chromosome extracopying. Dr. S. E. Mamaeva passed away while the chapter was under preparation and her coworker, Dr. T. R. Sukhikh, completed the manuscript. All correspondence should be directed to Dr. T. R. Sukhikh at the author's address. Dr. S. E. Mamaeva wished to dedicate the chapter to Dr. Nikolai Mamaev, her husband, friend, and physician. lnrernarional Review of Cyrology, Vol. 178

0074-7696/98$25.00

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Copyright 0 1998 by Academic Press. All rights of reproduction in any form reserved.

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S. E. MAMAEVA

1. Introduction For the past few years, extensive use of permanent cell lines in various fields of biology, medicine, and biotechnology has stimulated intensive cytogenetic studies of cells in culture. This is connected with a necessity of developing reliable criteria of purity and stability of cell lines as well as of their authenticity. Studies on the permanent cell lines as an autonomous living system with certain karyotypic and genotypic characteristics are of a special interest. From this point of view, of a fundamental importance is analysis of regularities of the karyotypic variability which are both common to cells growing in vitro and in vivo and are specific to permanent line cells; also important is to reveal peculiarities of karyotypic evolution during establishment of cell lines and under effects of various experimental factors.

II. Factors of Karyotypic Destabilization and Criteria for Purity and Stability of Cell Lines During long-term cultivation cell lines may change some of the original characteristics, including the karyotypic ones. Unfortunately, these events often result in a loss of important cell line properties for which the cell line was maintained. Such changes can be due not only to genetic peculiarities of cultured cells but also to conditions of their cultivation, particularly if different media, growth factors, serum factors, high doses of antibiotics, etc. are used for cultivation of the same line. When working with cell cultures, there is another problem for long-term cultivation: both intraspecies and interspecies contamination of some cell lines can occur (Nelson-Rees et al., 1974, 1981; Heneen, 1976; Lavappa, 1978; Pathak and Hsu, 1985; Glukhova et al., 1991; Chen, 1993). Such contamination can abolish many years of effort of specialists using such cell lines as the main object of study. For the past 10 years, more than 50 cases of interspecies and intraspecies contamination of cell lines have been found. Thus, researchers must develop reliable criteria for the authenticity, purity, and stability of populations of permanent cell lines and of control of the stability. At present, two criteria for the authenticity, purity, and stability of cell lines are used: DNA fingerprinting (D. A. Gilbert et al., 1990) and cytogenetical analysis (Mamaeva, 1984,1988; Chen, 1992). The DNA fingerprinting makes it possible to prove relation of cell lines compared or their origin from different sources (Thacker et al., 1988;van Helden et al., 1988; Gilbert et al., 1990; Stacey et al., 1992). Both methods can also be useful to reveal

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

3

specific peculiarities of the cell line and to establish its purity (Mann et al., 1989; Chen, 1992). However, the cytogenetical analysis is undoubtedly a better method because it allows individualization of any human and animal cell line and/or cell subline (Chen et al., 1982,1983; Mamaeva, 1984; Filatov and Mamaeva, 1985; Savelyeva and Mamaeva, 1987, 1988; Chen, 1988; Savelyeva, 1988).

111. Cytogenetic Approach to Analysis of Cultured Cells The cytogenetical method commonly employed for chromosome analysis provides for information on the species origin of cells as well as the modal number of chromosomes, the variability interval of the number of chromosomes, and presence of a number of marker chromosomes in cells. However, this information is not sufficient; it is often subjective and limits the area of problems that can be successfully solved using modern cytogenetical methods. T o get an objective karyotypic characteristic of the permanent cell line, we have developed an original approach (algorithm) for cytogenetical analysis of a cell line. The algorithm consists of nine consecutive stages.

A. Preparation of Metaphase Chromosomes Peculiarity of this stage is a departure from the traditional procedure (which is considered mandatory by many experimentators) of accumulating mitoses in cell cultures with mitotic drugs or a brief (for 5-10 min) treatment of cells with colcemide (0.02pg/ml). In this way it becomes possible to achieve a higher quality of metaphase-chromosome preparations while preserving the amount of metaphase plates sufficient for analysis (Djomin and Mamaeva, 1993; Abramova et ul., 1993). Equally important is to prepare chromosome preparations using two steps: by air-drying and by burning the fixative out after dropping a fixed cell suspension on the slide. The first step yields mitotic cells with intact cytoplasm, which is necessary for reliable estimation of the modal chromosome number and of the number of cell clones.

B. Staining of Metaphase Chromosomes Air-dried preparations of metaphase chromosomes have to be stained by the routine method. Preparations of metaphase chromosomes obtained by

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S. E. MAMAEVA

burning out the fixative are stained differentially for G- and C-bands as well as with silver nitrate to reveal Ag-positive NOR-bearing chromosomes.

C. Analysis of Chromosome Number To determine the variability interval of the number of chromosomes and the modal chromosome number in the cell line, at least 100 stained metaphase plates are analyzed. To establish the percentage of polyploid cells, 1000 to 3000 metaphase plates are analyzed.

D. Preparation of the Pool of Normal Chromosome Samples To distinguish and identify normal chromosomes and to establish origin of structurally changed chromosomes of human and animal cell lines, it is worth using a representative set of G-stained normal chromosomes of a different condensation degree (at least 50 photoexamples). To form such pool of normal chromosomes, material from cultures of blood, bone marrow, and embryonal tissue cells can be used (Fig. l). E. Karyotypic Analysis of Metaphase Plates To determine the typical karyotype of a cell line, it suffices to analyze 15 metaphase plates stained for G-bands. Examined metaphase plates should correspond to the following requirements: (1)their number of chromosomes should not be lower than the minimal number established when analyzing 100 stained metaphase plates; (2) metaphase plates should not have an overlapping of chromosomes, which handicaps interpretation of structurally changed chromosomes; (3) a part of the metaphase plates (at least 3-5) should have prometaphase chromosomes suitable for more accurate localization of chromosome breakpoints during marker formation. When analyzing cell lines with 20 structurally changed chromosomes or more, one should karyotype 30-40, rather than 15, metaphase plates, depending on the complexity of the line karyotype. F. Composing of Rows of Identical Marker Chromosomes Rows of identical marker chromosomes are formed from additional photocopies of all structurally changed chromosomes revealed after karyotyping

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

5

FIG. 1 Sample of the pool of human normal chromosomes different in the condensation degree.

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S. E. MAMAEVA

of metaphase plates stained for G- and C-bands and Ag-NORs, marker chromosomes of each cell being oriented as vertical rows in such a way that horizontal rows are composed of similar chromosomes but of different cells (Fig. 2). Preparing such rows is time-consuming but important for cell line karyotyping, in the course of which a great number of similar marker chromosomes are compared simultaneously and their origin is specified. This stage provides for information about morphological types of markers, frequency of their occurrence, and the fragment composition of 70-80% of marker chromosomes revealed.

G. Normal and Marker Chromosome Numbers To summarize results of analysis of chromosome sets in all cells of a line, a table is formed which presents the total number of chromosomes of each cell, the number of normal homologs of each chromosome, the number of markers, and the number of their copies (Table I). Simultaneous comparison of chromosomes from all cells allows identification of cells both with identical chromosome sets and with normal and marker chromosomes differing in composition. In this way it is possible to establish the degree of variability and stability of individual chromosomes as well as the number of homologs of each normal chromosome (i.e., the nulli-, mono-, di-, trisomy, etc.). The table shows interrelation between changes in the number of copies of certain normal and of marker chromosomes, which results in additional information about origin of some markers. In cases where cytogenetic analysis does not allow specification of the origin of all fragments of marker chromosomes and precise localization of chromosomal breaks, to establish origin of the markers, it is possible to apply in situ hybridization with specific DNA probes suitable for delineation of derivative fragments or whole chromosomes (Ruess et al., 1993). However, at present libraries of DNA probes available for routine use are limited to human DNA, and the application of this method for analysis of animal chromosomes is impossible so far. In addition, since in situ hybridization remains rather expensive, this handicaps a wide use of the method for identification of the origin of marker chromosomes of permanent cell lines.

H. Localization of Chromosome Breakpoints during Marker Formation The points of chromosome breaks ("hot spots") resulting from marker formation and rare structural rearrangements are noted at G-banded chromosome ideograms in bands in which breaks occur. The distribution of hot

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

7

FIG. 2 Fragmentary rows of identical marker chromosomes of the human cell line HEp-2.

TABLE I Fragment of the Summarized Table of Normal and Marker Chromosome Numbers of the Human Cell Line HEp-2

Cells

(N) 1 2 3 4

Number of chromosomes

Number of markers

1*

2

3

70 68 69 70

31 29 31 30

1 1 1 1

1 2 1 1

-

1 1 1 1

-

1 1 1 1

2 1 2 2

69

30

1

1

-

1

-

1

2

Normal chromosomes 4* 5 6* 7 8*

9

... 22

1 1 1 1

3 2 3 3

2 1 2 2

1 1 1 1

1 1

1

3

2

1

1

X

M1* 1

1

M2*

M3

M4

1 1 1 1

1 1 1

1 1

1

1

Markers M5* ...

Mn

1

1 1 1 1

2 1 1 1

1

1

1

-

USR 2 1 2

-

15

Note. USR, number of unique structural rearrangements. * Stable by the number normal chromosomes and markers.

1

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

9

spots on chromosomes serves as an important characteristic of the cell-line karyotype to reflect nonstability of certain chromosome regions (Fig. 3).

I. Reconstructed Karyotype of the Cell Lines The final goal of all stages of chromosome analysis is to obtain a highly informative characteristic of a cell line, its generalized reconstructed karyotype (GRK). Owing to identification of fragments of all marker chromosomes of a cell line, it is possible to reveal the real amount of chromosomal material in cells for each normal chromosome. For this purpose, in accordance with the commonly accepted scheme of representation of normal karyotypes, ideograms of entire chromosomes and/or their fragments reconstructed from markers are placed in a scheme like those normal chromosomes from which material they originate. Thus, it looks as if normal homologs are reconstructed from fragments of marker chromosomes, and such karyotype is called reconstructed karyotype (RK). By summing up reconstruction results of chromosome sets of individual cells or cell clones, the GRK of a cell line is obtained. Designing GRKs of permanent cell lines makes it possible to get objective information on changes in cell lines of the number of copies of normal chromosomes or their fragments with respect to the diploid chromosome set of human or animal, on the character of involvement of individual chromosomes in rearrangements, and on the degree of their variability. Comparison of reconstructed karyotypes of individual cells allows evaluation of the extent of balance of chromosome complements in cells of the same population, whereas by comparing the GRK of a cell line and/or its sublines, similarities and differences between them can be established according to their total chromosome material (Figs. 4 and 5 ) (Mamaeva ef al., 1983a, 1986; Mamaeva, 1984, 1988; Mamaeva and Tsvileneva, 1985;Filatov and Mamaeva, 1985;Savelyeva and Mamaeva, 1971, 1981; Grabovskaya et al., 1993).

IV. Karyotypic Evolution of Cell Lines during Their Establishment An important peculiarity of karyotypic variability of cells in culture (which was first revealed by our Cytogenetics Group) is that two qualitatively different stages corresponding to the establishment stage and the stabilization stage of a cell line can be separated in its evolution. The karyotypic evolution of cell lines at the stage of their establishment is the least studied. In fact, nothing is known about the effects of tissue-

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FIG. 3 Localization of chromosome breakpoints during marker formation in the human cell line HEp-2.

specific,karyotypic, and genotypic peculiarities of initial cells on the character of karyotypic evolution of a cell line during its establishment. This stage of evolution of a cell line is characterized, as a rule, by karyotypic heterogeneity of the cell population and selection of cell clones that are the most adapted for existence in vitro. It is at this stage that significant

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

markers

FIG. 4 Typical G-banded karyotype of the human cell line HEp-2. 68,X, 31, markers.

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S. E. MAMAEVA

I

. r--I

1

i 4 mI

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

13

numeral and structural rearrangements of chromosomes occur in cells. Duration of the establishment stage of a cell line is determined both by the amount of karyotypically different cell clones in the initial material and by the duration of karyotypic evolution of dominating cell clones that results in a balanced karyotype stabilization. Main karyotypic features of a cell line that has reached the stabilization stage are a minimal karyotype heterogeneity of cells, particularly a small variability of the number of chromosomes and a clearly defined modal class of the chromosome number, as well as balance of the karyotype and a number of other important features to be reviewed below.

V. Regularities of Karyotypic Variability of Cells in Culture Using the approach we have developed, an analysis was performed of karyotypes of more than 150 human and animal permanent cell lines that had reached the stabilization stage. This has allowed us to establish the following regularities of karyotypic variability of cells in culture: 1. The nonrandom character of numerical and structural chromosome changes in cell lines of different histogenesis. 2. Loss of one of the sex chromosomes (X or Y) during prolonged cultivation. 3. Constancy or a minimal variability of the number and distribution of Ag-positive NORs in chromosomes. 4. Balance of the chromosome set in cells in culture: a similarity of the total chromosome material in all cells of the line despite their karyotypic heterogeneity. 5. Retention of, as a minimum, disomy on all autosomes in most human and animal cell lines. Each of these regularities will be separately considered below, with a comparison of karyotypic variability of cells in vivo and in vitro.

A. Nonrandom Character of Numerical and Structural Chromosome Changes It is well known that numerical and structural rearrangements of chromosomes in tumor and leukemia cells in vivo are not random. More than 70

FIG. 5 Generalized reconstructed karyotype (GRK) of the human cell line HEp-2. The chromosomes and their fragments reconstructed from the markers are surrounded with a continuous line. The variable (by the number of copies) chromosome homologs are marked with a dashed line.

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S. E. MAMAEVA

specific or characteristic chromosome aberrations have been described in various human malignant tumors (Heim and Mitelman, 1987; Bigner et al., 1988, 1989; Nagarajan, 1994). Most commonly these are translocations of reciprocal and nonreciprocal types, deletions of definite chromosome regions and monosomies of entire chromosomes, inversions, and trisomies of definite chromosomes (Heim and Mitelman, 1987; Mitelman, 1994). Cytogenetical analysis reveals large deletions of chromosomes. Chromosome microdeletions that result in loss of heterozygocity of certain genes due to specific chromosome aberrations can be revealed only by molecular methods (Brodeur and Chin-to Fong, 1989; Goddard and Solomon, 1993). The chief proof in favor of a nonrandom character of numerical and structural chromosome changes in permanent cell lines is the maintenance in vitro of many specific chromosome aberrations found in tumor or leukemia material from which the particular cell line was obtained. First of all, these are specific translocations t(8;14), t(2;8), and t(8;22) found in patients with Burkitt lymphomas as well as in Burkitt lymphoma cell lines (McCaw etal., 1977;Shade et al., 1980; Bernheim et al., 1981). In cell lines established from hematopoetic cells of patients with chronic myelogenous leukemia, the Philadelphian chromosome is often retained (Kubonishi and Miyoshi, 1983; Pegoraro et al., 1983; Raskind et al., 1987). In most neuroblastoma cell lines, deletions of the short arm regions of chromosome 1 are present (Gilbert et al., 1982; Biedler et al., 1983), while in most small cell lung cancer cell lines, they occur on chromosome 3 (Whang-Peng et al., 1982; Whang-Peng and Lee, 1985; Graciano et al., 1987). In permanent cell lines, many other chromosome rearrangements are found that are similar to those in human tumor and leukemia cells (Hozier et al., 1980; Mark et al., 1981; Denny et al., 1986; Barker et al., 1987; Nacheva et al., 1987). Another convincing proof in favor of nonrandomness of structural chromosome rearrangements is the different distribution of chromosome breakpoints during marker formation in cell lines of different histogenesis. An example of this can be a pattern of distribution of chromosome breakpoints during formation of 98 markers in 7 sublines of HeLa cell line. In these sublines, centromere regions of chromosomes 1,3, and 5 are predominantly damaged, and less commonly, those of chromosomes 7,9, and 10. It should also be noted that chromosomes 8 and 11 do not actually take part in the marker formation. Out of 98 marker chromosomes, only one chromosome 8 with a damaged centromere region was found, and chromosome 11 was not involved in the formation of markers (Mamaeva, 1984). At the same time, the pattern of distribution of the chromosome breakpoints was quite different during marker formation in the Raji cell line established from B-lymphocytes of a patient with Burkitt lymphoma. Centromere regions of all chromosomes, except for chromosome 18, never participated in marker formation, whereas chromosomes 8 and 11, unlike

KARYOTYPIC EVOLUTION OF CELLS IN CULTURE

15

chromosomes of the HeLa line, turned out to be the most “hot”: they were included in 8 of 23 discovered markers (Savelyeva and Mamaeva, 1988). The same pattern of distribution of chromosome breakpoints was obtained when analyzing a large sample of human B-cell leukemia lines. Telomere rather than centromere regions were involved more often in structural rearrangements of chromosomes, a large number of chromosome breakpoints being present in chromosomes 7,8,9,11, and 14 (Shade et al., 1980). Quite a different character of the chromosome breakpoint distribution during marker formation was observed in seven human glioma cell lines (Mark et al., 1977). Breaks occurred predominantly in proximal regions of the long arm of chromosome 12 and in the middle of long arms of chromosomes 13 and 14. Although there are fewer animal permanent cell lines obtained than human cell lines, and the former are mainly from laboratory animals such as mouse, rat, and Chinese hamster, nevertheless these cell lines also have specific chromosome aberrations characteristic of different neoplasms and maintained or formed de novo during cell cultivation. In murine cell lines, structural rearrangements were observed most often in chromosome 15 and less often in chromosomes 1, 6, and 12. In rat cell lines those were in chromosomes 1 and 2, while in Chinese hamster cell lines, they occurred in chromosome 3 (D. A. Miller et al., 1979; Shepard etal., 1979;Wiener etal., 1980;Mamaeva, 1984). Among various nonrandom changes in the number of chromosomes found in initial tumor and leukemia material as well as in human and animal permanent cell lines, multifold copying of chromosome 7 is noticed in human cell lines, regardless of their histogenesis. Extracopying of chromosome 7 is described in cell lines of normal (Steel et al., 1980), leukemia (Heneen, 1976; Berger et al., 1979), and tumor origin (McCulloch et al., 1976; Chen and Seman, 1981; Chen et al., 1982). The number of copies of this chromosome can reach five to eight in cells of some glioma cell lines (Bigner et al., 1986, 1987; Henn et al., 1986; Westphal et al., 1988). Data on nonrandom changes in the number of chromosomes in animal cell lines are scarce so far. Trisomy has been noted only on chromosome 15 in most murine cell lines (de Both et al., 1981; Dofuki et al., 1975; Hagemeijer et al., 1982; Sasaki, 1982), on chromosomes 2 and 7 in rat cell lines (Levan et al., 1974; Levan and Mitelman, 1975; Sugiyama et al., 1981; Brett et al., 1986), and on chromosomes 3 and 5 in Chinese hamster cell lines (Bloch-Shtacher and Sachs, 1977; Connell and Ockey, 1977; Trewyn et al., 1979; Deaven et al., 1981). The pattern of distribution of hot spots in chromosomes and changes in the number of certain chromosomes are morphological manifestations of molecular-biochemical processes connected with changes in the number and structure of different genes, including oncogenes. This makes use of cell lines suitable for studying roles of specific chromosome changes in

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malignant cell transformation (Collins and Groudine, 1983; Selden et al., 1983; Croce and Nowell, 1985; Denny et al., 1986; Westbrook et al., 1987; Naoe et al., 1988).

6 . Loss of One of the Sex Chromosomes Loss of one of the sex chromosomes is a frequent event in human blood cells, bone marrow, and various other malignant tumors (Sanada et al., 1985; Heim and Mitelman, 1987; Bigner et al., 1988; Hayashi et al., 1989a; Thiel et al., 1992; Mitelman, 1994; Pandis et al., 1995). In addition, sex chromosomes can also be lost in normal cells in the course of aging of an organism (Pierre and Hoagland, 1971). The loss of X- or Y-chromosomes is a regularity in prolonged cultivation of human and animal cell lines. It can occur both at the early and later stages of establishment of permanent cell lines (Zakharov et al., 1966; Mark et al., 1977; Quinn et al., 1977; Filatov and Mamaeva, 1985; Ohyashiki et al., 1986; Bigner et al., 1987; Urmanova et al., 1989). Two-thirds of the human Y-chromosome is heterochromatic, and the number of genes localized on this chromosome is small. These are the genes responsible for determination of the sex and Y-antigen histocompatibility (Human Gene Mapping 11, 1991). Much of the X-chromosome in some animals, for instance the long arm of the Chinese hamster X-chromosome, is also heterochromatic (Filatov and Mamaeva, 1985). Cells which lose the X- or Y-chromosome with large blocks of heterochromatin and genes insignificant for the viability of cultured cells may have a proliferative advantage over the initial cells that contain the entire set of sex chromosomes. The inactive X-chromosome which is lost first of all, the second, active homolog can be extracopied (Camargo and Wang, 1980; Wang et al., 1990). Now the regularities of karyotypic variability of cultured cells that distinguish them from cell populations growing in the organism should be considered in detail; these are specific to permanent cell lines.

C. Constancy of the Number of Ag+-NORs and of Their Distribution in Chromosomes of Normal and Tumor Cells The NOR Ag-staining of chromosomes can significantly vary both in cells of different tissues of the same organism and in cells of the same tissue (Kano-Tanaka and Tanaka, 1982;Mamaev et al., 1984; Sozansky and Terekhov, 1984;DeCapoa et al., 1985; Lyapunova et al., 1988). This is true not only for the number and distribution of AgC-NORsin different chromosomes but

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also for intensity of their Ag-staining (Zakharov et al., 1981, 1982). For instance, the number of Ag+-NORsof chromosomes in cells from the same bone marrow biopsy of a healthy person or a patient with leukemia can vary from 1 to 8, this number being the highest (6-8) in the least differentiated cells (blast elements), while the lowest (1-3) in more differentiated cells (myelocytes and polychromic normoblasts) in which the Ag+-NORs is totally absent (Smetana and Likovsky, 1984; Mamaev et al., 1985; Yan and Stanley, 1988; Mamaev and Mamaeva, 1990). Some authors have also found heterogeneity on the number of Ag+-NORs in normal lymphocytes and skin fibroblasts of the same individuum (Mikelsaar and Schwarzacher, 1978; Ferraro ef al., 1981; Sozansky and Terekhov, 1983). Such intercellular variability of NOR Ag-staining of chromosomes is accounted for by different functional activity of ribosomal genes owing both to intertissue differences and to differences in the differentiation level of cells of the same histogenesis (Mamaev et al., 1985; Hall er al., 1988; Mamaev and Mamaeva, 1990; Grotto and Lorand-Metze, 1991). Quite a different pattern of NOR Ag-staining of chromosomes is observed in cell lines. Although the number of active (Ag-positive) NORs in cells of a given line may vary, a typical pattern of NOR Ag-staining of chromosomes can be found easily. Since cells are similar in the number and Ag-staining intensity of the NORs as well as in the distribution of Ag+NORs on chromosomes (Heneen, 1978; Henderson and Megraw-Ripley, 1982; Mamaeva, 1984; Mamaeva et al., 1990), it is easy to individualize any cell line even in the absence of marker chromosomes. In human cell lines the NORs on marker chromosomes can be located both interstitially and in telomeres, in regions of the secondary constrictions corresponding to NORs of normal chromosomes (Mamaev et af., 1980; DeLozier-Blanchet et af., 1986; Mamaev and Mamaeva, 1990; Pathak et al., 1992). This is often accompanied by a heteromorphism of Ag+-NORs, which indicates amplification of ribosomal genes. The general pattern of NOR localization, not only in normal chromosomes but also in marker chromosomes, as well as their Ag-staining character also can be considered a unique characteristic of a given cell line (Kislyakova and Mamaeva, 1987; 0. J. Miller et af., 1979; Tantravahi et al., 1982; Holden et al., 1985, 1986, 1987). The approach we have developed for karyotype analysis of a cell line, with final reproduction of a reconstructed karyotype, provides information on the total number of NOR-bearing chromosomes in the cell line; therefore, it is important to account for such a characteristic of the cell line as the quota of active (Ag-positive) NORs of chromosomes. According to our preliminary data, this parameter reflects the cell differentiation level in cell lines. It can be, for instance, minimal (4 Ag+-NORsout of 15 NORs in the cell) in such a highly differentiated cell line as the U-937 and maximal

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(10 Ag+-NORs out of 10 NORs in the cell) in poorly differentiated IMR32 and Raji (Mamaeva et al., 1990). Similarity of cells of the same line in the pattern of NOR Ag-staining of metaphase chromosomes confirms their monoclonal origin, but it reflects their close level of differentiation. Thus, available information on NOR Ag-staining of chromosomes indicates that constancy of the number of Ag+-NORsand their distribution on chromosomes in cells of the same population are characteristic of cells of permanent cell lines, whereas in an organism these characteristics can be variable and dependent most often on the differentiation level of cells being analyzed.

D. Balance of the Chromosome Set of the Leukemic and Tumor Cells in Culture Progression of leukemias and tumors in the organisms is accompanied by a permanently evolution of tumor cell karyotypes both in the tumor itself and particularly in its metastases (Sonta et al., 1977; Heim and Mitelman, 1987; Christiansen and Lampert, 1988). At initial stages of disease, simple structural chromosome rearrangements are usually found in malignant cells, such as reciprocal translocations, inversions, deletions, for instance, t(8;14), t(2;8), t(8;22) in Burkitt lymphoma; t(9;22) in chronic myelogenous leukemia; inv(l4) in T-cell lymphoma; t/del(3) in renal cell carcinoma; t(12) in lipoma; del(1) in neuroblastoma; and del(3) in the small cell lung carcinoma. However, as tumors develop, the complexity of chromosome aberrations increases. In tumor cells, multiple numerical and structural chromosome changes often occur which result in appearance of new cell clones (Heim and Mitelman, 1987). The karyotype heterogeneity of tumors in vivo is also due to the fact that tumors can have both monoclonal and polyclonal origins; i.e., they appear from karyotypically different, nonrelated cell clones (Shapiro et al., 1981; Hayashi et al., 1989b; Heim, 1993; Gorunova et al., 1995; Pandis et al., 1995). Thus, measurements of the DNA content in tumor cells by flow cytofluorimetry and analysis of their karyotypes often reveal several cell clones that differ both in the chromosome composition and in the DNA content, which indicates the absence of a genome balance in the cells studied (Peterson and Friedrich, 1986; Remvikos et al.; 1988; Merkel and McGuire, 1990; Dressler et al., 1993). The most important regularity characteristic of cells in culture, unlike tumor cells in an organism, is the balance of the chromosome material in permanent cell lines that have reached the stage of stabilization in their evolution. We mean by the balance of the chromosome material a similarity of reconstructed karyotypes of line cells, i.e., the similarity of the total chromosome material of different cells within the same

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clone, of cells of different clones of the same line, and, accordingly, of cells of the line as a whole despite their karyotypic heterogeneity (Mamaeva et al., 1986; Savelyeva and Mamaeva, 1987, 1988). We found similarity of the GRK in different sublines with the identical or close modal chromosome number in human HeLa, Raji, and Namalwa cell lines. For instance, cells of two HeLa sublines, M-HeLa and HeLa LChM, with the equal modal chromosome number of 51, differed significantly in the composition of normal and marker chromosomes, whereas the GRK of these sublines differed only in the number of copies in the short arms of chromosome 5 and the total amount of material of chromosome 12 (Mamaeva, 1984; Savelyeva, 1988). In a number of human cell lines there is also a negligible variability in the number of normal chromosomes in different clones, most often in chromosomes 5 and 7 (Mamaeva, 1984,1988; Savelyeva and Mamaeva, 1987, 1988). The genome balance in permanent cell lines is similar in the flow cytometrically determined DNA amount in cells (Kraemer et al., 1971, 1972; Mamaeva et al., 1983b; Mamaeva, 1984; Rozanov et al., 1984). As a rule, in any cell line that has reached the stabilization stage, one dominating cell clone which is characterized by a low variability coefficient of cell DNA content and a polyploid variant of this clone are found.

E. Retention of, as a Minimum, Disomy on All Autosomes in Most Leukemic and Tumor Cell Lines The karyotypic peculiarities of leukemic and tumor cells in vivo,apart from specific chromosome translocations, are partial or complete monosomies on certain autosomes (Heim and Mitelman, 1987; Brodeur and Chin-to Fong, 1989; Thiel et al., 1992; Mitelman, 1994). Heim and Mitelman (1987) found most often monosomies on chromosomes 1, 3, 5, 6,7, 9, 10, 11, 12, 17, 20, and 22 in human neoplasias. Moreover, more than 20 cases of tumors with nearhaploid cell clones characterized by monosomies on many autosomes and the modal chromosome number 23-34 have been described (Misawa et al., 1985; Kristoffersson et al., 1986). One of the most important regularities of karyotypic variability of cells in culture is retention of disomy on all autosomes in cells of most human and animal cell lines. Of all such cell lines, according to our estimation based on analysis of the GRK of the cell lines, this amounts to at least 85%. This means that to exist in vitro, permanent line cells must have at least two homologs of each autosome. Establishment of such lines is achieved either by selection of a preexisting original stem cell with an increased proliferative potential and with the diploid autosome set or by a gradual evolution of the karyotype of initial hypodiploid cells in v i m , with

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chromosomes undergoing both structural and numerical rearrangements in the course of evolution. An example of such evolution is the establishment of the K-562 cell line obtained from cells of the pleural effusion of a patient with chronic myelogenous leukemia in the blast crisis. During early passages, the modal chromosome number amounted to 45, and cells contained the Ph' chromosome and an unidentified translocation of chromosomes t(D;E). During culture, they gradually became hyperdiploid: as early as at the 10th passage, cells with 50-52 chromosomes predominated, while at the 84th passage, cells had near-triploid and tetraploid chromosome numbers. As the karyotypic heterogeneity of the cell line rose, new marker chromosomes appeared in cells (Lozzio and Lozzio, 1975; Kirsch et a/., 1985). The gradual karyotypic evolution of the cell line has resulted in the selection and stabilization of its hypotriploid cell clone with 12 marker chromosomes (Mamaeva, 1984; Chen, 1985). Retention or acquisition by initial cells of a gene balance characteristic of diploid cells is not the only condition for the survival of cells in vitro and establishment of a cell line. Many authors have found specific functional properties of initial cells important, including their differentiation level (Nilsson, 1979, 1994; Zang, 1982; Pahlman et al., 1990). The easiest way to get cell lines is by cell transformation, either spontaneous or induced by one or several viruses, particularly in cases where initial leukemia or tumor cells contain specific chromosomal translocations that provide growth advantages to these cells as a result of activating certain oncogenes. Most of such cell lines are virus-containing (Nilsson and Ponten, 1975; Hoshino et al., 1983; Miyamoto et al., 1984; Agrba and Timanovskaya, 1988; Artsybasheva and Ignatova, 1989; Lestou et al., 1993). Meanwhile, attempts to establish permanent cell lines from tumors by prolonged culture in vitro are successful in less than 10% of the cases. The cell lines are obtained most often from cells of highly malignant or metastatic, poorly differentiated tumors, such as establishment of multiple myeloma cell lines as described by Nilsson (1977). It is impossible to obtain cell lines from highly differentiated cells of benign solid tumors. For instance, the permanent cell line has never been observed to appear spontaneously from human meningioma, a usually benign tumor characterized by a specific monosomy on chromosome 22 (Zang, 1982). At the same time, there are only reports of three human meningioma lines which were obtained only by malignant transformation of human meningioma cells via their transplantation to nude mice (Tanaka et al., 1989). There is no permanent cell line reaching the stabilization stage in its evolution, which would have been proven to have cell clones with the nearhaploid chromosome number or with nullisomies on some chromosomes. Although Kohno and co-authors (1980) reported the NALM-16 cell line with the nearhaploid chromosome number obtained from bone marrow of a female with acute lymphoblastic leukemia, a cell population studied by

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authors at various time periods after the beginning of its culture have been found to have a high karyotypic variability of cells. First, almost all initial cells (98.2%) of the NALM-16 cell line had the near-haploid chromosome number (23-27), and only 1.8% of cells were hyperdiploid (54 chromosomes). During 15 months of culture, near-haploid cell clones were gradually replaced by hyperdiploid ones, so that the former amounted to 5.4% in the population and the latter to 91.2%. These data show that the survival and existence in culture of a cell line with the haploid chromosome set is impossible. In the course of their establishment in v i m , NALM-16 cells undergo a karyotypic evolution by selection of the most adapted, neardiploid cell clones, but the process of karyotypic stabilization of the cell line has not yet been completed. Drouin and co-authors (1993) obtained a similar cell line from a primary tumor of a patient with the squamous cell lung carcinoma, in which cells contained the near-haploid chromosome number (27) and chromosomes had no visible structural rearrangements. During culture, their chromosome number doubled, and near-haploid cells were gradually replaced by hyperdiploid cells with 54 chromosomes. This cell line will probably undergo further karyotypic evolution. Meanwhile, transplantation of human glioblastoma cells containing a cell clone with the near-haploid chromosome set (25-26) to the nude mice resulted in appearance of tumor cells developed in mice, with the initial near-haploid chromosome set which remained unchanged during 10 passages in vivo (Bigner et al., 1985). As to the possibility of establishing of the cell line with nullisomy on chromosome 13 described by Fisher and co-authors (1985), these authors most likely interpreted erroneously the marker chromosomes in cells with the material of absent homologs of chromosome 13 as normal homologs of chromosome 3. It should be noted that all the above-mentioned regularities of karyotypic variability of cells in culture resulted from investigations on karyotypes of a wide spectrum of cell lines both from human cells and from cells of various animals: monkey, rat, mouse, and Chinese hamster (Mamaeva, 1984; Mamaeva and Tsvileneva, 1985; Filatov and Mamaeva, 1985; Urmanova et al., 1989; Araviashvili et al., 1994; Yartseva et al., 1994). Thus, at least two homologs of each autosome are present in more than 85% of cell lines studied. This evaluation resulted from analysis of karyotypes of more than 150 human and animal cell lines investigated at our Laboratory and of karyotypes of about 350 lines studied by other authors.

VI. Cell Lines with Autosomal Monosomies and Compensation for the Loss of Genetic Material What are the rest (15%) of the cell lines, which are characterized by complete or partial monosomies on individual autosomes? In some of them

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monosomies were maintained in the course of prolonged cell culture, not uncommonly for several years. Such are most cell lines of the neurogeneous origin, for instance, neuroblastomas, retinoblastomas, etc. In other cell lines monosomies appeared only at the stage of their establishment, while by the period of a final karyotypic stabilization in vitro, cells with monosomies were replaced by cells without this genetic defect. Such lines are represented by most lines from cells of melanoma, lines of the glioma origin, lines from cells of patients with T-cell leukemia, most lines from various organs of patients with small cell lung carcinoma, and some other cell lines. The question naturally arises as to by what means cells of these lines are able to grow in vitro. There are three possible ways of “compensating” the loss by cells of the genetic material of some autosomies to allow cells to maintain their viability and to adapt to the growth in culture: (1) polyploidization of cells with monosomy; (2) amplification of oncogenes, predominantly of the myc family; and (3) extracopying of whole autosomes or their fragments.

A. Polyploidization of Cells with Monosomy on One or Several Chromosomes In this case, an increase in the number of all chromosomes in cells results in restoration of disomy on autosomes, of which one of the homologs was lost. Of the three ways of compensating chromosome monosomies, this is observed most often in cell lines because any initial tumor population has polyploid cells, more often with a number of chromosomes that exceeds twice the modal number (Muleris et al., 1990; Rege-Cambrin et al., 1992). Therefore, in case of monosomy in cells growing in vitro, the polyploid variant of such cells can get selective advantages. Replacement of clones of near-haploid cells with near-diploid and near-diploid near-tetraploid ones occurs gradually, starting with the earliest passages (Kohno et al., 1980). For instance, in the course of establishment of human T-cell leukemia cell lines, of which the initial tumor cells contain, as a rule, numerous deletion of chromosomes, more often de1(6)(q15q21), the polyploidization of such cells can be found already at early stages of establishment (More et al., 1985). During subsequent karyotypic evolution of the tetraploid variant of these cell lines, the modal chromosome number may be reduced to near-triploid (Ohno et al., 1988) or can rise up to hypertetraploid (Westbrook et al., 1987; Naoe et al., 1988). Unfortunately, no data are found in the literature about karyotypic evolution of cells in the process of establishment of human myeloma lines such as U-266 (with the modal chromosome number 44 and 87-88), U-1996 (82), the OPM-1 line (76-79), the OPM-2 line (65-75), and RPMI-8226

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(67). The modal chromosome number found in these cell lines and the double set of some markers in their karyotypes indicate that polyploidization occurred in the initially hypodiploid myeloma cells which were probably monosomal on many chromosomes (V. J. Turilova, unpublished data). Three groups of authors (Mark et al., 1977; Rey et al., 1983,1987; Bigner et al., 1987) described in detail karyotypic evolution of human glioma cell lines characterized initially by monosomies on chromosomes 9, 10, and 22. The authors followed changes in karyotypes from early passages in vitro to the 100th passage and further. Polyploidization of cells was found to occur in the process of establishment of the lines. Near-diploid cells were replaced by near-tetraploid ones during early stages of karyotypic evolution (30-40 passages). Later on, elimination of “unnecessary” and extracopying of “necessary” chromosomes for these cell lines (more often, chromosome 7) occurred, and the marker chromosome composition changed insignificantly: some initial markers disappeared while new ones appeared. As a result, after reaching a karyotype stabilization state (90-100 passages), the lines were represented by near-triploid cells with balanced sets of normal and marker chromosomes. Such is typical karyotypic evolution of polyploid cell lines obtained from solid tumors characterized by autosome monosomies. During establishment of some glioma cell lines, an even greater increase in the chromosome number occurred in cells so that the modal chromosome number was near-pentaploid (Bigner et al., 1986). Apart from this, a case was described in which a near-triploid cell line was established by passing the “tetraploidy stage.” In this case, selection of near-triploid cells that had been in the population of initial tumor cells occurred. This cell line reached the karyotype stabilization state also via numeral and structural changes of certain chromosomes (Rey et al., 1989).During karyotypic evolution of many human melanoma cell lines characterized by a deletion of the long arm of chromosome 6, cells also had the polyploidization stage as most of these lines were near-triploid or near-tetraploid (Semple et al., 1982; Pathak et al., 1983). In the study of compensation of monosomy, of great interest are cell lines established from cells of patients with small cell lung carcinoma. The deletion of the short arm of chromosome 3, de1(3)(p14p23) (Heim and Mitelman, 1987) characteristic of this tumor is present in 90% of tumors in vivo and retains in most small cell lung cancer lines (Whang-Peng and Lee, 1985; Waters et al., 1988). About half of these cell lines are characterized by appearance and then dominance of near-triploid and near-tetraploid cell clones (Zech et al., 1985). Unfortunately, evolution of karyotypes of these lines at the stage of their establishment was not studied in detail but the presence of at least two modal classes, near-diploid and near-tetraploid, in most of the cell lines indicate incompletion of the process of the line

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establishment (Waters et af., 1988). In this case it appears premature to state that the cell line has reached a stable karyotypic status, i.e., that a specific deletion in polyploid cells is maintained. More detailed studies of karyotypes of small cell lung cancer lines which completed the establishment are necessary. Based on available data, it is evident that near-diploid cell clones tend to be replaced by polyploid ones. The karyotypic evolution of cell lines that have lost monosomies on autosomes as a result of polyploidization of initial cells is frequently accompanied by changes in their phenotypic and growth characteristics as well as in the degree of malignancy. For instance, study of glioma cell lines has shown that polyploid line cells that appear during prolonged culture differ from the initial near-diploid tumor cells by the absence of a number of specific proteins, particularly of the glial fibrillar acid protein, by a threeto fivefold increase in the doubling time of the cell population and by the loss of malignancy (Pfeiffer et af., 1977; Ponten and Westermark, 1978; D. D. Bigner et af., 1981; S. H. Bigner et af., 1986).

B. Amplification of Oncogenes in Cells with Monosomies Amplification of oncogenes is known to result in the appearance of either double minichromosomes (DMs) or chromosomes with homogeneously stained regions (HSR) and chromosomes with differentially stained regions (DSR). Cells with one of these morphological markers of gene amplification usually predominate in cell populations characterized by amplification of oncogenes. Whereas the number of DMs in different cells of the same line can vary significantly (from 10 to 300) (Biedler et af., 1983; Brodeur and Seeger, 1986), the localization of HSR and DSR in a chromosome as well as the length of HSR and DSR are more stable (Bahr et af., 1983; van der Hout et al., 1989). The most demonstrative example of cell lines with the chromosome monosomy compensated by amplification of oncogenes are human neuroblastoma cell lines that are characterized by a specific deletion of the short arm of chromosome 1:p13 or p31.2 like the initial tumor cells in vivo (Balaban-Malenbaum and Gilbert, 1980; Biedler et af., 1980; Savelyeva et af., 1994). In tumor cells of approximately 30% of patients with neuroblastomas, particularly at the third and fourth stages of the disease, amplification of N-myc oncogene (from 3 to 300 copies) occurs: apart from deletion of chromosome 1, it is present in cells as different number of DMs (Brodeur and Chin-to Fong, 1989; Schwab and Amler, 1990). More than 40 neuroblastoma cell lines have been described. In cells of most of these lines, deletion in the short arm of chromosome 1 and DMs or, more often, HSR was also found (Biedler et af., 1983; McRobert et af.,

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1992). It is interesting to note that in cell lines with no such deletion, neither DMs nor HSR have been found; only two copies of N-myc oncogene have been found. One such line is the SK-N-SH line in cells of which two normal homologs of chromosome 1 are present (Biedler et al., 1983; Pahlman et al., 1990). Similarly, the N-myc oncogene is represented only by two copies in cells of the SKN-KANR line obtained from bone marrow cells of a patient with neuroblastoma; the DMs and HSR are absent, and both homologs of chromosome 1 are normal (Biedler et al., 1983). It appears that when neuroblastoma cell lines are established, selection of the original cells with deleted chromosome 1 and amplified myc oncogene occurs most often. Neuroblastoma cell lines retain the near-diploid modal number of chromosomes in cells, a gradual replacement of cells with DMs by more stable cells with HSR (Brodeur et al., 1981), and in a number of cell lines also doubling of the number of marker chromosomes that contain HSR, i.e., the amplified oncogene (Biedler et al., 1983; McRobert et al., 1992). An interesting atypical example of karyotypic evolution is found in a neuroblastoma line, Gi-ME-N. Although the short arm of chromosome 1 was deleted in cells of this line, no amplification of the N-myc oncogene was observed, and even early passages were accompanied by polyploidization of the initially pseudodiploid cells and their replacement by near-tetraploid ones. The complex chromosome rearrangements that occurred additionally in the initial cells of the line seemed to compensate the loss of the short arm of chromosome 1 by cell polyploidization rather than by amplification of the oncogene (Donti et al., 1988). Only a few cell lines have been described that have monosomies on certain autosomes and amplified oncogenes. Apart from neuroblastoma cell lines, a retinoblastoma cell line, Y-79, is known, of which cells are characterized by a partial monosomy on chromosome 5 and complete monosomy on chromosome 17 as well as by a 100-fold increase of copies of the myc oncogene that was revealed by in situ DNA hybridization and was present as HSR of the short arm of chromosome 1 (Inazawa et al., 1989). No retinoblastoma cell lines were found to have deletion of chromosome 13, de1(13)(q14) specific of this tumor (Gilbert et al., 1981; Seshadri et al., 1986). In leukemia cell lines, except for T-cell leukemia lines, chromosome aberrations such as monosomies occur much more seldom. The HL-60 cell line was obtained from peripheral blood promyelocytic cells of a female with acute myeloblastic leukemia whose cells are characterized by a partial monosomy on chromosomes 5, 9, and 14 (Yakovleva et al., 1992) and by amplification of the c-myc oncogene either as numerous DMs or as HSR (Nowell et al., 1983; Misawa et al., 1987). Amplification of the c-myc oncogene (up to 30 copies per cell) is also discovered in cells of two sublines of colon carcinoma: Colo 320DM and Colo 320HSR (Alitalo et al., 1983).

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Besides, cells of these sublines contain amplification of a DNA nucleotide sequence different from the c-myc oncogene (Hubell et a!., 1987). In cell lines obtained from patients with small cell lung carcinoma mentioned previously, deletion of the short arm of chromosome 3 was compensated by polyploidization of cells. In about 40% of other small cell lung cancer cell lines characterized by this specific deletion, amplification of the myc oncogene family (c-myc, N-myc, and L-myc) occurred (Nau et al., 1985; Bergh, 1990; Johnson et al., 1992). In this case a cell line had amplification of only one of these myc oncogenes (Nau et al., 1985, 1986; Waters et al., 1988; van der Hout et al., 1989). The oncogene amplification occurred in cells either as DMs the number of which varied from 10 to 260 or as HSR of different chromosomes (van der Hout et al., 1989). The myc family oncogenes in permanent cell lines of small cell lung carcinoma are amplified much more often than in the initial tumor cells (Wolman and Henderson, 1989; Bergh, 1990). The small cell lung cancer cell lines can be divided into the classic and variant types on the basis of expression of four biomarkers (Carney et al., 1985). The variant cell lines are characterized by a quicker cell multiplication and a higher efficiency of cloning. Most of small cell lung cancer cell lines characterized by amplification of the myc oncogenes are of the variant type (Little et al., 1983;Gazdar et al., 1985). A neuroendocrine differentiation of cells should be noted to be characteristic of most cell lines obtained from solid tumor and showing amplification of the myc oncogene family (Wolman and Henderson, 1989; Bergh, 1990). The ML-1, ML-2, and ML-3 sublines of the ML cell line obtained from a patient with acute myeloblastic leukemia are often considered to be characterized by oncogene amplification. In cells of these sublines, amplification of the c-myb oncogene occurred (Pelicci et al., 1984). A detailed analysis of karyotypes of the sublines showed that cells of near-diploid ML1 and ML-2 sublines and of the near-tetraploid ML-3 subline had a deletion of chromosome 6 in the q23 region. In situ DNA hybridization studies established that the slight increase of c-myb oncogene copies is a result of increase in the number of the normal and marker chromosome 6 copies rather than of amplification of this oncogene (Henderson and Wolman, 1988; Wolman and Henderson, 1989). In two colon carcinoma cell lines, Colo 201 and Colo 205, a small increase in the number of the c-myb oncogene copies was found: by 8 and 10 times, respectively, the c-myb oncogene is considered to be amplified in cells of these lines (Alitalo et al., 1984). However, such a view is doubtful because the karyotypic markers of amplification as DMs or HSR are not characteristic of cells of these lines. The Colo 201 and Colo 205 lines are near-triploid (the modal number of chromosomes 69 and 73, respectively), and the number of chromosome 6 copies increases in their cells. Most likely, the increased number of c-myb oncogene copies results from an increase in

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the number of chromosome 6 copies rather than from amplification of this oncogene. In some cell lines of human carcinomas of different tissue origin, a simultaneous amplification of myc oncogenes and Ki-ras and erbB-2 oncogenes was found (Watanabe et al., 1992; Fukumoto et al., 1993). Fukumoto and co-authors (1993) studied in detail karyotypes of two Lu-65 and KHC287 lines obtained from cells of patients with large cell lung carcinoma and showed convincingly a compensatory effect of coamplification of myc and Ki-ras oncogenes in multiple autosome monosomies of cells of these near-diploid lines. The authors also established that after the first passages of these lines the oncogene amplification occurred in the form of DMs, which later became HSRs. Simultaneous amplification of c-myc and erbB-2 oncogenes (up to 10 copies of each oncogene) was found in the BSMZ line established from a human breast carcinoma (Watanabe et al., 1992). Unfortunately, the authors did not analyze in detail the karyotype of this cell line. As human breast cancer cell lines are characterized by a high heteroploidy, the absence of data about karyotype of this line makes it difficult to conclude whether the increase in the number of oncogene copies is due to their amplification or if it results from an increase in the number of copies of normal and marker chromosomes that contain these genes. There are data on amplification of the erbB-2 oncogene in human breast tumor cells, but karyotypes of cells of these tumors have not been studied (Slamon et al., 1987; Smith et al., 1993). There are reports on karyotypic markers of oncogene amplification in the form of DM in tumor cells and in cell lines obtained from human breast carcinoma (Barker and Hsu, 1979), but the oncogenes and the number of their copies in these cells were not studied. It is hoped that the current incomplete information about gene amplification in different cell lines of tumor origin will be completed in future. To summarize, the amplification of oncogenes (predominantly myc oncogenes) may be one of the chief mechanisms for gene activation, which allows tumor cells in culture to compensate loss of genetic material as a result of intra- and interchromosomal rearrangements. Most likely, the increased expression of oncogenes owing to their amplification provides growth advantages to malignant cells. The accumulated data are not sufficient to clarify the nature of interconnections between gene amplification and specific chromosome aberrations; however, the role of myc oncogenes is strongly proved for unrestricted proliferation of cells, for their malignant transformation and a progressive development of tumors in vivo, and particularly for the possibility of existence of permanent cell lines obtained from tumor cells (Wolman and Henderson, 1989; Bergh, 1990; Gibson and Croker, 1992; McRobert et al., 1992;von Hoff et al., 1992; Ozaki et a/., 1993).

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C. Extracopying of Whole Autosomes or Their Fragments This mode of compensating monosomies of cell lines occurs much more seldom than the other two ways described above. As an example, results of analysis of the karyotype of a hypodiploid SW-837 line obtained from cells of a patient with rectum adenocarcinoma can be referred to. All cells of the GRK cell line have a complete monosomy on chromosomes 13 and 18 and a partial monosomy on chromosomes 1, 3, 6 , 8, 11, 17, 19, and 22. At the same time, material of long arms of chromosomes 5 and 20 were extracopied in cells of this line (Gorunova et al., 1992). Analysis of the GRK HeLa-Tk- subline deficient in thymidine kinase has shown monosomies in these cells on chromosomes 11 and 18 and extracopies of chromosome 20 and of the short arm of chromosome 5 (Mamaeva, 1984). Cell lines with monosomies in autosomes often contain an increased number of copies of chromosome 7. The extracopying of chromosome 7 is a characteristic feature of karyotypes of glioma cell lines (Mark et al., 1977), melanoma cell lines (Chen and Shaw, 1974; Sozzi et al., 1990), and some lung cancer cell lines (Fukumoto et al., 1993). Thus, it appears that the increase of the number of chromosome copies, which results in a rise in activity of the growth-associated genes localized in these chromosomes, promotes increase of the cell proliferative potential and thus determines selective advantages of cells in culture. Compensation of autosome monosomies by extracopying material of some normal chromosomes is most likely characteristic of both human and animal cell lines. From this point of view, of interest is the L6J1 line obtained from rat myogenic nonmalignant cells with the hyperdiploid number of chromosomes. The GRK cells contain only one homolog of chromosome 17 and additional homologs of chromosomes 1 and 12 (Fedortseva et al., 1983). Only three modes of compensating monosomies of cells in permanent cell lines have been considered. It cannot be ruled out that further search for lines- “exceptions”-and a detailed analysis of their karyotypes as well as molecular-biological characteristic of each cell line will make it possible to reveal new ways of compensating monosomies in cell lines from different tissues and species.

VII. Concluding Remarks Attention is to be focused on several aspects of the karyotypic variability of permanent cell lines, these aspects being important to understand properties of cell lines as autonomous living systems. The data considered in this review allow stating that knowledge of general regularities of karyotypic

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variability of cells during establishment and prolonged culture of cell lines makes it possible to forecast the behavior of each particular line early in its establishment. Such prognosis is important for the use of cell lines in various molecular-genetic experiments and in biotechnology. The cytogenetic control of permanent cell lines, particularly those that are characterized by chromosome monosomies, should be carried out at each stage of experiment, simultaneously with analysis of such characteristics of cultured cells as the growth rate, expression of specific markers, and degree of malignancy. To forecast behavior of a particular cell population in culture, it is necessary to understand which karyotypic peculiarities of initial cells determine character of the karyotypic variability of cells in the process of establishment of the cell lines as well as the maintenance of their stable, balanced state. The general regularities found in the karyotypic variability of tumor cell in vivo and in v i m , such as the nonrandom character of numerical and structural chromosome changes, loss of one of the sex chromosomes, nonrandom character of distribution of breakpoints on chromosomes during establishment of markers, and extracopying of certain chromosomes, indicate similarities of karyotyic evolution of cells in organisms and in culture. It is not less important to study distinct peculiarities of the karyotypic variability of cultured cells, which are associated with adaptation and existence of cells in vitro. The chief regularities of the karyotypic variability characteristic only of cells in culture are the balance of the chromosome set of cells and retention in most cell lines of disomy on all autosomes. The described peculiarities of the karyotypic evolution of cell lines-"exceptions'' characterized by autosome monosomies, such as polyploidization of a stem cell line, amplification of oncogenes, and a selective extracopying of certain chromosomes-are seldom observed in tumor cell population in vivo and are characteristic to karyotypic evolution of permanent cell lines. The initial generalizations provide a basis for a promising direction in which investigations on peculiarities of karyotypic variability of cells in culture could be developing.

Acknowledgments The author thanks Dr. Tatyana R. Sukhikh for valuable discussion and help in preparing this work for publication as well as all researchers of the Cytogenetics Group of the Laboratory of Cell Morphology of the Institute of Cytology of the Russian Academy of Sciences for their constant assistance in composing the manuscript.

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Sucrose Transport in Higher Plants John M. Ward, Christina Kuhn, Mechthild Tegeder, and Wolf B. Frommer Institut fur Botanik, Universitat Tubingen, D-72076 Tubingen, Germany

Presumably due to its physicochemicalproperties, sucrose represents the major transport form of photosyntheticallyassimilated carbohydrates in plants. Sucrose synthesized in green leaves is transported via the phloem, the long distance distribution network for assimilates in order to supply nonphotosyntheticorgans with energy and carbon skeletons. At least in Solanaceae, sugar export seems to be a tightly regulated process involving a number of specific plasma membrane proteins. Significant progress in this field was made possible by the recent identification of plasma membrane sucrose transporter genes. These carriers represent important parts of the long-distancetransport machinery and can serve as a starting point to obtain a complete picture of long-distance sucrose transport in plants. A combination of biochemical studies of transporter properties together with expression and localization studies allow specific functions to be assigned to the individual proteins. Furthermore, the use of transgenic plants specifically impaired in sucrose transporter expression has provided strong evidence that SUT1 transporter function is required for phloem loading. Physiologicalanalyses of these plants demonstrate that sucrose transporters are essential components of the sucrose translocation pathway at least in potato and tobacco. KEY WORDS: Sugar, Transport, Phloem, Vascular tissue, Active transport, Plasmodesmata, Apoplastic, Symplastic.

I. Introduction The multiplicity of biochemical reactions proceeding within an organism and even within a single cell is only possible due to compartmentation created by multiple intracellular and surrounding membranes. Basically membranes consist of two types of molecules: (i) lipids that prevent free exchange of ions, metabolites, and macromolecules, and (ii) proteins enabling controlled exchange of molecules and information between compartInternational Review of Cyrology, VoL 178

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ments or with the environment. Despite their importance, very few of these membrane proteins from plants have been characterized at the molecular level. For example, none of the carriers involved in photorespiration have been cloned yet. Due to the strategic position of these proteins at the beginning or end of metabolic pathways, they might play important regulatory roles that can only be addressed when sequence information is available. In plants, biochemical approaches to identify plasma membrane carriers involved in long-distance transport of assimilates have not been successful and recent advances have been primarily due to molecular and genetic approaches as described in this review. Many unicellular organisms such as green algae take up solutes such as glucose from the solution across their plasma membrane by carriermediated processes. The hexose transporters have been identified by molecular genetic approaches (Sauer and Tanner, 1989). In multicellular organisms such as higher plants, the functional differentiation of cells made the development of specific transport vessels necessary. Especially in higher land plants, remarkable distances of up to 100 m have to be passed in order to allow an exchange of metabolites between different cells at the distal ends of the plant, e.g., leaves and roots or flowers of trees. For the longdistance transport of sugars, amino acids, and ions from source organs (net exporting tissues) to sink organs (net importing tissues), plants have developed a veinal network, i.e., the phloem. The most abundant compound in the phloem of most plant species is sucrose, whereas hexose concentrations are very low (Zimmermann and Ziegler, 1975). Sugars are synthesized in the mesophyll cells, especially the palisade parenchyma. From there they have to be translocated across several cells to reach the veinal network. On their way, several cell types have to be passed-neighboring mesophyll cells, bundle sheath cells, and phloem parenchyma or companion cellsbefore the sugars reach the actual conduits, the sieve elements. From there sugars can be translocated to their destination within the sieve tubes. Plants do not utilize a muscle-based pump able to create the driving force for long-distance transport. Nevertheless, the velocity of translocation in the phloem is high, ranging from 0.5 to 3 m/hr (Kockenberger et al., 1997). The carriers located at the distal ends of the phloem are assumed to be directly involved in creating the driving force. Higher plants have developed an osmotic pump consisting primarily of three types of plasma membrane proteins with distinct functions: sucrose H+/symportersthat are able to accumulate osmotically active sucrose in high concentrations in the phloem, H+-ATPases that are necessary for energization of the active transport and, presumably, water channels that allow the passive influx of large amounts of water into the conduits thus driving the phloem translocation by mass flow. Of course, ATP is necessary to energize transport and has to be supplied as well. An essential prerequisite for this osmotic pump

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43

is that the conduits (phloem cells) are osmotically isolated from their neighboring cells. Thus, it is necessary that either phloem cells are not connected to neighboring mesophyll cells or that connections are tightly regulated. One possible mechanism for sucrose delivery to the phloem involves export from mesophyll cells followed by active uptake into phloem cells. Besides this protein-mediated traffic across the plasma membrane, intercellular connections such as plasmodesmata might allow direct exchange and equilibration of solutes in what is called the symplasm. The term symplasm is meant to indicate that the cytoplasm of several cells is continuous and that metabolites can be freely exchanged. -The connections between plant cells, called plasmodesmata, are structures connecting the majority of cells within a leaf and might be seen as analogous to ring canals or gap junctions. However, it is a matter of intensive discussion whether free exchange is possible between all interconnected cells or whether gating through plasmodesmata is regulated as has been described for gap junctions (Lew, 1994; Kwak and Jongsma, 1996; Fishman et al., 1995; van Be1 and Oparka, 1995; Overall and Blackman, 1996). Thus, two principal routes can be envisaged: (i) carrier-mediated transport across the plasma membrane and diffusion through the cell wall (apoplastic) and (ii) direct diffusional cell-to-cell transport via plasmodesmata (symplastic). To clarify the actual pathways, molecular approaches directed at a better understanding of both plasmodesmal functioning and plasma membrane transport processes of sugars are required. During the past 5 years molecular approaches have enabled significant progress in the understanding of transport of sucrose across the plasma membrane. This review will concentrate on sucrose transport itself and try to highlight unresolved questions in the area of sugar transport and more specifically in the understanding of phloem loading. For readers interested in details of the closely related topic of Hf-ATPases, refer to Sussman (1994) or for a review of glucose transport see Tanner and Caspari (1996).

A. Anatomy and Function of the Phloem To understand long-distance transport in plants, it is necessary to briefly introduce the structure of the veinal network involved in long-distance translocation of metabolites in vasculated land plants. Similar to animals, plants utilize defined long-distance transport systems, i.e., the phloem and the xylem. The two systems are closely entangled and rarely are found on their own (Aloni et al., 1986). The phloem is responsible for long-distance transport of assimilates from leaves, the sources in which assimilation takes place, to sink tissues that are dependent on the import of metabolites generated by photosynthesis in leaves. The actual conduits are living cells

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named sieve elements. These cells are highly specialized and their structure and function have been reviewed in much more detail elsewhere (Behnke, 1989; Behnke and Sjolund, 1990). The phloem of dicotyledonous plants consists of a number of different cell types, the main conduits being sieve elements (SEs). The sieve elements are in direct contact with companion cells (CCs) and phloem parenchyma. In some species, phloem contains transfer cells in which the plasma membrane surface is increased by 3- to 10-fold due to cell wall invaginations (Pate and Gunning, 1972; Wimmers and Turgeon, 1991). Transfer cells are either modified phloem parenchyma cells (type B) or modified companion cells (type A). The presence of transfer cells may indicate the position of intense transport of solutes across the plasma membrane. This is further supported by an asymetric distribution of the plasma membrane-bound H+ATPase (BouchC-Pillonet al., 1994). Proton pumps were more numerous in the area of plasma membrane infoldings in which active nutrient uptake is assumed to take place. Companion cells contain a dense cytoplasm and comparatively high number of mitochondria, indicating that they might be involved in supplying the energy for loading and transport. This is supported by the finding that plasma membrane H+-ATPases are specifically expressed in the phloem and that antibodies detect the highest amount of ATPase protein in the phloem (Parets-Soler et al., 1990). Specifically, the H+-ATPaseprotein was detected in CCs of Arabidopsis thaliana and Vicia faba (BouchC-Pillon et al., 1994; DeWitt and Sussman, 1995). In potato, veins down to the sixth order are bicollateral and consist of the major abaxial phloem surrounded by bundle sheath cells and the minor adaxial phloem strands accompanying the central xylem vessel cells (McCauley and Evert, 1989). The seventhorder minor vein complex is surrounded by a bundle sheath. During development of the phloem conduits, division of a mother cell leads to two closely associated cell types, the sieve elements and the companion cells. Due to their close ontogenic and functional association the two cells are often addressed as SE/CC complex. In many plant species including Solanaceae, the SE/CC complex is symplastically isolated from surrounding cells, but the CC and SE cells are linked by a specific type of plasmodesmata (PPU). These differ from plasmodesmata (PD) between parenchyma cells in organization and diameter and possibly also size exclusion limit (SEL). Whereas the SEL of other plasmodesmata is approximately 800-1000 Da and is similar to that reported for animal gap junctions (Spray and Bennett, 1985), the SEL of PPUs seems to be much higher. The SEL of PPUs in the fascicular phloem of V. faba has been determined by use of fluorescent dextrans to be at least 10 kDa (van Bel, 1996). During maturation, SEs lose their nuclei and many organelles, retaining only a modified endomembrane system (sER) and few plastids and mito-

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45

chondria. The plasmodesmata of the walls interconnecting individual sieve element members dilate to macrochannels allowing a living tube to be built up. Thus, long-distance transport of sugars takes place within living cells rather than extracellularly as in animals. There are other important differences in long-distance transport between plants and animals: (i) in plants the disaccharide sucrose is the major transported sugar, whereas in animals it is the monosaccharide glucose; and (ii) in animals specialized cells circulate and are transported throughout the organism, whereas in plants, cells are not known to be transported. Sieve elements remain active over a whole growth period, i.e., several months in annual plants or up to decades, e.g., in palm trees. Due to the lack of nuclei, active SEs must retain proteins stable enough to function for the lifetime of the cell or mRNAs and proteins must be imported from neighboring CCs. Analyses of phloem sap, which can be isolated with the help of aphid stylectomy, have raised many important questions regarding the trafficking and transport activities involved in phloem loading. The sap is composed mainly of sugars, but malate, amino acids, or ureides and various ions, especially potassium, are also found (Riens et al., 1991; Lohaus et al., 1995). Several observations support the hypothesis that SEs are supplied with new proteins or mRNAs from the neighboring CCs: (i) Phloem sap contains proteins and enzymatic activities (Kennecke et al., 1971), (ii) some phloem proteins exhibit rapid turnover rates (Nuske and Eschrich, 1976), and (iii) phloem is capable of a constitutive efflux of an identical spectrum of polypeptides for a period of days (Nakamura et al., 1993). This is further supported by the finding that mRNA of a typical SE protein, the P-protein, is localized in CCs, indicating protein movement into mature SEs via plasmodesmata (Bostwick et al., 1992). 6 . Sucrose as a Transport Metabolite

Sucrose is a sugar typically found in plants. In contrast to the animal kingdom, most plants use sucrose rather than glucose as the major transport form of carbohydrates for long-distance translocation. Green algae are also able to synthesize and accumulate sucrose, possibly indicating that sucrose first served as a storage form of assimilated carbon and was adopted later as the transported form of carbohydrates in higher plants. Glucose is either absent from phloem sap or present only in minute amounts. The phloem may even be unable to take up hexoses as shown in transgenic plants in which apoplastic sucrose concentrations were artificially increased (Heineke et al., 1992). The phloem thus displays a high selectivity for sucrose. In a number of cases, besides sucrose, other sugars or sugar alcohols were found in phloem saps such as raffinose, stachyose, verbascose or

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mannitol, and sorbitol (Bieleski, 1969; Zimmermann and Ziegler, 1975). The concentrations of sucrose in the phloem sap of sucrose translocating species ranges from 200 to 1600 mM (Kallarackal et af., 1989; Winzer et al., 1996). The sugar concentrations in the vascular system are thus much higher compared to those of mammalian systems. It is assumed that the physicochemical properties of sucrose, including the low viscosity of concentrated solutions and its chemical stability as a nonreducing disaccharide, are key properties that made sucrose an ideal compound for long-distance translocation. The nonreducing nature is a unifying principle of all sugar compounds translocated in the phloem (Lucas and Madore, 1988). Furthermore, sucrose can easily be interconverted from the two key carbohydrates (glucose and fructose) by several enzymes such as sucrose phosphate synthase in leaves and sucrose synthase and invertases in importing tissues. Energy-minimized model predictions for the hydrated sucrose molecule may help in understanding how enzymes and transport proteins can recognize sucrose. Structural requirements indicate that related recognition and interaction mechanisms might also be involved in the case of sucrose receptors in taste papillae (Immel and Lichtenthaler, 1995).

II. Sugar Transport A. Symplastic Transport via Plasmodesmata In plants, most cells are separated from each other by cell walls. Despite this separation, the majority of cells are interconnected by plasmodesmata, which might be considered analogous to gap junctions (Lucas et al., 1993b; Epel, 1994). The function of these organelles in intercellular trafficking of metabolites remained obscure for many years. The finding that plasmodesmata allow the trafficking of both endogenous and viral mRNAs and proteins has led to a new understanding of their functions (Lucas et d.,1995). However, very little is known about plasmodesmal structure at the molecular level. Plasmodesmata provide a second system that allows direct transfer from cell to cell in addition to solute exchange across the plasma membrane by carrier-mediated mechanisms (Robards and Lucas, 1990). Fluorescent dyes with a molecular mass of up to 1000 Da can move freely between mesophyll cells. As mentioned previously, the SE/CC complex is especially dependent on plasmodesmal transport. However, the continuity from mesophyll to the sieve element companion cell complex differs depending on the species. In some species, classified as potential symplastic loaders, the density of plasmodesmata at the interface between the mesophyllhascular paren-

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chyma to the companion cells is high, whereas in those with only few plasmodesmata in this region apoplastic transport is likely to be required. This hypothesis has stimulated an analysis of multiple species regarding the density of plasmodesmata and the classification into different types of plants according to the density of plasmodesmata at this interface (Gamalei, 1986; van Bel, 1993). Several lines of evidence have been used to support symplastic routes for phloem loading in certain species (van Be1 et al., 1994). First, anatomical analyses using the electron microscope have elucidated the structure and distribution of plasmodesmata. As pointed out by van Be1 and Oparka (1995), the frequency of plasmodesmatal connections cannot be used to determine symplastic continuity and the open or closed state of plasmodesmata must be considered. Second, as mentioned previously, the permeability of plasmodesmata for fluorescent dyes was used to demonstrate that molecules below a certain molecular weight threshold can move from cell to cell. However, these studies have been criticized (Delrot, 1989).Third, the sensitivity of carrier-mediated sucrose transport to thiol group-modifying agents [p-chloromercuribenzenesulfonic acid (PCMBS)] was used to determine whether sugar transport is independent from carrier-mediate processes. Two methods were used to determine the effects of PCMBS on phloem loading. The efflux of sugars from the petiole of detached leaves can be measured and differences in the sensitivity of exudation to PCMBS were found to correlate with the classification based on anatomical studies, i.e., symplastic loaders are not affected by PCMBS. Experiments using other pharmacological or antisense inhibition of active transporters could be used to further test this hypothesis. Microautoradiographs of leaves were analyzed for the accumulation of sugars in the veins after labeling with I4CO2(Fritz et al., 1983). The lack of sensitivity to PCMBS was used as an indication for symplastic loading (van Be1 et al., 1994). However, all these methods are indirect and their interpretation may be influenced by contention that observed pits are simple holes through which metabolites can diffuse. Furthermore, recent studies have elegantly demonstrated that plasmodesmata are complex structures (Overall and Blackman, 1996) and that the permeability can be regulated not only by external factors but possibly also by endogenous factors (Lucas et af., 1993a). One of the major contributions to the understanding of the role of plasmodesmata in metabolite transport comes from recent observations of a maize mutant (sedl) blocked in plasmodesmal transport (Russin et al., 1996). The mutant plants were drastically impaired in growth and development. The leaves of sedl plants accumulate anthocyanins and starch. Whole leaf autoradiography of mutant leaves showed that export of sucrose was reduced. Electronmicroscopic analysis showed that the plasmodesmata at the bundle sheathhascular parenchyma interface of minor veins were struc-

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turally modified, in that the plasmodesmata were distorted and covered with cell wall material. Provided that this plasmodesmal defect is the reason for reduced sucrose export, in maize, sucrose translocation from mesophyll into vascular tissues occurs primarily on a symplastic route. However, until more details are known it is not possible to exclude that the effects observed are secondary. Even if plasmodesmata were required on the path to the SE/CC complex, one major problem concerning the hypothesis of symplastic transport of sucrose into phloem is that it is difficult to explain on a thermodynamic basis. In Cucurbira pep0 callus cultures, an osmotic gradient was observed between SEs and CCs, indicating that phloem loading may occur between these cells (Lackney and Sjolund, 1991). Thus, also for symplastic models sucrose transport through plasmodesmata has to occur against a concentration gradient. Nevertheless, active transport through plasmodesmata is conceivable. For example, an interesting mechanistic model for active loading through plasmodesmata was developed by Turgeon and Gowan (1990). The synthesis of raffinose takes place mainly in intermediary or companion cells (Beebe and Turgeon, 1992). This idea for an active transport through plasmodesmata is based on the assumption that the size exclusion limit is sufficient for the passage of sucrose in the direction of the SE/CC but insufficient to allow a reflux of raffinose. An alternative theory for a mechanism of symplasmic phloem loading has been proposed by Gamalei et al. (1994). It would be possible to keep the concentration gradient low between the cytoplasm of mesophyll cells and phloem cells by subcellular compartmentation of transport sugars. The energy needed for accumulation of sugars in intracellular compartments would be comparable to that required for apoplasmic loading (van Bel, 1996). It is possible that both symplastic and apoplastic pathways are present within one species (van Bel, 1993). A testable proposal could therefore be that under some conditions the plant exchanges small molecules in an nonselective manner through plasmodesmata connecting SEs with other phloem cells. However, under other conditions the plasmodesmata might be closed and then only those metabolites for which respective carriers are present can be transported. Regulation of the permeability of plasmodesmata by changes in turgor support this possibility (Oparka and Prior, 1992; Schulz, 1995). Under certain conditions, molecules larger than 1000 Da can also move through plasmodesmata. The best studied case is the movement of a number of viral proteins and RNAs. The use of viral movement proteins has proven to be an invaluable tool to study plasmodesmal transport (Citovsky, 1993; Lucas et al., 1993a). Recently, it could be shown in an elegant study that endogenous proteins such as transcription factors can also move through plasmodesmata and that they can even mobilize the transport of their own

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mRNA (Lucas el al., 1995). Further indications for a function of PD in signaling derive from experiments in which viral movement proteins were overexpressed stably in transgenic plants leading to alterations in carbohydrate metabolism (Balachandran et al., 1995). One may therefore assume that the enucleate sieve elements are constantly supplied not only with metabolites but also with RNAs and proteins from their companion cells. The components involved in trafficking of macromolecules through plasmodesmata are unknown. In fact, not a single gene encoding a plasmodesmatal protein has been cloned at this time (Epel et al., 1996). Recent studies could, however, demonstrate that cytoskeletal structures are involved in targeting of viral movement proteins to the plasmodesmata (Heinlein et al., 1995; McLean el al., 1995; Padgett et al., 1996). 6 . Apoplastic Transport via Plasma Membrane Transporters

Due to the lack of knowledge regarding the actual open status of plasmodesmata (even where plasmodesmal connections are frequent they may be closed or nonfunctional), carriers may be required for phloem loading. At least in species with low symplastic connectivity between the SEKC complex and the surrounding cells, carrier-mediated apoplastic phloem loading seems highly probable. Support for the hypothesis of apoplastic transport derives from the block of sugar export from leaves observed in plants that overexpress an invertase in the cell wall (von Schaewen et al., 1990;Heineke et al., 1992). For at least the past 40 years, there has been great interest in characterizing sucrose transport at the plasma membrane. For sugarcane, the kinetics of active sucrose transport were described by Bieleski as early as 1960. As is the case for most transport processes, the uptake kinetics are complex and consist of multiple components. The problem in interpretation of these kinetics is that the materials used, such as leaf discs or plasma membrane vesicles, are not homogenous and represent multiple cell types. It is therefore not possible to judge whether a single protein, several proteins in different cells, or several proteins within a single cell are responsible for the complex kinetics. Theoretically, a minimum of two distinct transport activities are required for phloem loading-one responsible for the export of sucrose and other solutes into the cell wall in the vicinity of the phloem and a second one responsible for the import into the veins. 1. Biochemical Studies

During the past few decades, sucrose transport activities have been studied in whole tissues, protoplasts, and, since the development of efficient meth-

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P '

P 0

FIG. 1 Predicted topology for StSUT1. Amino acid residues that are identical in all eight of the SUTl transporters (see Table I) are shown in bold, other positions are shown in shadow. The single charged amino acid (D152)predicted to be in the membrane (span 4) is circled. The conserved sequences conforming to the [RK]X2_3[RK] motif following membrane spans 2 and 7 (see text) are circled.

ods for purification, in plasma membrane vesicles (Larsson, 1985). Transport activities were mainly detected in leaves; however, some activity was also found in sink tissues (Delrot, 1989). Normally the uptake consists of at least three kinetic components, e.g., in V.faba two saturable (low and high affinity) and one linear component were identified (Delrot and Bonnemain, 1981).The best studied carrier systems correspond to the saturable component and are proton symporters (Giaquinta, 1977,1983; Komor et al., 1977; Delrot, 1981;Buckhout, 1989;Bush, 1989,1993;Lemoine and Delrot, 1989; Williams et al., 1992). The transport is active and has been described as a sucrose proton cotransport with a 1:l stoichiometry (Bush, 1990). The substrate specificity is high and is restricted to sucrose and phenylglucosides (Fondy and Geiger, 1977;Hitz et al., 1986;Lucas and Madore, 1988;Delrot,

N

L

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1989).the activity is sensitive to thiol-group modifying agents and to diethylpyrocarbonate. Comparison of the transport activity in developing versus mature leaves has shown that the sucrose/proton cotransport is differentially active and develops during maturation of the leaf (Lemoine et af., 1992). In sugar beet, a protein of 42 kDa was identified as a potential candidate for the sucrose transporter and was partially purified (Li et af., 1992). An antiserum directed against the 42-kDa fraction from the plasma membrane was able to specifically inhibit sucrose transport in vesicles (Gallet et af., 1992). However, attempts to isolate the carrier gene by screening cDNAexpression libraries with this antiserum have been unsuccessful (W. B. Frommer, R. Lemoine, and S. Delrot, unpublished results). 2. Identification of Transporter Genes Complementation of yeast mutants has proven to be an excellent tool to identify heterologous genes based on their function, especially in the case of transporters (Frommer and Ninnemann, 1995).At first sight, complementation of a Saccharomyces cerevisiae mutant deficient in sucrose uptake appears to be unsuitable for isolating plant sucrose transporters due to the capability of budding yeast to metabolize sucrose extracellularly. However, a modified strain deficient in secreted invertase, but able to metabolize imported sucrose due to expression of a sucrose-cleaving activity, was used as an artificial complementation system to isolate sucrose transporter cDNAs from spinach and potato (Riesmeier et af., 1992,1993). Due to the high levels of expression in leaves and the high conservation at the DNA level, heterologous screening has proven to be a useful tool to isolate respective genes from other species, e.g., tobacco, tomato, Arabidopsis, and Plantago (Gahrtz et af., 1994; Sauer and Stolz, 1994;Burkle et al., 1997; Weig and Komor, 1996; H. Buschmann, B. Hirner, J. M. Ward and W. B. Frommer, unpublishd results). This approach has also identified the presence of more than one carrier within a single species. Detailed sequence comparisons and phlogenetic studies show that, in most cases, gene amplifications must be relatively old, thus predicting specific functions for the individual members of the family (Rentsch et af., 1997). 3. Structure of Transporter Proteins

The sucrose transporter (SUT) genes encode highly hydrophobic proteins. In Table I eight homologs of SUTl from different plant species are listed. The SUTl proteins have a calculated molecular mass of around 55 kDa, whereas the apparent molecular mass on SDS-polyacrylamide gels was found to be approximately 47 kDa (Kuhn et af., 1997). The number and position of membrane spanning domains for the SUTl family of transport-

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TABLE I Family of SUT1 Sucrose Transporters in Plants

Identity Name

Source

(%)

StSUTl LeSUTl NtSUTl SoSUTl BvSUTl PmSUC2 RcSCRl AtSUC2

potato tomato tobacco spinach beet Plantago Ricinus Arabidopsis

100 94.7 85.8 65.9 65.1 64.7 64.5 64.1

Length Accession (amino acids) Number 516aa 511aa 507aa 525aa 523aa 510aa 533aa 512aa

542087 575299 1076644 549000 1076257 1086253 542020 481131

Reference Riesmeier et al., 1993 Frommer et al., unpublished Frommer et al., unpublished Riesmeier et al., 1992 Frommer et al., unpublished Gahrtz et al., 1994 Weig et ab, 1996 Sauer and Stolz, 1994

ers was evaluated using the TMAP program (Person and Argos, 1994). Alignments were first constructed using the Clustal algorithm for all SUTl sequences currently available in the databases (references and accession numbers are listed in Table I). Twelve membrane spanning domains were predicted by the TMAP program using this sequence alignment. The TMpred program (Hofmann and Stoffel, 1993) was then used to predict the beginning and end of transmembrane segments of SUTl from potato. The resulting topological model for StSUTl is presented in Fig. 1. Amino acid residues that were found to be identical (45% of amino acid positions) in all eight of the sucrose transporters are presented in bold lettering. The majority of conserved positions were found within the putative membrane spanning domains. A single charged amio acid (D152) is found within the membrane (transmembrane span 4) located at the center of the sequence GFWILDVANN which is fully conserved within all SUTl homologs. The suggested orientation of the protein in the membrane (Fig. 1) is based on two loop regions following membrane spans 2 and 7 where homology to the [RK]X2-3 [RK] motif (circled in Fig. 1) of bacterial H+-coupled sugar transporters is found (Henderson, 1990; Marger and Saier, 1993; Prive and Kaback, 1996). This supports the hypothesis that the SUTl structure, consisting of 12 membrane-spanning domains, arose from an ancestral gene duplication event. Other sequences that are characteristic of the bacterial H+-coupled sugar transporters are missing or are weakly conserved in SUTl. The only homology with a PESPR sequence following the 6th transmembrane span of bacterial transporters (Henderson, 1990) is a conserved E in this position in SUT1. The hallmark PETK motif following the 12th transmembrane domain in bacterial sugar transporters (Henderson, 1990) is not found in SUTl and only two conserved proline residues are found at this location. Although no extensive sequence homologies were found

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53

to the prototype of sugar transporting proteins, the lactose permease from Escherichia coli, the two subfamilies of sugar transporters seem to be related not only in being disaccharide transporters but also in their structure with 12 membrane-spanning domains separated by a large central loop (Marger and Saier, 1993). Further dispersed homology in the N-terminal regions between SUT proteins and bacterial sugar:cation cotransporters, especially the E. cofi melibiose permease were recently reported (Naderi and Saier, 1996). The general topology of members of the MFS superfamily, especially lactose permease, has been determined using a variety of techniques. The general topology should, in principle, hold true for SUT1. Specific features of the model presented in Fig. 1 would require actual experimentation to verify. For example, whether the N- and C-termini are in the cytoplasm of in the apoplastic space needs to be tested. Mutational studies on the sucrose transporter in conjunction with the yeast expression system represent efficient tools to analyze which regions of the proteins are important for function and which amino acids are involved in substrate recognition. The model presented here indicates conserved regions which could be targets for directed mutagenesis. Purification of the protein from yeast together with crystallization studies might represent a way to obtain a detailed model of the protein and its functions (Cyrklaff et al., 1995; Stolz et al., 1995; Deisenhofer et al., 1995; Ostermeier et al., 1995; Prive and Kaback, 1996). 4. Biochemistry and Biophysics of SUT1-Mediated Transport The yeast expression system has also allowed the analysis of the biochemical properties of the transporters because the uptake of radiolabeled sucrose can be measured directly in the presence of competitors and inhibitors. The properties of SUTl from spinach and potato are very similar regarding the inhibition of transport by protonophores, thiol-group modifying agents, and DEPC. The K,,, value of the sucrose carriers was estimated to be approximately 0.3-1 mM, and the specificity toward other sugars is consistent with the data described for the sucrose carrier as determined in pfanfa (for reviews, cf. Delrot, 1989; Bush, 1993). Inhibitor studies indicated that a proton gradient is required to allow sucrose transport into yeast cells. As a typical example from this family, the potato StSUTl sucrose transporter has been characterized electrophysiologically after expession in Xenopus faevis oocytes by measuring carrier currents using two-electrode voltage clamp (Boorer et af., 1996). The biochemical properties were again similar to those obtained for StSUTl expressed in yeast and for sucrose uptake in leaf plasma membrane vesicles (Riesmeier et al., 1993; Lemoine et al., 1996). The stoichiometry of H+/sucrosetransport was approximately 1: 1, confirming values obtained in plasma membrane vesicles from sugar beet

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JOHN M. WARD ET AL.

leaves (Bush, 1990;Slone et al., 1991).SUTl thus corresponds to the protonophore-sensitive high-affinity component of sucrose uptake kinetics measured in plants.

5. Low-Affinity Transport Recently, a potential candidate responsible for the low-affinity uptake system was identified. Using photoaffinity labeling techniques, a sucrose binding protein (SBP) has been isolated from soybean cotyledons (Ripp er al., 1988; Warmbrodt et al., 1989; Grimes et al., 1992). Because the protein is localized in the phloem, the authors speculate that this protein might be involved in sucrose transport. Interestingly, the SBP is able to mediate sucrose transport on its own in the yeast expression system described previously (Overvoorde et al., 1996). The biochemical properties may indicate that SBP encodes the linear component of sucrose uptake observed in many transport studies. Heteromeric structures have been reported for mammalian amino acid transporters (Bertran et al., 1992). Therefore, SBP may have a regulatory role in phloem sucrose transport through interaction with SUTl. Consistent with this, SBP was colocalized with sucrose/H+ cotransporters in the plasma membrane of V.faba transfer cells (companion cells) in developing seeds (Harrington et al., 1997). Coexpression of SUTl and SBP in yeast or in oocytes might be a suitable approach to test this hypothesis.

6. EMlux of Sucrose into the Apoplast Before sucrose can be loaded into the phloem, an efflux into the apoplast is required either from mesophyll cells or directly at the interface to the SE/CC complex. Mechanistically, the release carrier could be a facilitator or an antiporter. An efflux system was characterized by Delrot (1989) and Laloi et al. (1993). The characteristics obtained argue for the existence of a passive sucrose efflux system that is different from sucrose/H+ symport responsible for phloem loading. A similar situation regarding efflux exists in unloading processes in which sink cells take up sugars from the apoplast. Also in this case the existence of efflux carriers has been postulated (Patrick et al., 1995; X. D. Wang et al., 1995; McDonald et al., 1996). It is still a matter of debate whether these carriers function as facilitators or as sucrose proton antiporters. To date, none of these carriers has been identified at the molecular level (Patrick and Offler, 1995). 7. Targeting of Transporters to the Plasma Membrane

Very little is known about how the sucrose transporters are targeted to the plasma membrane. In analogy to membrane protein targeting in yeast, one

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might assume that it involves import into the endoplasmic reticulum (ER) membrane and transfer via the Golgi apparatus and secretory vesicles to the destination at the plasma membrane. Because several genes for plasma membrane carriers complement yeast mutants, the proteins must either be recognized correctly by the yeast targeting machinery or they reach their destination via unspecific mechanisms (Frommer and Ninnemann, 1995). Possibly, only a fraction of the protein reaching the plasma membrane is sufficient for function. Similar assumptions can be made in case of the Xenopus oocyre expression system. Immunogold labeling and GFP fusions in combination with yeast mutants defective in targeting should help answer these questions. In the case of amino acid transport, the amino acid permeases AAP1-6 and ProT, which are unrelated to their yeast counterparts at the sequence level, d o not seem to be affected by the shr3 mutation that blocks correct targeting of endogenous amino acid permeases (Rentsch er af., 1996).

8. Regulation of Transporters

Very little is known about the regulation of sugar transport in either source or sink tissue. Diurnal fluctuation of sugar export has been observed in a number of plant species. In potato, the majority of export (approximately 70%) occurs during a 12-hr day (Heineke et af., 1994). Changes in the phasing of diurnal export were found as compensatory mechanisms either when export of triosephosphates from chloroplasts was blocked by antisense inhibition of the triose phosphate translocator (TPT) (Riesmeier et al., 1992) or when starch biosynthesis was decreased by ADP glucose pyrophosphorylase (AGPase) antisense inhibition (Leidreiter et af., 1995). Apparently, this cycling correlates with the maximal rates of photosynthesis and sucrose biosynthesis. Therefore, it currently remains unclear whether the fluctuations are a property of the biochemical reaction cycles involved or of the transport system itself. The export from chloroplasts might represent one possible determinant because it could be shown that the expression of the TPT gene of potato is light dependent (Schulz et af., 1993). The expression of the sucrose transporter SUTl is also diurnally regulated at both the mRNA and protein levels, indicating that both mRNA and protein turnover are regulated and contribute to determine sucrose transport activity level. Inhibition studies using cycloheximide show that the half-time of protein turnover is in the range of a few hours (Kiihn et af., 1997). This high turnover rate points to a specific mechanism involved in controlling the number of active carriers in the plasma membrane and may suggest involvement of endocytosis as in the case of mammalian glucose transporters or yeast amino acid permeases (Hein et af., 1995; Thorens, 1996).

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It could be shown that the steady-state level of SUTl mRNA is under developmental and hormonal control (Harms et al., 1994). Active sucrose transport activity in leaves of sugar beet develops upon maturation of the leaves (Lemoine et al., 1992). As might have been expected, the expression profile of the sucrose transporter mRNAs from potato and Arabidopsis follow the sink-to-source transition (Riesmeier el al., 1993; Truernit and Sauer, 1995). Further mechanisms might also play a role in the regulation of sucrose transport. Various physical and chemical treatments are capable of inhibiting phloem loading. Typical examples are anaerobiosis, which probably acts at the level of ATP supply for the Hf-ATPase, whereas uncouplers directly inhibit energization (Sowonick et al., 1974; Giaquinta, 1977; Servaites et al., 1979; Thorpe et al., 1979; Maynard and Lucas, 1982). Hormones have multiple effects on plant growth and development. One possible mechanism could be through stimulation or inhibition of transport. Hormones such as auxins and cytokinins have long been known to increase the rate of phloem loading (Lepp and Peel, 1970; Patrick, 1976). Fusicoccin or auxins can rapidly promote sucrose uptake, whereas abscisic acid acts as an inhibitor (Malek and Baker, 1978; Sturgis and Rubery, 1982; Vreugdenhil, 1983). In broad bean, a direct promotion of assimilate export by the application of gibberellic acid was reported (Aloni et al., 1986). Phloem loading in isolated bundles of celery seems to be directly affected by gibberellic acid and auxins (Daie et al., 1986). However, a principal problem with such studies is the difficulty to differentiate between effects operating at the different points of regulation. SUTl mRNA and protein levels can be induced by the addition of auxins and cytokinins to detached leaves (C. KLihn and W. B. Frommer, unpublished results), whereas no such effects was observed on the two major H+-ATPase genes expressed in potato leaves (Harms et al., 1994). Furthermore, a number of environmental factors have been found that also affect phloem loading. Metals in high concentrations reduce sugar transport (Peterson, 1979; Samarkoon and Rauser, 1979; Rauser and Samarakoon, 1980; Delrot, 1989). Also, deficiencies in phosphorous, potassium, or magnesium lead to a block of sucrose export from leaves (Cakmak et al., 1994). SO2 can also strongly inhibit phloem loading possibly by interfering directly with the sucrose transport due to its sensitivity to sulfhydrylmodifying agents (Teh and Swanson, 1982;Minchin and Gould, 1986;Maurousset et al., 1992). C. Localization of Transporter SUTl in the Phloem

Several possibilities exist as to where the sucrose transporter SUTl might be localized. The primary assumption is apparently that the carrier is in-

FIG. 2 (A) Immunolocalization of the sucrose transporter StSUTl in sieve elements (stem cross sections of potato) and costaining of nuclei with DAPI (as described by Kuhn et al., 1997). (B) Bright field image of the same section shown in (A). Bar = 15 pm.

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volved in loading and thus is present at the plasma membrane of phloem cells. RNA in situ hybridization experiments actually demonstrated that SUTl is phloem associated (Riesmeier et af., 1993). The data regarding phloem localization in leaves and stems are supported by an analysis of tomato SUT promoter-reporter gene fusions and of fusions with the analogous transporter gene SUC2 from Arabidopsis (B. Hirner and W. B. Frommer, unpublished results; Truernit and Sauer, 1995). SUTl or SUC2 (as the Arubidopsis thafiana homolog was named) thus might play a role not only in phloem loading but also in retrieval of sucrose leaking from the conduits along the transloation pathway. Low levels of SUTl gene expression were also found in sink tissues, where the carriers might be involved in unloading processes. As mentioned previously, the phloem itself consists of multiple cell types. Furthermore, mesophyll cells might also utilize sucrose retrieval functions. The resolution of the in situ hybridization and promoter GUS studies were insufficient to determine the expression at the cellular level (Riesmeier et af., 1993). Therefore, immunolocalization studies were required. These studies were carried out in parallel by two groups and covered five species: potato, tomato, tobacco, Plantago, and Arabidopsis (Kuhn et af., 1997). In Plantago and Arubidopsis, immunofluorescence with specific antibodies detected the SUTl homologs named SUC2 in companion cells (Stadler et af., 1995; Stadler and Sauer, 1996). In contrast, immunolocalization using immunofluorescence and silver-enhanced immunogold staining detected SUTl in plasma membranes of enucleate (SEs) of tobacco, potato, and tomato (Fig. 2; Kuhn et af., 1997). This localization coincides with the osmotic gradient observed between SEs and CCs (Lackney and Sjolund, 1991).SUTl was found in both adaxial (inner) and abaxial (external) phloem in leaves and stems, respectively. The protein was found not only in minor veins of source leaves but also in stems, petioles, and roots. In situ hybridization at the EM level also showed that SUTl mRNA localizes mainly to SEs and is preferentially associated with the orifices of the plasmodesmata. To determine where SUTl mRNA is produced, antisense inhibition experiments using a CC-specific promoter ( R o f C )were performed. The inhibition led to strong phenotypic effects in the transgenic plants that are due to inhibition of sucrose export from leaves (Kuhn et af., 1996). This and the localization of the mRNA indicate tha transcription of SUTl mRNA occurs in CCs. Together with the turnover of mRNA and protein, this provides strong evidence for targeting of plant endogenous mRNA and potentially SUTl protein through phloem plasmodesmata and for sucrose loading at the plasma membrane of SEs. Two potential pathways exist for SUTl biosynthesis. The RNA may be guided along the cytoskeleton through the PPU and translated in SE. Alternatively, translation already occurs in CCs and the protein is guided to the plasmodesmal orifices and enters the SEs either as part of the E R or

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as part of the plasma membrane, both of which are continuous through the plasmodesmata (Overall and Blackman, 1996; Fig. 3). Interestingly SUTl protein is present in developing SEs that still contained a nucleus (Kuhn et al., 1997). It is not known if SUTZ transcription occurs in SEs at this early stage in development and, conversely, we do not know at which point in development SUTZ mRNA or protein import into SEs from the CCs begins. However, this finding fits nicely with reports that these cells may already be active in sugar transports (Schumacher, 1933; Lackney and Sjolund, 1991). Various groups have identified proteins in the phloem sap (Fisher et al., 1992; Schobert et al., 1995, Nakamura et al., 1993, Bostwick et al., 1992). Some proteins were found to be specific for sieve elements Wang et al., 1995). At the plasma membrane of sieve elements, large numbers of particles probably representing membrane proteins were detected (Sjolund

(a.

Inn, -

cc

RNA

binding

protein

Golgi

apparatus

PM

nucleus

FIG. 3 Potential routes of sucrose transporter mRNA and protein. Pathway on the left: mRNA produced in the companion cell nucleus is transported along the cytoskeleton via RNA binding proteins to the plasmodesmata, mRNA trafficks via a specific target mechanism and must be translated in the sieve elements. Pathway on the right: the protein is made at the ER in companion cells, traverses the golgi, and is targeted by vesicles along the cytoskeleton to the plasmodesmata. The protein trafficks via a specific target mechanism to the seive element plasma membrane.

59

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and Shih, 1983). The function of these proteins in the phloem is unknown; their biosynthetic pathway, whether or not they originate in the CCs, is unclear. In addition, the stability of these proteins in the phloem should be ascertained and will provide much needed information concerning general protein turnover rates in the phloem sap (and provide a clue concerning protein import rates). Transport of mRNA between cells may seem to be a very unusual proposal. However, polarized transport of mRNA within and even between cells has been observed during Drosophilu oogenesis and in neurons (St. Johnston, 1995). Trafficking of RNA is also involved in viral transport in phloem facilitated by movement proteins and in transport of RNA for the transcription factor KnZ (Lucas et ul., 1995). In several cases both protein and RNA are transported. It remains to be shown whether, in addition to SUTZ mRNA, the protein is also transported. If SUTZ mRNA traffics from CCs to SEs, it must be translated in SEs.However, the majority of ribosomes are lost during SE maturation. Nevertheless, SEs are able to synthesize proteins (Neumann and Wollgiehn, 1964). The purification of SEs from callus cells may represent a way to directly determine translation of specific mRNAs Wang et al., 1995). It will therefore be essential to reevaluate the paradigm of the absence of ribosomes in SEs using, e.g., immunolabeling techniques, because direct proof of SUTZ synthesis in SEs is currently not possible. An important question associated with the localization of SUTZ is how SUTl protein can be targeted to the plasma membrane of sieve elements if dictyosomes are lacking. The high turnover of SUTl indicates that specific mechanisms are involved requiring possibly ubiquitination and intracellular degradation as found for several yeast plasma membrane transport proteins (Hein et ul., 1995). Recently, ubiquitin was found to be one of the major proteinaceous components of phloem sap (Schobert et ul., 1995). It will also be interesting to understand how this system can function in enucleate cells. The differences in SUTl localization observed in Arubidopsis and Pluntugo compared with tomato, potato, and tobacco may be due to differences in loading mechanisms. Alternatively, more than one carrier might be present in all plants analyzed and sucrose loading may take place in a stepwise manner involving different carriers in the CCs (SUC2) and SEs (SUT) as suggested by physiological analyses (Roeckl, 1949). In tomato, related genes that have been isolated could serve such a function (B. Hirner, H. Buschmann, J. M. Ward, and W. B. Frommer, unpublished results).

(a.

D. Models for Active Phloem Loading in Different Plant Species Geiger (1975) suggested that the release of assimilates from photosynthetically active cells occurs in the vicinity of the phloem. This hypothesis seems

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attractive for two reasons: (i) Export from all cells on the way from synthesis to the site of loading would be in the opposite direction of the transpiration stream, and (ii) the ubiquitous presence of large amounts of sugar in the apoplast could serve as a substrate for the growth of microorganisms. Despite the high concentrations of sucrose in the mesophyll(20-200 m M ) and the phloem (500-1500 mM), only very low concentrations of sugars were found in the apoplast (0.07-5 mM; Ntsika and Delrot, 1986; Delrot, 1989). The reason for this could be that apoplastic transport is restricted to the interface between the phloem parenchyma and the companion cells and that otherwise sugar moves along the symplast. This is supported by the finding of a steep osmotic gradient at the boundary of the conducting complex (as reviewed in Delrot, 1989). The localization clearly shows that in several species the final step occurs directly at the SE/CC complex. An epitope-tagged plasma membrane proton pump (Ht-ATPase) was immunocytologically localized in phloem companion cells. The authors suggest that sucrose must be taken up either into CCs or SEs (DeWitt and Sussman, 1995). Thus, in Arabidopsis, SUC2 and the Ht-ATPase colocalize in companion cells (Stadler and Sauer, 1996; DeWitt et al., 1996). In potato the localization of the ATPase has not been determined. Thus, different models for energization of sucrose loading into sieve elements can be envisaged (Fig. 4). One might speculate that the companion cell is electri-

JT 1

Solanaceae

Arabidopsis, Plantago type 1 plants

type 2 plants

FIG. 4 Three hypothetical models for phloem loading: (A) apoplastic in type 1 plants by SLIT1 at the SE plasma membrane, (B) apoplastic in type 1 plants by SUC2 at the CC plasma membrane, and (C) symplastic loading using the trap in type 2 plants via plasodesmata.

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cally coupled to the sieve elements through the plasmodesmata and that the membrane potential and the proton gradient generated by the H+ATPase located either at the companion cell or at the sieve element should be able to drive the transport against a cocnentration gradient across the common outer membrane (Fig. 5).

E. Evidence for the in Vim Function of SUTl Transporters The best way to study the actual function of a protein is the analysis of mutants. One very elegant approach was used by screening T-DNA tagged mutants of Arabidopsis by PCR with gene-specific and T-DNA primers (Krysan et al., 1996). This allowed to identify several H+/ATPase mutants. However, so far no sucrose transport mutants have been described. One possibility is that such mutants could be lethal, as will be seen in the next paragraph. To create plants with reduced sucrose transport activity, potato plants were transformed with the StSUTl gene in antisense orientation (Riesmeier et al., 1994; Kiihn et al., 1996). If sucrose transport mediated by this transporter is essential for phloem loading, a reduction in transport activity should affect carbon partitioning and photosynthesis. As expected, SUTl antisense plants show drastic phenotypic effects. The plants display retarded growth and the leaves are crinkled, show local bleaching, and accumulate anthocyanins. Development of the phenotype depends on light period and intensity (Kiihn et al., 1996). An analysis of metabolites shows a 5- to 10fold increase in leaf sucrose and starch content and an even higher increase in hexoses. A similar accumulation of soluble carbohydrates was found when petioles of potato leaves were cold-girdled, a treatment supposed to block phloem translocation (Krapp et al., 1993). Enhanced partitioning into insoluble carbohydrates was also found in a number of studies trying to block the export by heat girdling (see Grusak et aL, 1990, and references therein). Efflux measurements with excised leaves from the antisense plants indicate a strong reduction in phloem transport. The reduced efflux strongly affects the supply of sink organs with sucrose because the plants have a reduced root system and reduced tuber yield. The similarities to the phenotype of transgenic potato plants overexpressing a yeast invertase in the cell wall of leaves are striking (Heineke et al., 1992; Riesmeier et al., 1994). Comparable effects were observed in potato plants in which SUTl was expressed in antisense orientation under control of the companion cellspecific RolC promoter (Kiihn et al., 1996). It was, however, not possible to estimate the control coefficient of SUTl because low levels of SUT expression presumably in mesophyll cells masked the actual amount of SUTl protein and sucrose transport activity in the phloem (Lemoine et al.,

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Companion Cell

Sieve Element

A

H+

SUCROSE

FIG. 5 Consequences of different cellular localizations of H+lsucrose cotransporters and corresponding H+/ATPasesfor the energization of sucrose loading of the phloem: (A) colocalization in companion cells as the simplest mode and subsequent symplastic movement of sucrose through deltoid plasmodesmata, (B) colocalization in sieve elements requiring supply of ATP in sieve elements (as observed by Kluge et al., 1970), and (C) differential localization of the ATPase in companion cells and the H+/sucrosecotransporters in sieve elements requiring diffusion of protons from the ATPase to the transporter and/or electric coupling of both cell types to supply electron motive force.

1996). Due to the high amount of biomass transferred to potato tubers, loading in Solunum tuberosum might represent a special case. The effect of an antisense inhibition was therefore analyzed in tobacco, in which the

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dry matter in seeds in relation to that of leaves is at least 10-fold lower compared to the ratio for tubers and leaves in potato. Also in tobacco (minor vein configuration type IIa), inhibition of SUTl led to dramatic growth retardation and accumulation of carbohydrates in leaves (Biirkle etal., 1997).Thus, apoplastic loading carriers seem to be generally important components at least in solanaceous species. The effects observed are there fore in agreement with expected results that SUTl gene expression in companion cells is essential for phloem loading. Apparently, no alternative pathway is present in potato that can compensate for the lack of SUTl molecules. Of course, sucrose has to be imported into sink tissues such as root cells, pollen, and seeds. SUTl/SUC2 expression has also been found in these tissues (Riesmeier et al., 1993; Truernit and Sauer, 1995). However, the function of phloem-associated sucrose/H+ symporters in sink tissues remains unclear. In several plant species, such as tomato, Arabidopsis, Plantago, and Vicia, additional sucrose transporter genes have been identified (Gahrtz et al., 1996). Several of these genedproteins show highly specific expression patterns. For example, in Plantago, PmSUCl is expressed in young ovules (Gahrtz et al., 1996). In Vicia seeds, cross-hybridization with sucrose transport genes could be detected in transfer cells of cotyledons (Harrington et al., 1997).

111. Conclusions Molecular approaches have provided the first hints concerning the mechanism of sucrose loading into the phloem. However, many questions, such as the role of plasmodesmata in phloem loading and unloading, have not been addressed at the molecular level. In addition, it is likely that another class of transporter, important for sucrose efflux from the phloem in sink tissue and also having a role in phloem loading, as discussed in this review, remain to be discovered. The availability of genes for members of the SUT family as well as phloem sap-specific proteins provides the tools to further explore and understand the mechanism and regulation of long-distance transport in plants.

Acknowledgments We are very much indebted to Alexander Schulz (Kiel), V. R. Franceschi (Pullman), and Remi Lemoine (Poitiers) for the excellent collaboration. We also thank V. Ulle-Schneider for preparation of the figures. This work was made possible by grants to WBF on “Saccharose Transport” from BMBF (1992-1996).

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Delrot, S. (1981). Proton fluxes associated with sugar uptake in Vicia faba leaf tissue. Plant Physiol. 68, 706-71 1. Delrot, S. (1989). Phloem loading. In “Transport of Photoassimilates” (D. A. Baker and J. A. Milburn, eds.), pp. 167-205. Longman Scientific, London. Delrot, S., and Bonnemain, J. L. (1981). Involvement of protons as a substrate for the sucrose carrier during phloem loading in Vicia faba leaves. Plant Physiol. 67, 560-564. DeWitt, N. D., and Sussman, M. R. (1995). Immunocytological localization of an epitopetagged plasma membrane proton pump (H+-ATPase) in phloem companion cells. Plant Cell 7, 2053-2067. DeWitt, N. D., Hong, B., Sussman, M. R., and Harper, J. F. (1996). Targeting of two Arabidopsis H+-ATPase isoforms to the plasma membrane. Plant Physiol. 112, 833-844. Epel, B. L. (1994). Plasmodesmata: Composition, structure and trafficking. Plant Mol. Biol. 26,1343-1356. Epel, B. L., Van Lent, J. W. M., Cohen, L., Kotlizky, G., Katz, A., and Yahalom, A. (1996). A 41 kDa protein isolated from maize mesocotyl cell walls immunolocalizes to plasmodesmata. Protoplasma 191, 70-78. Fisher, D. B., Wu, Y., and Ku, M. S. B. (1992). Turnover of soluble proteins in the wheat sieve tube. Plant Physiol. 100, 1433-1441. Fishman, G. I., Gao, Y., Hertzberg, E. L., and Spray, D. C. (1995). Reversible intercellular coupling by regulated expression of a gap junction channel gene. Cell Adhes. Commun. 3,353-365. Fondy, B. R., and Geiger, D. R. (1977). Sugar selectivity and other characteristics of phloem loading in Beta vulgaris L. Plant Physiol. 59, 953-960. Fritz, E., Evert, R. F., and Heyser, W. (1983). Microautoradiographic studies of phloem loading and transport in the leaf of Zea mays L. Planta 159,193-206. Frommer, W. B., and Ninnemann, 0. (1995). Heterologous expression of genes in bacterial, fungal, animal, and plant cells. Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 419-444. Gahrtz, M., Stolz, J., and Sauer, N. (1994). A phloem-specific sucrose-H’ symporter from Plantago major L. supports the model of apoplastic phloem loading. Plant J. 6, 697-706. Gahrtz, M., Schmelzer, E., Stolz, J., and Sauer, N. (1996). Expression of the PmSUCl sucrose carrier gene from Plantago major L. is induced during seed development. Plant J. 9,93-100. Gallet, O., Lemoine, R., Gaillard, C., Larsson, C., and Delrot, S. (1992). Selective inhibition of active uptake of sucrose into plasma membrane vesicles by polyclonal antisera directed against a 42 kD plasma membrane polypeptide. Plant Physiol. 98, 17-23. Gamalei, Y. V. (1986). Characteristics of phloem loading in woody and herbaceous plants. Sov. Plant Physiol. (Engl. Transl.) 32, 656-665. Gamalei, Y. V.. van Bel, A. J. E., Pakhomova, M. V., and Sjutkina, A. V. (1994). Effects of temperature on the conformation of the endoplasmic reticulum and on starch accumulation in leaves with symplasmic minor vein configuration. Planta 194, 443-453. Geiger, D. R. (1975). Phloem loading. In “Transport in Plants” (M. H. Zimmerman and J. A. Milburn, eds.), Encycl. Plant Physiol., New Ser., Vol. 1, pp. 395-431. Springer, Berlin. Giaquinta, R. T. (1977). Possible role of pH gradient and membrane ATPase in the loading of sucrose into the sieve tubes. Nature (London)267, 369-370. Giaquinta, R. T. (1983). Phloem loading of sucrose. Annu. Rev. Plant Physiol. 34,347-387. Grimes, H. D., Overvoorde, P. J., Ripp, K., Franceschi, V. R., and Hitz, W. D. (1992). A 62kD sucrose binding protein is expressed and localized in tissues actively engaged in sucrose transport. Plant Cell 4, 1561-1574. Grusak, M. A., Delrot, S., and Ntsika, G. (1990). Short-term effects of heat-girdles on source leaves of Vicia faba: Analysis of phloem loading and carbon partitioning parameters. J. EXP. Bot. 41, 1371-1377. Harms, K., Wohner, R. V., Schulz, B., and Frommer, W. B. (1994). Regulation of two p-type Ht-ATPase genes from potato. Plant Mol. Biol. 26, 979-988.

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Interaction of Cytoskeletal Proteins with Membrane Lipids G. Isenberg* and V. Nigglit

* Biophysics Department E-22, Technical University of Munich, D-85747 Garching, Germany; and t Institute of Pathology, University of Bern, CH-3010 Bern, Switzerland

Rapid and significant progress has been made in understandinglipidlprotein interactions involving cytoskeletal components and the plasma membrane. Covalent and noncovalent lipid modifications of cytoskeletal proteins mediate their interaction with lipid bilayers. The application of biophysicaltechniques such as differential scanning colorimetry, neutron reflection, electron spin resonance, CD spectroscopy, nuclear magnetic resonance, and hydrophobic photolabeling,allow various folding stages of proteins during electrostatic adsorption and hydrophobic insertion into lipid bilayers to be analyzed. Reconstitutionof proteins into planar lipid films and liposomes help to understand the architecture of biological interfaces. During signaling events at plasma membrane interfaces, lipids are important for the regulation of catalytic protein functions. ProteinAipid interactions occur selectively and with a high degree of specificity and thus have to be considered as physiologically relevant processes with gaining impact on cell functions. KEY WORDS: Cytoskeleton, Plasma membrane, Protein-lipid interactions, Lipid modifications,Signal cascades, Biological interfaces, Model membranes.

I. Introduction Cells and tissues are embedded in a three-dimensional gel continuum of biopolymers ranging from the extracellular matrix via a cytoplasmic lattice to the nuclear matrix. A second system is a membrane continuum that spans itself through this gel reticulum from the nuclear envelope via the endoplasmic reticulum (ER) and the Golgi system to the plasma membrane. These two worlds, the one being primarily composed of lipids and the Inrernntbnal Review of Cytology, Vol. 178

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other being mainly constituted from proteins, superimpose and give rise to different cell compartments. Wherever the two systems make contact, they generate interfaces, which are complex in both architecture and functions. The literature is rich in studies of proteins that cross the hydrophobic lipid barrier, such as pore proteins, transporters, and receptors, but the detailed mechanism of binding, oligomerization, and transmembrane insertion has to be thoroughly investigated for each case of protein-lipid interaction. Investigation of the interaction of cytoskeletal components with membranes and in more detail with membrane lipids started late in the 1980s. This may in part be due to the peculiar situation that protein chemists hesitate to deal with lipids and vice versa. Second, most of the current methods had to be developed because no straightforward reaction kits were available and this holds true today. Most important, the progress in research on protein-lipid interactions at interfaces has been achieved by applying biophysical techniques involving Fourier-transformed infrared spectroscopy (FTIR), neutron reflection, electron spin resonance (ESR), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), and other techniques in combination with the more classical biochemical approach of protein purification and reconstitution. To efficiently apply these techniques, a common language between physicists and biologists had to be found, a process that also takes time. Significant progress has been made in studying protein-lipid interactions with simplified membrane models. Unilamellar, small (100 nm) and giant liposomes (2500-1000 nm) can be reproducibly generated by the extrudor technique. Low-scale two-dimensional lipid monolayers may now be produced with a miniaturized Langmuir technique (Wilhelmy system), saving precious ingredients needed for the experiments. Finally, coating on solid substrates offers a new technology for studying various layers of interfaces. Because biological membranes seem to be rather complex (Fig. l), model systems are indispensable for investigating (i) the reconstitution of membrane proteins, (ii) the individual way of lipid interaction, and (iii) the functioning of proteins in an oriented configuration dependent on a limited number of controllable parameters. There have been a small number of reviews about cytoskeleton-lipid binding over the past decade (Niggli and Burger, 1987; Isenberg, 1991; Janmey, 1994; Isenberg and Goldmann, 1995; Sackmann, 1995). This article summarizes the rapid progress in development and application of new investigation methods as well as the growing insights into the biological significance of cytoskeleton-lipid interactions.

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INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

Extracellular

FIG. 1 The plasma membrane: the complex trilayer architecture of this biological interface consists of fibrous extracellular matrix proteins, a lipid-protein bilayer, and intracellularly anchored cytoskeleton proteins. This scheme implies that interactions between the membrane components occur many-fold. Reprinted from Sackmann, Biological membranesarchitecture and function, 1995, pp. 4-63, with kind permission from Elsevier Science-NL, Sara Burgerhartstaat 25, 1055 KV Amsterdam, The Netherlands.

II. Lipids and Posttranslational Lipid Modifications By their nature, lipids are amphiphilic molecules with a high tendency of self-assembly into ordered structures. In aqueous solutions, this selfassembly results in the formation of vesicles or micelles and, at aidwater interfaces, in the spontaneous arrangement of lipid monolayers. The netto charge of a phospholipid is determined by the substitution of its polar head group. These charges are stable over a wide pH range (pH 2-10). Meanwhile, phospholipids can be synthesized with a purity 299%. Some of the most convenient lipids used for the formation of model membranes are shown in Fig. 2. L-a-Dipalmitoylphosphatidylcholine(DPPC) and L-a-dimyristoylphosphatidylcholine (DMPC) are neutral phospholipids, whereas (L-a-Dimyristoylphosphatidylglycerol(DMPG) and L-adimyristoylphosphatidylserine (DMPS) represent negatively charged phospholipids. Tensides as represented by C12E4, a molecule with 12 carbon atoms and four hydrophilic ethylene glycol residues, serve as model systems for Gibb’s monolayers. For directly monitoring pattern formation of lipid films, fluorescently labeled probes (e.g., Texas red sulfonyllabeled DPPE) in low quantities (50.1 mol%) are mixed with a lipid composition of choice. Alternatively, 7-chloro-4-nitrobenzeno-2-oxa-1,3diazole (NBD) or fluorescein-labeled lipid or proteins may be incorpo-

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0

HO

P

DMPG (667)

8 0

AA(312.5) HO

FIG. 2 Some of the most commonly amphiphilic lipids used for lipid monolayers and liposomes.

rated into the lipid membrane (Dietrich et al., 1993) to directly visualize changes in packing order and mobility of membrane constituents (Mohwald, 1990). Myristoylation and palmitoylation are the major covalent modifications of cytoskeletal proteins with long fatty acid chains (acylation) (Schmidt, 1989). Candidate for these types of modifications are the histidine-rich, pH-sensitive protein hisactophilin, MARCKS, and Dictyostelium talin, which has palmitic acid covalently bound (G. Gerisch, personal communication). Myristic acid consisting of 14 carbon atoms is amide linked to N-terminal-linked glycine residues by a cytosolic N-myristoyl transferase

77

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS CH,(CH2) &ONH-Gly,,).

Myristoylation Acylation Palmitoylation

:

Cys

,cys Isoprenylation

Glycosylphosphatidylinositol anchor (PIG-tail)

.......SerO). . .. .

0

- S - CIt -

(CH2),&CH,

- s -f-yq-y

OCH,

Protein

\

\

\

+Farnesyly 1-Geranylgeranyl-

-

0 II

C

- Eth - P - Glycan - Inos I

FIG. 3 Covalent lipid modifications that occur in cytoskeletal proteins (for details see

text).

(Fig. 3). Substitution of the methylene group by 0 or S atoms will result in a partial redistribution of the membrane-bound form to the cytoplasmic fraction. Palmitoylation occurs by a thioester binding of C-16 palmitic acid to cysteine residues via a membrane-bound palmitoyltransferase. It has been observed that palmitoylation occurs most readily at hydrophobic sites close to the membrane spanning region of proteins (Schmidt, 1989). However, otherwise cytoplasmic proteins, such as the spectrin-binding protein ankyrin (Staufenbiel, 1987), get palmitoylated. In contrast to methylation, which is a stable modification, palmitoylation has a rapid turnover, which may be of regulatory influence for the reversible insertion of proteins into lipid bilayers. Isoprenylation consists of either farnesol or geranylgeranyl linked to cysteine residues (Rilling et al., 1990). Binding of isoprenoid to proteins is highly dependent on the CAAX motif. It is interesting that the nuclear membrane skeleton comprising lamins seems to use a combination of CAAX farnesylation and a nuclear targeting signal (Holtz et al., 1989) to target the protein to the inside of the nuclear membrane. Finally, there exists the possible modification by glycosylphosphatidylinositol(GPI), the GPI anchor (Low, 1989; Ferguson and Williams, 1988). By substituting individual residues in the basic cascade of amide-linked ethanolamine phosphodiester-linked glycan phosphoinositol (PI), a wide spectrum of variations exhibiting different physical and biochemical properties becomes apparent. The actin-binding protein 5’-nucleotidase is linked by this type of anchor via the receptor to extracellular fibronectin and laminin (Stochaj et al., 1989).

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G. ISENBERG AND V. NlGGLl

111. Methods of Choice to Study Cytoskeleton-Lipid Interactions

A. Differential Scanning Calorimetry Our laboratory has introduced high-sensitivity differential scanning calorimetry (DSC) (Mason et al., 1981) to monitor the interaction of various actin-binding proteins with lipid mixtures (Heise et al., 1991). With rising temperature, lipids arranged in a bilayer undergo phase transitions from a crystalline phase (L,) to a gel phase (L p t ), to a ripple phase (PF)and finally to a liquid phase (La). These phase transitions are endothermic reactions that consume energy from the environment. In a reaction chamber the loss of heat during this reaction is compensated by an excess of specific heat. The energy difference is recorded as a function of temperature. Phase transitions appear as peaks in the “thermograms” (Fig. 4) and integration over the area below the DSC signal yields the difference in enthalpy. Suppression of the pretransition peak, the onset of chain melting, and a shift of the liquidus temperature. T1(the endpoint of main melting in the fluid phase) to higher temperatures indicates stable interactions of the protein with lipid bilayers. With increasing protein-lipid ratios, binding becomes saturable. DSC is a noninvasive technique that operates in a physiological temperature range between 10 and 35°C. It has been successfully applied to monitor the binding and insertion of talin (Heise et al., 1991; Goldmann et al., 1992), talin-vinculin complexes (Goldmann et al., 1992), and filamin into lipid bilayers (Tempe1 et al., 1994b).

B. Film Balance Technique Two-dimensional lipid monolayers spread onto an air-water interface are ideal model systems to study insertion and pattern formation of proteins within membranes, especially for those that electrostatically adsorb to the membrane surface and penetrate only into one-half of the hydrophobic lipid leaflets. Much progress has been achieved in developing a miniaturized Langmuir trough combined with fluorescent optics, allowing recording of domain formation and lateral diffusion processes of lipids and proteins by epifluorescent light microscopy (Mohwald, 1990; Heyn et al., 1991; Dietrich et al., 1993). Pressure-area diagrams of lipid mixtures independent of adsorbed or inserted proteins after injection into the subphase, controlled by lateral pressure, temperature, ionic strength, and pH, will then allow a precise documentation of the insertion behavior.

79

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

161412-

lo-/\

a

8b 6-

2d 0-

I

I

12

16

I

I

20

~

I

24

‘

28

I

‘

I

I

32

Temperature [ “C ]

301 f--

28

241

22

0.0 0.4 0.8 1.2 1.6 2.0 2.4 2 8 ProteiMLipid [molar ratio *10--3] FIG. 4 Typical “thermograms” obtained by DSC reflecting the phase transition of lipid layers on protein incorporation (here, filamin) with rising concentrations (a-d). The pretransition peak (approximately 17°C) gets suppressed and the liquidus temperature T I is shifted to higher temperatures (25-32°C). Electrostatic and hydrophobic interactions are saturable with increasing protein-lipid molar ratios (bottom).

C. Hydrophobic Photolabeling

Hydrophobic photolabeling is a powerful technique to site specifically mark various reactants of interest. Highly reactive lipid analogs, most conveniently photoactivatable phosphatidylcholine derivatives (e.g., [3H]PTPC/ 1l), have been synthesized (Brunner, 1993) that carry their photoreactive

80

G. ISENBERG AND V. NlGGLl

function in one of the fatty acid acyl chains. These labels are straightforwardly incorporated into any vesicle size with a lipid composition of choice (Fig, 5 ) . Upon photoactivation, a highly reactive carbene is generated, a powerful electrophile, resulting in an effective reaction with nucleophiles and C-H bonds in the immediate neighborhood of the label. Thus, in the case of a hydrophobic lipid label, only proteins that insert into at least one-half of the lipid membrane can react. According to Brunner (1993) a photoactivatable group should at least satisfy the following criteria: (i) It should be small enough to avoid any steric perturbation in the system, (ii) it should be stable in the dark and susceptible to light at a wavelength that does not influence photolytic damage of chemical bonds, and (iii) the reaction should result in stable products that then may be isolated, purified, and analyzed. In each case, labeling efficiency should be controlled and be compared with control proteins. In our hands, this method proved to be highly specific. The actin-binding proteins a-actinin (Niggli and Gimona,

\ kl hux

x-Q-gcF3

nucleophiles w-addition I insertion P u -u .I

products

FIG.5 Hydrophobic photolabeling: photolabeling groups and reactive intermediates as demBy a nucleophilic reaction the singlet caronstrated for 3-trifluoromethyl-3-phenyl-diazirine. bene efficiently inserts into C-H bonds. Because the lipid analog carries the photoactivatable group at the end of the hydrophobic acyl chain (O), only protein domains that insert into the hydrophobic inner lipid core of membranes get labeled.

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

81

1993), vinculin (Niggli et al., 1986), talin (Goldmann et al., 1992; Niggli et al., 1994), and filamin (Tempel et al., 1994b) have been investigated by applying hydrophobic lipid labeling. Each of these proteins was significantly labeled after reconstitution into lipid vesicles. Control proteins, such as IgG or bovine serum albumin, incorporate significantly less label than do these cytoskeletal proteins (Niggli et al., 1986; Niggli and Gimona, 1993). Moreover, in a mixture of two domains of talin, only one domain reacts selectively with the photolabel (Niggli et af., 1994), indicating selectivity and specificity of hydrophobic photolabeling. A disadvantage of the technique is the relatively low labeling efficiency obtained with carbene-generating reagents due to a predominant reaction of the highly reactive intermediates with phospholipids. This does not allow accurate determination of the fraction of protein inserting into the bilayer.

D. Computer-Assisted Structure Predictions Many of the cytoskeleton-associated proteins known to interact with lipid interfaces also exist in a soluble cytoplasmic form. They may only transiently interact with membranes. In such a situation, a two-step mechanism involving binding by electrostatic attraction and insertion after refolding into a suitable configuration would be of advantage for a stable interaction with the bilayer (Fig. 6). Protein folding into a membrane-compatible configuration would involve mainly ordered structures such as a-helices or p-strands. Surface binding to the polar lipid head groups can easily be achieved by exposing amphipatic a-helices. Insertion into one-half of the hydrophobic bilayer would require P-barrels or the formation of hydrophobic a-helices. When the primary amino acid sequence is known, the methods proposed by Kyte and Doolittle (1982) and Chou and Fasman (1978) allow the prediction of such helices with high precision (Jahnig, 1990).The hydrophobicity index can be calculated for each amino acid and the probability for membrane spanning derived from hydrophobicity plots (Eisenberg et al., 1984). A matrix method has been developed in our laboratory (Tempel et af., 1995) to discriminate between surface-seeking and transmembrane configurations of protein a-helices. By applying this structure prediction method, we have been able to designate potential lipid-binding motifs along the primary sequence of the actin-binding proteins vinculin and talin (Tempel et af., 1995) as well as filamin (Tempel et af., 1994a). The predicted lipid-binding domain in the 32-kDa fragment of chicken vinculin, aa 935974, matches very well with the lipid-binding motif (aa 913-970; see Table I) determined by cosedimentation and hydrophobic photolabeling using various GST fusion proteins of vinculin domains (R. P. Johnson, V. Niggli, P. Durrer, and S. W. Craig, unpublished).

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G. ISENBERG AND V. NlGGLl

BINDING

FIG. 6 Protein interactions with phospholipid bilayers. Binding of proteins at lipid interfaces frequently occur in a two-step mechanism: Adsorption is facilitated by electrostatic binding. Amphipatic a-helices may bind to the surface of negatively charged bilayers (A). Hydrophobic a-helices may insert or penetrate the bilayer with different degrees (B-D). Clusters of charged residues close to an a-helix may help to orient the protein within the bilayer.

E. Neutron Reflection Neutron reflection, which offers the unique possibility of scattering contrast variation (Penfold and Thomas, 1989; Bayerl et al., 1990; Brumm et al., 1994; Naumann et al., 1994), facilitates measurements of molecular mass density distributions in the x/y direction along a lipid monolayer surface as well as in the perpendicular z-axis across the layer. Neutron reflection is a noninvasive technique that allows the reliable determination of thickness and the scattering length densities of the hydrocarbon and head group regions of the lipid monolayers as a function of lateral pressure and phase state of the lipid layers (Naumann et al., 1996). Accordingly, this method has successfully been applied to monitor the binding of hisactophilin to lipid surfaces and the insertion behavior of the natural and genetically modified form of this protein in lipid monolayers (Behrisch et al., 1995; Naumann et al., 1996). Because neutron reflection in particular can be applied to study the group of nonintegral but membrane-associated cytoskeleton proteins, which couple to lipid layers by a combination of electrostatic and hydrophobic forces in the presence of fatty acid chains, investiga-

83

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

TABLE I Lipid-Binding Domains of Cytoskeletal Proteins Protein

Sequence

Sequences of proteins interacting specifically with phosphoinositides a-Actinin TAPY RNVNIQNFHLSWK" 168-184 (Fukami et al., 1996) Cofilin WAPECAPLKSKM" 104-115 (Yonezawa et al., 1991) 13-22 (Yu et al., 1992) KVFNDMKVRK~ Gelsolin KHVVPNEVVVQRLFQVKGRR" 150-169 (Janmey et al., 1992) 135-149 (Yu et al., 1992) KSGLKY KKGGVASGF Consensus sequence for gelsolin (Yu et (WR)XXXKX(K/R)(WR) al., 1992) Profilin 126-136 (Yu et al., 1992) KCYEMASHLRRb FSMDLRTKST" 83-92 (Sohn et al., 1995) Villin 133-147 (Janmey et al., 1992) YNVQRLLHVKGKKNVC' Consensus sequence for villin (Yu et al., (WR)XXXX(WR)(WR) 1992) Seauences of uroteins interacting with acidic phospholipids Caldesmon 626-658 TSAVVGNKAAKPAKPAASDLPVPAEGVRNIKSM 669-710 (Bogatcheva et al., 1994) GGTGTPNKETAGLKVGVSSRINEWLTKTPEGK SPAPKPSDL' Filamin 49-71 FTRWCNEHLKCVSKRIANLQTDL~~~~~ 131-155 (Tempel et al., 1994a) DWQSGRALGALVDSCAPGLCPDWDS"b.d MARCKS KKKKKRFSFKKSFKLSGFSFKKNKK' 150-174 (Taniguchi and Manenti, 1993) 126-155 (Nakaoka et al., 1995) SSPKAEDGAAPSPSSETPKKKKKRFSFKKS (sequences for electrostatic interaction) Vinculin 913-940 SSKGNDIIA AAKRMALLMAEMSRLVRGG" 940-970 (Johnson et al., 1997) GSGNKRALIQCAKDIAKASDEVTRLAKEVAKY RLVRGGSGNKRALIQCAKDIAKASDEVTRLA~ 935-978 1020-1040 (Tempel et al., 1995) KEVAKQCTDKRIR~ TEMLVHNAQNLMQSVKETVRE~ Talin 21-39 PSTMVY D ACRMIRERIPE A~ 287-342 GQMSEIEAKVRYVKLARSLKTYGVSFFLVKE 385-406 (Tempel et al., 1995) KMKGKNKLVPRLLGITKECVMRVDE~ GEQIAQLIAGYIDIILKKKKSKb Domain overlapping with actin-binding region. Predicted phospholipid binding site. Domain overlapping with calmodulin-binding region. Based on sequence analysis of the authors, this domain may interact with lipids mainly via electrostatic forces.

''

84

G. ISENBERG AND V. NlGGLl

tions are currently extended with respect to the focal adhesion proteins talin and vinculin (D. Hess, G. Isenberg, and E. Sackmann, unpublished).

F. Miscellaneous Techniques and Binding Constants Circular dichroism measurements are often combined with the previously mentioned techniques to relate the CD spectra with the secondary structure of cytoskeleton proteins and peptides prior to and upon binding to lipids. Examples include studies on hisactophilin (Hanakam et al., 1996b), profilin (Raghunathan et al., 1992), gelsolin (Yu et al., 1992), and talin (Isenberg et al., 1997a). Actual numbers of binding and affinity constants with respect to cytoskeleton protein-lipid interactions are still scarce. One study uses light scattering to determine the molar affinity of talin and vinculin for lipid membranes (Goldmann et al., 1995) with K,,, = 2.9 X lo6 M-' for talin and 3.3 X lo5 M-' for vinculin. These numbers compare well with data obtained by hydrophobic lipid photolabeling (Goldmann et al., 1992). Using tryptophan fluorescence spectroscopy, Czurylo et al. (1993) report an association constant of K,,, = 1.45 X lo5 M-' for caldesmon-lipid binding. In general, these lipid-binding data are comparable with values obtained for stable protein-protein interactions, e.g., the binding of actinbinding proteins to actin (Goldmann and Isenberg, 1993).

IV. Interactions of Actin and Associated Proteins with Membrane Lipids A. Actin-Lipid Interactions Interactions of pure G-actin and F-actin with charged and uncharged lipid bilayers have been reported (Laliberte and Gicquaud, 1988; St. Onge and Gicquaud, 1989, 1995). It is, however, a common feature that positively charged phospholipid vesicles composed of phosphatidylcholine (PC) and sterylamine drive actin polymerization mainly by an electrostatic effect, as does poly-L-lysine and inert positively charged solid substrates. Therefore, it remains questionable if this observation is of physiological significance because an incorporation of cholesterol into such liposomes greatly abolishes their affinity for actin (Okimasu et al., 1987). On the other hand, such model systems may be used to attach and orient actin filaments on lipid films for a better electron microscopical observation and to study the effect of lipids and actin-associated proteins. Despite some hydrophobic amino acids on the surface of the actin molecule, the netto charge is negative for

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

85

actin monomers and actin filaments at physiological pH. This normally leads to a repulsion between vesicles containing negatively charged phospholipids and actin (G. Isenberg and V. Niggli, unpublished observations) so that generally the binding of actin to lipids under various conditions seems to be unspecific and of very low affinity. Moreover, most of the techniques discussed previously and also additional binding assays, such as differential sedimentation and gel filtration, fail to detect a major lipid-binding capacity of actin.

B. Actin Nucleating Proteins

1. Talin When we came across talin in 1990-1991, this protein was known to be one of the major proteins of focal adhesions (Burridge and Connell, 1983a,b; Hock et al., 1989; Beckerle and Yeh, 1990) and one of the constituants of the leading edge of moving cells that colocalizes with actin in nascent actin filament bundles formed during protrusion (Izzard and Lochner, 1980; DePasquale and Izzard, 1991). In fact, actin binding of talin was independently shown by two groups using different techniques: Muguruma et al. (1990) used gel filtration and Goldmann and Isenberg (1991) applied stopped-flow kinetics to show that talin binds G-actin with a binding constant of K D = 0.3 X M and a rate of 7 X lo6 M-' sec-' with a loss of two or three molecules per second. Using DSC, it was shown that talin binds to phospholipid membranes by exhibiting both a hydrophobic and an electrostatic component (Heise et af., 1991). This effect is greatly enhanced in the presence of negatively charged phospholipids (DMPS and DMPG). In the same study (Heise et al., 1991), it was shown by FTIR that in lipid mixtures containing DMPC and DMPG, with one binding partner deuterated (DMPC-dS4),talin preferentially binds to DMPG. Thus, there exists a high selectivity for negatively charged phospholipids. A similar behavior is monitored when measuring the insertion of talin into lipid monolayers with the film balance technique (Dietrich et al., 1993). Partitioning of talin was observed in mixed DPPC-DMPG lipid monolayers. Prior to viewing with epifluorescence light microscopy, talin was labeled with NBD (Detmers et al., 1981). For lipid labeling, 0.1% N-Texas red sulfonyl dipalmitoyl L-a-phosphatidylethanolamine was added. This kind of protein-lipid double-labeling revealed that talin always codistributes with regions into which negatively charged lipids are segregated. Talin, even at elevated physiological salt concentrations, penetrates into lipid layers at an amazingly high lateral pressure of 30 mN/m, which mimics the situation in biomembranes (Sackmann, 1995). Because only a combina-

86

G. ISENBERG AND V. NlGGLl

tion of various methodological approaches will give clear-cut answers, hydrophobic labeling with photoactivatable lipid markers (Niggli et al., 1986; Brunner, 1993) has been applied to investigate the binding of talin to lipids (Goldmann et al., 1992). [3H]PTPC/11,a photoactivatable PC derivative, selectively reacts with protein domains, which insert into the hydrophobic part of lipid membranes. Talin gets strongly labeled after spontaneous insertion into liposomes containing the lipid analog. Only a minor portion of the talin molecule penetrates into the hydrophobic membrane core, an interpretation that gets support by neutron reflection studies (Hess et al., 1997). Because a specific lipid interaction of talin has been reliably documented by various methods, particular attention is directed toward the identification of the lipid binding site in structural and functional terms. The primary sequence of mouse talin has been established by Rees et al. (1990). It has been speculated from the apparent sequence homology (>40%) with the membrane and actin-binding proteins 4.1 and ezrin, that the lipid-binding domain resides in the smaller (47-kDa) calpain I1 cleavage product (Beckerle et al., 1987). Indeed, we could confirm by functional and analytical assays that the 47-kDa head portion harbors the major lipid-binding domain in the talin molecule (Niggli et al., 1994). Though the 200-kDa talin rod domain exhibits a repetitive hydrophobic core pattern consisting of repeated motifs of amphiphilic helices (McLachlan ef al., 1994), it seems unlikely that further lipid-binding domains are exposed on the rod portion because out of a mixture of 47- and 200-kDa fragments only the 47-kDa domain binds to lipid vesicles (Niggli et al., 1994). By applying computerassisted structure predictions on the 47-kDa N-terminus, Tempe1 et al. (1995) have designated three regions along the primary sequence, residues 21-39,287-342, and 385-406, that potentially could bind to acidic phospholipids with high specificity: PSTMVVDACRMIRERIPEA

21-39

GQMSEIEAKVRYVKLARSLKTYGVSFFLV KEKMKGKNKLVPRLLGITKECVMRVDEK

287-342

GEQIAQLIAGYIDIILKKKKSK

385-406

According to these sequences, synthetic peptides have been synthesized and peptide-specific polyclonal antibodies have been raised in order to map the localization of these lipid-binding domains directly on the talin molecule by ultrastructural methods (Isenberg et al., 1997b). By analytical ultracentrifugation and chemical cross-linking of native purified talin, it has previously been proven that talin in its functional form exists as a dumbbellshaped homodimer, 51 nm in length with an antiparallel arrangement (Goldmann et al., 1994). Decoration with polyclonal antibodies designed

INTERACTION OF CYTOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

87

to recognize lipid-binding domains localize these at the opposite ends of individual talin molecules (Fig. 7). This clearly shows that (i) lipid binding sites are exposed on the surface of each half of the talin homodimer and that (ii) the opposed globular ends of the talin homodimer represent the 47-ka subunit. This interpretation is confirmed by viewing thrombin-cleaved head and tail portions of talin individually by rotary shadowing (Isenberg et al., 1997b). Lipid association of talin in a biological sense seems to be quite important: Although first proposed in 1978 (Isenberg et al., 1978; Small et al., 1978), it took almost 20 years before the idea was accepted that cell locomotion is directly coupled to actin polymerization. Evidence has accumulated that talin, in addition to serving as a cytoskeleton-lipid anchor, may have a

FIG. 7 Localization of the lipid-binding domain on the 47-kDa subunit of the talin molecule. Peptide-specific antibodies raised against the predicted lipid-binding sequences within the 47kDa membrane-binding domain of talin (see text) bind to the opposite ends of the talin molecule (b-e). Talin without bound antibodies is shown as a control sample (a); magnification 310,OOOX (Isenberg et al., 1997b).

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special role in nucleating actin filament assembly at lipid interfaces (for review, see Isenberg and Goldmann, 1992, 1995). Talin facilitates actin dimer formation, thus overcoming the rate-limiting step during actin polymerization. The rapid formation of actin filament nuclei (Kaufmann et al., 1991; Goldmann et al., 1992), reflected by an increase of filament number concentration over filament length, leads to a marked increase of the overall actin polymerization rate (about twice as fast). In addition, viscoelastic measurements of talin-induced actin gels show that talin induces an increase in actin filament stiffness (Ruddies et al., 1993). Such a reduction in chain dynamics may help to avoid repulsion between filaments due to undulation forces and thus favor their parallel arrangement (Goldmann et al., 1993a). An actin binding site has been designated to the 190-kDa tail fragment of talin (Niggli et al., 1994), which is capable of nucleating actin filament growth and reducing the viscosity of F-actin by shortening the average actin filament length as does intact talin. When purified talin is reconstituted into lipid vesicles, the functional properties persist, indicating that talin in its membrane-bound orientation is able to induce actin assembly right at the lipid interface (Kaufmann et al., 1992). A direct binding of talin to the cytoplasmic domain of platelet GP IIb-IIIa makes a regulatory funtion involving the integrin-talin-cytoskeleton pathway highly likely (Honvitz et al., 1986; Knezevic et al., 1996). It is tempting to speculate that the lipid interaction may be of importance for this signal cascade. 2. Ponticulin A membrane spanning glycoprotein identified in polymorphonuclear leucocytes and Dictyostelium that could also mediate actin binding and nucleation is the 17-kDa protein ponticulin (Wuesthube and Luna, 1989). Unlike talin, this protein failed to nucleate the polymerization of actin when added back to commercially lipids but was active when incorporated into native Dictyostelium membrane vesicles (Chia et al., 1993). Apparently, a specific lipid composition including diacylglycerol (DAG) (Shariff and Luna, 1992) is needed to guarantee actin nucleation by ponticulin. The authors, however, cannot rule out that in their system an augmentation of nucleation may be achieved by a dissociation of otherwise active capping proteins or by the activation of additional nucleation proteins.

C. Actin Cross-Linking Proteins 1. a-Actinin The structural and functional properties of a-actinin in general have been reviewed by Blanchard et al. (1989) and Vandekerckhove (1990). The selec-

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89

tion of Dictyostefium mutants, defective in a-actinin (Schleicher et al., 1988), and the design of a variety of monoclonal antibodies (Wallraff et af., 1986) have strengthened our knowledge that a-actinin is a homodimer, oriented antiparallel, and capable of cross-linking ( Jockusch and Isenberg, 1981) or bundling (Meyer and Aebi, 1990) actin into a supramolecular actin lattice. Recent data offer the explanation that bundling and cross-linking of aactinin are reflected by a sol-gel percolation transition occurring in crosslinked actin networks that is quite comparable to that in synthetic gels (Tempe1 et af., 1996). Because the association-dissociation equilibrium between actin and a-actinin changes with temperature (Goldmann and Isenberg, 1993), various cross-linking ratios may be produced by slight shifts in temperature, thus inducing a rheological switch, when operating close to a phase boundary between different gel states (Isenberg, 1996). A potential lipid interaction of a-actinin in vivo was discovered by the finding that, when platelets were activated, a 30-fold increase in bound palmitic acid (PA) and DAG was observed (Burn et af., 1985). Previously, Meyer et al. (1982) had noticed that the same two lipids, P A and DAG, bound to a-actinin from a total lipid extract at a molar ratio of 1:l:l. Because DAG results from hydrolysis of phosphatidyl 4,5-biphosphate (PIP-2), it was speculated that membrane binding of a-actinin in some way may be linked to PI turnover and signaling. Indeed, PIP-2 is bound to aactinin at a high molar ratio (20-30 mol/mol protein) in vivo and obviously is necessary for complete gelation activity under in vitro conditions (Fukami et af., 1992, 1994). The binding site of PIP-2 on a-actin has recently been identified (Fukami et af., 1996). Like talin, a-actinin can be reconstituted into lipid monolayer films (Fritz et al., 1993). Photobleaching in the evanescent field has allowed quantification of this binding and demonstrated that a-actinin in this lipid-inserted configuration remains accessible for actin binding from the bulk phase. a-Actinin with other lipid- and actin-binding proteins may form triple complexes with phospholipid bilayers. In the case of vinculin (Niggli and Gimona, 1993), this leads to an enhancement of vinculin binding and a suppression of a-actinin-lipid interaction. This example clearly points out the necessity that before evaluating a protein function in vivo, one should consider the effect of competing binding partners in the range of their physiological concentrations. a-Actinin, like talin, binds directly to the cytoplasmic site of integrin subunits (Otey et al., 1990; Pavalko et af., 1991). It would be interesting to evaluate the role of lipid insertion for this specific interaction. 2. Spectrin

Together with a-actinin and dystrophin, spectrin belongs to the protein superfamily that shares a repeatedly conserved sequence motif coding for

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a series of three a-helical domains as a building block for the elongated rod structure of these molecules (Parr et al., 1992). Evidence for an interaction with phospholipids has been obtained for brain (Diakowski and Sikorski, 1995) and erythrocyte spectrin (Michalak et al., 1993). Ankyrin inhibits the binding of erythrocyte spectrin to phospholipid vesicles (Bialkowska et al., 1994), whereas band 4.1 enhances binding (Takeshita et al., 1993). Evidence for electrostatic coupling of spectrin to charged phospholipids was obtained by analyzing the penetration into lipid monolayers and by evaluating the shifts of phase transitions and phase boundaries of lipid mixtures in the presence of spectrin (Mombers et al., 1980; Maksymiw et al., 1987). The selective binding of spectrin to negatively charged phospholipids phosphatidylserine (PS) and phosphatidylglycerol (PG) results from locally clustered positive charges along the folded spectrin polypeptide chain, although the whole protein has a net negative charge. Hydrophobic interactions with PC and (PE) are probable phosphatidylethanolamine because hydrophobic ligands have been shown to act as strong quenchers for intrinsic protein fluorescence (Isenberg et al., 1981). In situ labeling of erythrocyte spectrin was achieved by application of the hydrophobic label phenylisothyocyanate (Sikorski and Kuczek, 1985) and by ultrastructural methods. Interaction with lipid bilayers is facilitated by covalently bound palmitate; however, only a small fraction of spectrin was shown to be [3H]palmitoylated (Mariani et al., 1993). Spectrin contains a pleckstrin homology (PH) domain in its C-terminal region (aa 2198-2304) that has the capacity to interact with PIP-2 phosphate groups (Hyvonen et al., 1995). When expressed in cell lines, the PH domain of spectrin is targeted to the plasma membrane (Wang and Shaw, 1995; Wang et al., 1996). 3. Filamin

In smooth muscle cells, the high-molecular-weight (250 kDa) actin crosslinking protein filamin is arranged in an alternative pattern with vinculin and dystrophin in regions closely aligned beneath the plasma membrane. This prompted us to investigate the interaction of filamin with membrane lipids. When filamin is added to a lipid vesicle preparation (DMPG-DMPC, 1: l ) , this immediately induces vesicle-shape changes (Goldmann et al., 1993b), consistent with effects observed after loading PC and PE vesicles from the inside with actin-filamin mixtures (Cortese et al., 1989). The possiblity that actin-binding proteins influence shape and deformability of lipid layers becomes of importance for cellular shape changes and pseudopod formation (Stossel, 1989; Condeelis, 1992). The reliable and precise method of correlating structures with a given amino acid sequence suggested only two regions (aa 49-71 and 131-155) of a total number of 2647 aa for the binding of filamin with phospholipid membranes (Tempe1 et al., 1994a).

INTERACTION OF CMOSKELETAL PROTEINS WITH MEMBRANE LIPIDS

91

DSC measurements in combination with hydrophobic labeling and film balance studies verify an insertion of filamin into the lipid bilayer in reconstitution experiments (Tempe1 et al., 1994b). The lipid-filamin interaction is highly charge dependent because hydrophobic interactions are reduced with rising salt concentrations. It will be interesting to find out whether the designated lipid-binding domains also harbor the PIP-2 binding site, which when saturated (3 mol PIP-2/mol filamin) completely inhibit the filamin cross-linking activity, i.e., the binding to actin (Furuhashi et al., 1992).

D. Actin-Binding Shuttle Proteins A group of proteins, including profilin, hisactophilin, and MARCKS, are actin-binding shuttle proteins. They all can exist in a cytoplasmic and a membrane-bound form and they all bind actin. For a shuttle function, the protein has to carry a cargo (actin), which can be released upon a certain signal. It should also “lock in” at its final place of destination, the membrane, in a reversible manner. The major role of these proteins would thus be to target actin to certain sites at the plasma membrane where it is needed to exert a force by polymerization. Signals involved in locking in and release could be pH, electrostatic switches by changing protein conformations, covalent lipid modifications, and binding or release of phosphoinositides. For a detailed description, the following proteins with respect to their lipid interactions are reviewed. 1. Profilin The general structure and function of this important actin-binding protein has been reviewed in detail elsewhere (Machesky and Pollard, 1993; Pollard et al., 1994; Jockusch et al., 1995). Briefly, this small 12- to 17-kDa actinsequestering protein, together with thymosin p4,is believed to regulate the monomeric pool of actin inside cells. In addition, however, profilin stimulates nucleotide exchange on G-actin and as a profilin-actin complex binds to the membrane-apposed barbed fast-growing end of F-actin (Goldschmidt-Clermont et al., 1992; Pantaloni and Carlier, 1993). Thus, profilin action is complex. Dissociation of the profil-actin complex is achieved by the binding to phoshoinositol phosphates (Lassing and Lindberg, 1985). This interesting observation stimulated the search for regulatory mechanisms on the basis of phosphoinositides. Indeed, it was found that profilin binds to PIP-2 micelles with submicromolar affinity (Kd 5 0.1 X M ) (Goldschmidt-Clermont et al., 1990). The binding of profilin to PIP-2 not only liberates polymerization-competent G-actin but also affects hydrolysis of PIP-2 by phospholipase C-y (PLC-y) (Goldschmidt-

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Clermont et al., 1990,1991; Machesky et ab, 1990).Tyrosine phosphorylation of PLC-y by the PDGF receptor overcomes this inhibition. The binding site for PIP-2 has been mapped to a small region of five amino acids around residue 88 (Sohn et al., 1995; Table I). Binding of PIP-2 at this site results in a conformational change of profilin with an increase of a-helical content of 35% compared to 5-9% for profilin alone (Raghunathan et al., 1992; Lu et al., 1996). Interestingly, profilin has an almost 10-fold higher affinity to D-3 phosphoinositide (PtdIns 3,4)P2 than for (PtdIns 4,5)P2 (Lu et al., 1996) and its inhibitory effect on PLC-y-mediated PtdIns(4,5)P2 hydrolysis is overcome by PtdIns(3,4)P2 and PtdIns(3,4,5)P3. On the other hand, the a-helical content of profilin is much less (1.4%) upon binding to PtIns(3,4)P2 versus binding to PtIns(4,5)P2 (17.4%) (Lu et al., 1996). The implications that these new insights will have on understanding the PI turnover and actin shuttle inside cells are unknown. 2. Hisactophilin

A shuttle protein that reacts to small pH changes is the histidine-rich, 13.5kDa protein hisactophilin, first purified from Dictostelium (Scheel et al., 1989). Its structure has been resolved by NMR (Habazettl et al., 1992). The molecule is polarized with tightly packed @-barrelconformation, harboring N and C termini at one side and loosely organized @ hair pins, carrying almost all the histidine residues, at the opposite side (Fig. 8). In living cells, hisactophilin shuttles within minutes between the cytoplasm and the plasma membrane in a pH-dependent manner (Hanakam ef al., 1995; 1996a). At low ionic strength, the binding of hisactophilin to lipid monolayers is purely electrostatic (Behrisch et al., 1995). At physiological conditions, a synergistic binding involving hydrophobic insertion of the N-terminal-linked myristoyl chain into the lipid bilayer with a partition coefficient of K p = 1.1 X lo4 M-' and electrostatic interaction by a cluster of histidines (K,,, = 8 X lo5 W1)occurs (Hanakam etal., 1996b). Because histidines switch from cationic to anionic at pH 7.0, phospholipid binding also switches in a strongly pH-dependent manner (Fig. 8 ) . Constructs of Dictyostelium hisactophilin with and without bound myristic acid have been developed to investigate membrane insertion and actin coupling by the Langmuir technique and with the neutron reflection technique (Naumann et al., 1996). Gibb's equation;

allows the surface excess (r)to be directly related to the change in surface tension (d,) of the interface with the variation of the bulk solute concentration (cB). Remarkably, the myristoylated and the nonmyristoylated variant

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FIG.8 Model of the pH-sensor hisactophilin. The myristoylated P-barrels face the lipid membrane and histidine-rich loop structures are directed against the cytoplasm. Only the myristoylated native protein anchors into the hydrophobic lipid bilayer core, whereas the fatty acid-deficient mutated EC-His couples to mixed DMPC/DMPG monolayers electrostatically in an opposite direction. d,, hydrophobic lipid chains; d2, polar lipid head groups; d3. protein (modified from Naumann er af., 1996, with permission).

of hisactophilin both bind to lipid layers by an electrostatically driven process, resulting in a pronounced reduction of lipid mobility and hence in a substantial effect on membrane structure (Behrisch et af., 1995). However, only the myristoylated native protein penetrates into both the semipolar headgroups and the hydrophobic interior of the lipid layers, whereas the nonmyristoylated hisactophilin forms an adsorbed protein layer (Naumann et af., 1996).

3. MARCKS A protein that also makes use of a myristoyl electrostatic switch for reversible membrane interaction (McLaughlin and Aderem, 1995) is MARCKS, the myristoylated alanine-rich C-kinase substrate. MARCKS is an amphitropic protein that can be isolated from cytoplasm or in a membrane-bound form (Manenti et af., 1992, 1993). Its interaction with membrane lipids involves hydrophobic and electrostatic components (Taniguchi and Ma-

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nenti, 1993) The myristoyl tail inserts into the inner lipid leaflet (Vergkres et al., 1995) and charged basic residues help to anchor the protein to the acidic lipid bilayer surface (Kim et al., 1994; Nakaoka et al., 1995). Basic amino acids cluster around the myristoyl N terminus (Resh, 1994), interrupted by serine and threonine residues that, upon phosphorylation, switch off the lipid interaction (Kim et al., 1994; Swierczynski and Blackshear, 1996). MARCKS inhibits PIP-2 hydrolsis by PLC--y and sequesters PS and PIP-2 into lateral membrane domains. Phosphorylation of MARCKS reverses this inhibition by producing a burst of IP3 and DAG (Glaser et al., 1996). Though MARCKS binds to F-actin with different affinity (Hartwig et al., 1992) and apparently is involved in phagocytosis and secretion, its precise cellular function is not known. Regulated by two important chemotactic signals, it may function as a protein kinase C (PKC) and calcium-calmodulin-regulated transducer molecule during cell stimulation.

E. Actin Capping and Severing Proteins None of the known actin filament capping and severing proteins have been shown to interact with phospholipid bilayers directly. However, most if not all of them can be regulated by PIP-2. Consequently, there exist polyphosphoinositide binding sites, which have partially been identified and sequenced. 1. Capping Proteins The name capping protein was introduced when the first candidate of this class of proteins was isolated in 1980 from Acanthamoeba (Isenberg et al., 1980a). Capping protein is heterodimeric (32- and 34-kDa subunits), binds exclusively to the barbed fast-polymerizing end of actin filaments, nucleates actin filament growth, and is ubiquitously found in vertebrate brain (Kilimann and Isenberg, 1982), Dictyostelium (Schleicher et al., 1984), in the z lines of skeletal muscle (Casella et al., 1986), yeast (Amatruda and Cooper, 1992), and platelets (Nachmias et al., 1996). The binding of PIP-2 was found to be inhibitory for the association of capping proteins to actin filament barbed ends (Heiss and Cooper, 1991;Haus er al., 1991). Because uncapping in vitro occurs slowly with a half-life time of capped barbed ends of 30 min and more (Schafer et al., 1996), the hypothesis has been put forward that PIP-2 will cause efficient uncapping and the rapid formation of free growthcompetent barbed actin filament ends in vivo (Hartwig et al., 1995). PIP2 binding will also dissociate CAP-100 (Hofmann el al., 1992) and g-Cap

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39 (Yu et af., 1990; Onoda and Yin, 1993) from actin filament ends (for more references, see Weeds and MacIver, 1993; MacIver, 1995).

2. Severing Proteins The Ca2'-sensitive F-actin severing proteins gelsolin ( Janmey and Stossel, 1989; Yin et af., 1988), villin (Janmey et al., 1992), severin (Eichinger and Schleicher, 1992), adseverin (Maekawa and Sakai, 1990), and destrin and cofilin (Yonezawa et af., 1990, 1991) all bind PIP or PIP-2. For gelsolin, the phosphoinositide binding sequence has been localized in the actin monomer binding domain (Yu et af., 1992). PIP-2 binding and actin interaction are thus exclusive and consequently PIP-2 inhibits severing, nucleation, and actin monomer binding. By applying deletional mutagenesis and synthetic peptides (Yu et aL, 1992) one phosphoinositide binding site was identified in residues 135-149 with the following sequence: KSGLKYKKGGVASGF. A second binding site resides in residues 150169 with the following sequence: KHVVPNEVWQRLFQVKGRR ( Janmey et af., 1992). Both sites bind PIP-2 with equal affinities. Cofilin, the PIP-2 binding site also responsible for actin binding, has been designated to the sequence, WAPECAPLKSKM (Yonezawa et al., 1991), which is distinct from the gelsolin sequence. There may, however, exist more than one binding site for PIP-2 because a motif similar to that of gelsolin has been recognized in residues 13-22 of cofilin: KVFNDMKVRK (Yu et af., 1992; cf. Table 1).

F. Focal Contact Proteins The biological significance of focal adhesions and molecular interactions has been discussed and reviewed in detail (Jockusch et af., 1995; Bockholt and Burridge, 1996). Here only those components will be mentioned whereas interactions with lipids have been investigated. For talin and aactinin, see sections IVB and IVC. 1. Vinculin Vinculin is one of the few proteins for which binding to lipids has been demonstrated in vitro (Niggli et af., 1986) and in vivo (Niggli et af., 1990). Lipid binding occurs with negatively charged phospholipids (PA, PI, and PG) but not with neutral lipids (PC and PE) (It0 et af.,1983; Niggli et af., 1986). Vinculin has been successfully integrated into planar lipid monolayers (Fringeli et af., 1986;Meyer, 1989).The Kd for vinculin-phospholipid to 5.3 X lo-'' M) depending interaction can vary considerably (1.2 X

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on temperature, surface pressure, and lipid composition. Whether myristate (Kellie and Wigglesworth, 1987) or palmitate (Burn and Burger, 1987) as covalently attached posttranslational modifications play a role for bilayer anchoring is not clear. However, the degree of covalently bound lipid may determine the state of phosphorylation. For a long time it was under debate whether vinculin could bind to actin. The initial observation, that vinculin cosediments with actin upon binding (Isenberg et al., 1982), has now been unequivocally confirmed by identifying an actin binding site within the vinculin sequence (Menkel et af., 1994). However, this actin binding site may be masked by a head to tail interaction within the vinculin molecule (Johnson and Craig, 1995a). Similarly, the lipid binding region is also masked by head to tail interaction (Johnson and Craig, 1995b). On the other hand, phosphoinositides (Gilmore and Burridge, 1996) and acidic phospholipids are able to inhibit the intramolecular association between the N- and C-terminal regions of vinculin (Weekes et af., 1995), exposing actin binding and PKC phosphorylation sites. The actin binding domain resides in residues 893-1016 (for a model based on sequences, see Goldmann et al., 1996), whereas the lipid-binding domain can be clearly designated to residues A A 913-970 (Tempe1 et af., 1995; Johnson et af., 1997), which is within the same region. Because actin binding and a site for PKCmediated serine-threonine phosphorylation (Schwienbacher et af., 1996) are opened up by acidic lipid associations, the functional properties of vinculin seem to be directly regulated by binding to acidic phospholipids and thus to membrane bilayers. Vinculin, therefore, serves as a good example of how signals get transmitted by conformational changes of actin-binding proteins at cell boundaries (Isenberg, 1996).

2. Ezrin

Ezrin belongs to the ERM protein family (ezrin, radixin, and moesin) and occurs widespread. Its sequence homology to band 4.1 protein and talin as well as its localization suggest that the protein could act as a membrane cytoskeletal linker (Algrain et al., 1993). Overexpression leads to accumulation of ezrin at the plasma membrane (Andreoli et af., 1994). Ezrin cosediments with PS-containing liposomes at low ionic strength. At physiological salt conditions (130 mM KCI) ezrin binds strongly to PC liposomes when >5% PIP-2 is included (Niggli et af., 1995). A fusion protein, containing the N-terminal residues 1-309, shows similar binding characteristics to liposomes compared to those of the intact protein, thus binding of ezrin to lipid membranes occurs, as expected, via its N-terminal domain (Niggli et al., 1995).

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G. Myosins Myosin I isolated from Acanthamoeba (Adams and Pollard, 1989; Miyata et al., 1989) and intestinal brush border (Hayden et al., 1990) has been shown to interact with anionic phospholipids. It is the positively charged carboxy-terminal tail of the myosin I heavy chain that mediates the association with phospholipid bilayers (Swanljung-Collins and Collins, 1992). Myosin I binds to phospholipids with a higher affinity than that for actin. Apparent dissociation constants were found to range between 0.5 and 3.0 X lo-' M (Adams and Pollard, 1989; Hayden et al., 1990). Binding of myosin I to neutral phospholipids (PC) is unsuccessful but is accelerated in liposomes containing PG, PS, and phosphoinositolphosphates. A major fraction of myosin I, including its associated kinase, is linked to membranes in situ (Kuleszka-Lipka et af., 1991; Baines et al., 1992). The kinase that is activated by a phospholipid-enhanced autophosphorylation in vitro (Brzeska et al., 1990,1992) is not activated in a mernbrane-bound form, whereas, lipids still stimulate myosin I phosphorylation by its kinase (Kuleszka-Lipka et al., 1991). The stable incorporation of myosin I into lipid vesicles has prompted the use of this system for in vitro motility assays (Zot et af., 1992; Zot, 1995). Bound to a solid phospholipid-coated substrate, myosin I is able to move actin filaments. This may have implications on actin-based intracellular motility (Isenberg et al., 1980b). The affinity of myosin I for negatively charged phospholipids has enabled Celia et al. (1996) to grow two-dimensional myosin crystals on planar lipid films from dioleylphosphatidy1 serine. Myosin 11, the conventional two-headed isoform, can also bind to lipid bilayers, provided that anionic phospholipids (PS) are present (Li et af., 1994; Murakami et al., 1994). Myosin I1 binds to PS liposomes via its COOH terminal of the heavy chain. This binding to phospholipids is prerequisite for phosphorylation of the heavy chain by PKC (Murakami et al., 1994). Hence, a specific lipid-modulated function of myosins becomes evident.

H. Others [Caldesmon, Synapsins, and Annexins] Caldesmon is a smooth-muscle actin-binding protein that is believed to be involved in actin assembly regulation. Phospholipid binding was first reported by Vorotnikov and Gusev (1990; 1992). With PS vesicles, caldesmon forms a strong complex (K,,, = 1.5 X lo5 M - ' ) to which hydrophobic and electrostatic interactions contribute (Czurylo et al., 1993). Using various calmodulin expression fragments, the lipid binding site was predicted to reside within the A A sequence 626-710 of caldesmon (Bogatcheva el al.,

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1994), which is in agreement with lipid-binding sequences derived from computer modeling (Bogatcheva and Gusev, 1995). In the nervous system, the vesicle-specific phosphoprotein synapsin I is a lipid- and actin-binding protein. The proline-rich hydrophobic head of synapsin inserts into the hydrophobic bilayer core and simultaneously reacts with acidic phospholipids (Benfenati et al., 1993). Finally, there is the large group of annexin isoforms, several of which have been reported to bind to phospholipids (Meers and Mealy, 1993, 1994; Evans and Nelsestuen, 1994; Plager and Nelsestuen, 1994; Junker and Creutz, 1994; TravC et al., 1994; Wang and Creutz, 1994). Multiple actinbinding motifs suggest that actin binding is a common property of annexins. Among the multiple functions that have been attributed to the annexins, a coupling of the cytoskeleton to membranes is the most likely.

I. Regulation by Phosphoinositides In this review it has frequently been documented that a great number of actin-binding proteins bind to and are regulated in their function by phosphoinositides. It has been argued that protein binding to PIP-2, as to other charged lipids, may be unspecific because PIP-2, as a highly charged molecule producing a high charge density when clustered in micelles, will attract proteins in solution. However, phosphoinositides as low physiological concentrations have been incorporated in combination with other lipids into unilamellar vesicles, thus mimicking a biological membrane with defined parameters. Acting at low stoichiometry, they have been found to react rather specifically with actin-binding proteins, either inhibiting or stimulating certain catalytic functions. On capping and severing proteins, phosphoinositides generally act inhibitory. Even a small amount of 100 FU.M of PIP-2 is enough to release capping proteins bound with very high affinity to actin filament barbed ends (Kd 2 10”M-’) quantitatively. PIP-2 regulation in general may be of significance because it may act at crossroads between cell signaling events that are mediated by small GTP-binding proteins and signaling events influencing assembly mechanisms of the actin cytoskeleton. In permeabilized platelets, as an in vivo model system, it was recently shown that activated rac causes the uncapping of barbed actin filaments by upregulation of PIP-2 synthesis (Hartwig et al., 1995). Similarly, rho and Cdc 42, by their binding and stimulation of PtdIns-4-P 5-kinase, may have immediate influence on the assembly events of the cytoskeleton near membranes (Carpenter, 1996). The discovery of these new signaling pathways (Gutkind and Vitale-Cross, 1996) points against unspecific binding effects and the role of PtdIns (4,5)P2 being solely a precursor to IP3 and DAG formation by PLC-.)I. Also, PIP-2 binding to actin-associated

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proteins occurs selectively and with specificity, e.g., the binding of cx-actinin to actin is PIP-2 enhanced, whereas the actin cross-linking activity of filamin is inhibited. Talin (Isenberg et af., 1996) and ezrin (Niggli et al., 1995) are recruited to liposomes containing low amounts of PIP-2 under physiological conditions. The functional relevance of this interaction is not yet clear. Profilin interacts with a 10-fold higher affinity with PtIns (3,4)P2 compared to PtIns(4,5)P2, indicating the presence of a highly specific binding pocket. Finally, the rather specific opening of an actin-binding site on vinculin induced by binding to PIP-2 may stress the general importance of phosphoinositide regulatory pathways. More information on phosphoinositide regulation, which in general seems to act antagonistically to Ca2+-signals, is given in two reviews (Janmey, 1994, 1995).

V. Interactions of Microtubules and Associated Proteins with Membrane Lipids Tubulin and a number of tubulin-associated proteins, tau, microtubuleassociated protein-2 (MAP-2), dynein, and dynamin, have the capacity to interact in vitro with lipids. At least for tau, MAP-2, and dynamin, part of the binding of acidic phospholipids occurs via the highly basic, tubulinbinding domain. As expected from this finding, acidic phospholipids interfere with microtubule assembly in the presence of MAPS. MAP-2 and dynamin contain additional high-affinity binding sites for PI and for PIP/ PIP-2, respectively. Whether these direct lipid interactions play a role in tubulin-membrane linkage and in situ regulation of microtubule dynamics is unclear. Recent studies suggest a physiological role of direct bilayer interactions in retrograde organelle transport, where dynein may retrogradely transport organelles bound directly to the lipid bilayer, a process possibly regulated by phosphoinositides.

A. Tubulin and Microtubules Purified bovine brain tubulin cosediments with unilamellar PC vesicles in Ficoll density gradients (Caron and Berlin, 1979). Moreover, tubulin induces release of a fluorescent dye from PC vesicles, indicative of a disturbance of the bilayer as a result of a close interaction. This interaction was observed to occur at the transition temperature of the lipid that may lead to structural fluctuations necessary for tubulin insertion. Other proteins, such as albumin and myosin, are inactive in this assay. The interaction does not appear to be primarily nonionic because it is unaffected by up to 4 M NaCI (Klausner

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et af.,1981).The observations that trypsin digestion of PC-associated tubulin occurs slower than that of free tubulin and that different cleavage products are obtained (Kumar et af., 1981) also suggest a direct lipid interaction. Moreover, PE has been reported to enhance microtubule assembly (Hargreaves and McLean, 1988). Interestingly, phosphorylation of tubulin may modulate tubulin-bilayer association. Hargreaves et af. (1986) observed that in vitro phosphorylation of purified tubulin by the calmodulin-calciumdependent protein kinase enhances its association with PC vesicles, as shown using gel filtration chromatography. The interaction appears to be reversible because treatment of the tubulin-vesicle complex with alkaline phosphatase leads to a partial release of the bound tubulin. The interaction very likely does not directly involve the C-terminal phosphorylated domain of tubulin because the latter is accessible to proteases in vesicle-associated tubulin. A capacity of microtubule protein to interact with acidic phospholipids, such as PS or PG, has also been observed using free-flow electrophoresis or Ficoll density gradient centrifugation (Joniau and De Cuyper, 1984; Caron and Berlin, 1987). Moreover, cardiolipin and IysoPC (2 nmol lipid/ mol tubulin dimer) have been identified in highly purified porcine brain tubulin preparations (Hargreaves and McLean, 1988). In contradiction to these reports, tubulin does not interact significantly with PI liposomes in mixtures of MAP-2 and tubulin (Surridge and Burns, 1992). Ding et af. (1992) suggest a capacity of tubulin to interact with a negatively charged lipid. Mouse brain tubulin interacts in vitro with bacterial lipopolysaccharide (LPS). This was shown using gel filtration of LPS-tubulin mixtures and cross-linking of a photoactivatable LPS derivative to tubulin. This interaction is not inhibited by 2 M NaCl and very likely involves the lipid moiety of LPS because lipid A prevented cross-linking of the LPS derivative. Lipid A is P-1-6-linked disaccharide of glucosamine, acylated with R-2-hydroxymyristate in positions 2,3,2',3' and phosphorylated at positions 1 and 4' (Raetz, 1990). This lipid carries four negative charges at neutral pH. The microtubule-associated protein MAP-2 also reacted with the LPS derivative (Ding et al., 1992), in agreement with its capacity to interact with negatively charged phospholipids (Surridge and Burns, 1994). The findings by Ding et af. (1992) may explain why taxol, which stabilizes microtubules, has the same effect on macrophages as LPS after uptake via pinocytosis. Direct tubulin-bilayer interactions have not been further investigated. Neither the lipid binding site nor the possible physiological relevance of such interactions are known. A capacity for direct bilayer interactions could be one reason why tubulin has been identified as a membrane component in a number of systems (for reviews, see Stephens, 1986; Niggli and Burger, 1987). It is to date not clear whether this observation is due to artifacts occurring during the fractionation process or whether a fraction of tubulin possibly differing from "cyto-

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plasmic tubulin” is indeed associated in situ with membranes. The observation that labeled soluble tublin included during membrane purification does not become associated with membranes speaks against an artifactual association (Bhattacharyya and Wollf, 1975). Especially in cilia, a fraction of tightly membrane-associated tubulin appears to exist, exhibiting behavior different from that of axonemal tubulin isolated from B-tubule plus central pair (Stephens, 1981, 1985; Stephens et af., 1987). Analysis of amino acid composition shows that purified membrane tubulin is uncharacteristically low in cysteine and histidine and, especially in the &chain, unusually high in isoleucine and thus more hydrophobic. Membrane tubulin appears to interact more strongly with both anionic and cationic detergents than axonemal tubulin. However, both membrane a and /3 tubulins exhibit the same PI in two-dimensional electrophoresis as the axonemal proteins, and react equally well with anti-tubulin antibodies (Stephens, 1981; Stephens et al., 1987). Membrane tubulin, but not axonemal tubulin, reassociates with lipids and other membrane proteins after detergent solubilization, removal of detergent, and freeze-thaw cycles. Moreover, in molluscan gill cilia most membrane tubulin antigenic sites appear to be buried within the bilayer, although axonemal and membrane tubulin react equally well with a rabbit polyclonal anti-tubulin antibody on nitrocellulose blots, suggesting in situ insertion into the bilayer (Stephens et af., 1987). According to Regula et al. (1986), membrane tubulin derived from bovine brain differs from cytosolic proteins in its enrichment in a Triton-X 114rich phase. Triton X-114 condenses to a micellar phase above its “cloud point,” carrying along integral membrane proteins. Beltramo et al., (1992) have addressed the question whether membrane tubulin enriched in the Triton phase differs from cytosolic tubulin in its posttranslational modifications. Interestingly, using specific antibodies against a-tubulin, acetylated tubulin, and tyrosinated and detyrosinated tubulin, these authors demonstrated a 20-fold higher proportion of acetylated tubulin in fractions of membrane tubulin. In contrast, tyrosinated and detyrosinated tubulin were somewhat lower in the membrane fractions. Membrane tubulin appears to be restricted to neutral tissues because it could not be detected in kidney, lung, and liver. Enrichment of membrane tubulin in the Triton-rich phase may, however, be due to interaction with a membrane component rather than due to its increased hydrophobicity, according to a recent report (Beltramo et aL, 1994). Bovine brain plasma membrane-associated tubulin can be completely extracted from membranes with 8 M urea or 0.1 M Na2C03, suggesting interaction with the plasma membrane as a peripheral protein. Dissociated tubulin is not further enriched in the Triton phase, possibly due to dissociation from a component conferring hydrophobicity. This component is very likely not phospholipid because phosphatidylcholine-associated tubulin does not partition into the

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detergent-rich phase (Beltramo et al., 1994). The enigma of the putative existence of membrane tubulin thus necessitates further investigations.

B. MAP-2 and Tau MAP-2 (200 kDa) is present in dendrites and dendritic processes, whereas MAP-2C (70 kDa), a differential splice product, is located in axons, dendrites, and glial cells (Avila and Nido, 1995). Tau represents a family of closely related proteins of 55-68 kDa located primarily to the axon. Sense and antisense approaches indicate the involvement of tau in neurite extension and stability of neurite microtubules and a role of MAP-2 in the initial establishment of neurites (Esmaili-Azad et al., 1994; Caceres et al., 1992). Both proteins contain a highly basic carboxy-terminal, proline-rich region including three or four tandem repeats containing homologous 18 amino acid repeats (with a net negative charge of 12 for the tau domain and 18 for the MAP-2 domain). This domain is involved in microtubule binding (Lewis et al., 1988). Both MAP-2 and tau promote tubulin polymerization in vitro (Avila and Nido, 1995). One of the starting points for the study of direct phospholipid interaction of these proteins was the observation that acidic phospholipids, especially PI, inhibit self-assembly of microtubules in the presence of microtubule-associated proteins, independently of the presence of GTP (Yamauchi and Purich, 1987; Surridge and Burns, 1992). In these studies, neutral phospholipids, such as PC, PE, or diacylglycerol, are inactive and PG or PS are much less efficient than PI. PS and PG are only active at concentrations >50% (mol/mol) in liposomes, with the remainder being PC. PI, in contrast, is active down to concentrations of 1%.The observation that PI does not affect polymerization of tubulin in the absence of MAPS suggests that the latter proteins confer sensitivity to phospholipids. Indeed, MAP-2A and MAP-2B, but not MAP-1 or tubulin, cosediment with sonicated PI liposomes. PI thus dissociates MAP-2 from microtubules (Yamauchi and Purich, 1987; Surridge and Burns, 1992). PI also prevents the enhancement of F-actin viscosity by MAP-2 (Yamauchi and Purich, 1993). Baudier and Cole (1987) provide some evidence suggesting a direct lipid interaction of tau because PE and PI, but not PS, induce smearing of the tau band on SDS-PAGE. Moreover, phosphorylation of this protein by a Ca*+/calmodulinkinase is enhanced by PS and PE but inhibited by PI (Baudier and Cole, 1987). In a detailed study, Surridge and Burns (1994) analyzed the affinity of lipid interaction for tau, MAP-2, and MAP-2C using cosedimentation of these proteins with multilamellar liposomes. The authors conclude that MAP-2 contains both a highaffinity (Kd = 51 nM) and a low-affinity phospholipid binding site (& = 1.5-2.5 p M ) . The high-affinity site confers specificity for PI, whereas the

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low-affinity site allows unspecific interaction with acidic phospholipids such as PS. Tau and MAP-2C show only low-affinity binding ( K d = 1.4-2.4 p M ) to acidic phospholipids. Low-affinity binding of MAPS and tau may be mediated by the highly cationic homologous tubulin-binding sequence present in these proteins. Indeed, a C-terminal peptide of MAP2C containing this domain binds to PI liposomes (Surridge and Burns, 1994). The highaffinity PI-binding site is very likely located in the 1372-amino acid sequence inserted in the middle of MAP-2 but not present in MAP-2C and tau (Burns and Surridge, 1995). This has not yet been demonstrated directly using the purified insert. The inhibitory effect of PI on MAP-2-tubulin interaction may be due to (i) a direct inhibition by occupancy of the tubulin-binding site or (ii) an indirect effect, possibly via induction of a conformational change as a consequence of PI binding to the 1372-amino acid insert. Sterical hindrance by the liposomes may also play a role. It is unclear why PI prevents MAP-2- but not tau-induced actin gelation (Yamauchi and Purich, 1993). The actin-binding domains of MAP-2 and tau are thought to be located in the tubulin-binding domain because a peptide corresponding to a repeat of this C-terminal domain of MAP-2 and tau also has the capacity to interact with actin (Correas et al., 1990). Based on these structural similarities, one would predict that PI should affect both tau- and MAP-2-induced actin gelation. However, this is not the case, and PS, which inhibits self-assembly of microtubules mediated by MAP-2, had little effect on MAP-2-induced actin gelation, suggesting the contribution of other domains in the latter event (Yamauchi and Purich, 1993). A number of questions concerning the physiological relevance of the interaction of tau and MAP-2 with phospholipids remain to be answered. According to a recent report, PIP-2 binds directly to recombinant human tau, as shown using electron microscopy, fluorescent labeling, and dynamic light scattering (Flanagan et al., 1996). The selectivity and affinity of tau-PIP-2 interaction have not yet been determined, and the binding site is as yet unknown. It is not clear whether MAP-2 also interacts with PIP2 and PtdIns(3,4,5)P3 and whether these lipids affect the function of MAP2 and tau. Interestingly, intact tau and an amino-terminal fragment of tau lacking the tubulin binding site, when transfected into PC12 cells, locate to the plasma membrane, as shown using immunofluorescence staining and subcellular fractionation (Brandt et al., 1995). PIP-2 may be a possible interaction partner of the amino-terminal domain of tau in the membrane. This domain protrudes from the microtubule surface about 19 nm in microtubule-bound tau. Tau thus has the capacity to stabilize microtubules close to membranes. In contrast to the findings for tau, interaction of MAP-2 with plasma membranes has not yet been reported. Possibly other factors may regulate

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or inhibit MAP-2 membrane association in intact cells not present in the in vitro system.

C. Dynein Cytoplasmic dynein (heavy chain of dynein, 510-540 kDa; accessory proteins, 53-74 kDa) is a microtubule-associated motor protein involved in retrograde organelle transport. It exhibits a microtubule-activated ATPase activity (Vallee, 1993; Zhang et al., 1993; Vallee and Sheetz, 1996). Indications for direct lipid interactions of dynein derive from observations by Lacey and Haimo (1994) that dynein binds to synaptic membranes digested with proteases with the same affinity compared to undigested membranes (& = 26-43 nM). However, a role of transmembrane or tightly membranebound proteins cannot be excluded because in another study binding of dynein to microsomal membranes was reduced upon protease treatment. Using large, unilamellar liposomes and flotation on sucrose, a high-affinity interaction ( K d = 80 nM) of purified bovine brain dynein with PS and PG, but not with PI, PC, or PE, was demonstrated (Lacey and Haimo, 1994). Ionic interactions appear to be involved because the interaction was decreased by addition of 0.2-0.5 M NaCl. Interactions of dynein with organelle membranes may thus at least partly be due to interaction with specific acidic phospholipids. An interesting question is whether the target organelles modify the functions of such motor proteins. Ferro and Collins (1995) studied the effect of small, sonicated, unilamellar liposomes on the ATPase activity of dynein purified from rat testis. They found that liposomes containing PC, PS, or a mixture of phosphatidylinositol phosphates, PI, and PS all stimulated dynein ATPase activity about 2.4-fold. No lipid specificity was observed, and 150 mM NaCl did not affect the response. The effect was specific for dynein because kinesin ATPase activity was not affected by phospholipids. The finding that PC also stimulated dynein ATPase activity is contradictory to the report on a specific interaction of dynein with PS and PG but not with neutral lipids such as PC or PE (Lacey and Haimo, 1994). Possibly the discrepancies are due to the use of dynein from different sources and to differences in the liposome preparation. It remains to be shown where the lipid binding site in dynein is located and whether dynein bound to liposomes is functional in motility studies. Concerning the former question, a 74-kDa cytoplasmic dynein subunit putatively involved in interaction with organelles exhibits repeated clusters of basic amino acids in its highly charged N-terminal domain, a possible lipid interaction site (Paschal et al., 1992). The latter question is difficult to assess because purified dynein is inactive in the absence of added cytosol in vesicle transport studies. However, work by Muresan et al. (1996) strongly suggests that dynein is

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indeed capable of transporting liposomes along microtubules. Using cytosol and vesicles isolated from squid axon, they showed that cytosol promotes the minus-end directed movement of acidic liposomes and of proteasetreated plus-end and minus-end vesicles. PC vesicles were also moved, in agreement with the stimulation of dynein ATPase activity by PC (Ferro and Collins, 1995). However, in sucrose gradients, dynein interacted more strongly with acidic phospholipids (PG, PS, and PI). Purified kinesin does not promote liposome movement (Muresan et af., 1996), consistent with its lack of interaction with lipids (Ferro and Collins, 1995). These studies do not exclude a role of another motor protein in liposome transport, but very likely, based on the in vitro studies, most of the activity may be due to dynein. Degradation of dynein with UVhanadate correlated with a loss of retrograde vesicle and liposome transport (Muresan et al., 1996). The direct interaction of dynein with the bilayer may thus be physiologically relevant in the retrograde transport of vesicles, although a role of membrane-bound docking proteins cannot be excluded (Vallee and Sheetz, 1996). Kinesin, bound to a specific receptor, may override the minus-end motor, which is bound with relatively low affinity. Vesicles without a specific receptor thus move to the minus end, whereas all vesicles with a kinesinspecific receptor move to the plus end (Muresan et al., 1996). Intriguingly, PI 3-kinase has been implicated in transport of membranes (De Camilli et al., 1996). Locally increased production of phosphoinositides with a high negative charge may recruit dynein or related molecules to specific membrane compartments. D. Dynamin I

Dynamin I (94 and 96 kDa) belongs to a novel family of GTP-binding proteins and is involved in the release of coated endocytotic vesicles from the plasma membrane. The 94-kDa dynein isoform in neural tissue from Drosophila has been shown to remain associated with membranes as a peripheral protein after cell fractionation (Gass et al., 1995). The protein contains a conserved, GTP-binding N-terminal domain, a pleckstrin homology (PH) domain, and a highly basic, carboxy-terminal domain (ca 100 amino acids; pl, 12.5). This domain binds microtubules and may carry a protein kinase C phosphorylation site (Vallee and Shpetner, 1993). Liu et al. (1994) showed that dynamin I binds to glass beads coated with a mixture of PC and PS, an interaction enhanced by 30-300 p M Ca2+but inhibited by Mg2+,GTP, and ATP. Ionic interactions play a role because 300 mM NaCl partially inhibited binding. Using sonicated liposomes containing 25% PS, PI, or PG, with the remainder being PC, Tuma et al. (1993) demonstrated a five- to seven-fold stimula-

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tion of dynamin GTPase activity by these lipids. PC was inactive. Endogenous brain membrane vesicles also accelerate dynamin GTPase activity as well as microtubules. Phospholipids do not lead to further activation in the presence of microtubules, possibly suggesting interaction at the same site. The basic C-terminal domain of dynamin may be involved in binding to both acidic C termini of tubulin subunits and acidic phospholipids. Indeed, an 80-kDa truncated form of dynein lacking the C-terminal domain shows no enhancement of GTPase activity by acidic phospholipids. The relevance of dynamin-microtubule association is unclear because dynamin appears to be membrane associated but not microtubule associated in intact cells. Further information on the possible mechanism of membrane- and phospholipid-induced activation of dynamin GTPase has been provided by Tuma and Collins (1995). They show, using a zero-length cross-linking agent, that a crude coated vesicle preparation from rat brain and PScontaining liposomes induce formation of dynamin dimers, trimers, and tetramers. The C-terminal domain appears to be required for membrane binding and subsequent oligomerization, although dimer formation also occurs in a fragment lacking this domain. Interestingly, cross-linking provided no evidence for the presence of a membrane receptor. The authors propose that cooperative associations between dynamin molecules bound to a polymeric surface are responsible for activation of GTP hydrolysis. Lin and Gilman (1996) tested the effect of phosphoinositides and of G protein Py subunit on dynamin GTPase activity. Sonicated liposomes containing 10% PIP-2 and 90% PC induce a 5.4-fold activation of dynamin GTPase, whereas PC, PE, PI, or PIP are inactive and PS induces only a very moderate activation. Salim et al. (1996) similarly found GTPase activation of dynamin by PIP and PIP-2 (10% phosphoinositides and 90% PC) but not by PS, PE, PG, PI, and PC. These findings are somewhat in contrast to those by Tuma et al. (1993), who found substantial effects of PI and PS. The discrepancies may be due to differences in protein and lipid concentrations. Other factors also regulate GTPase activity because G protein by subunit inhibits dynamin GTPase activity markedly at low dynamin concentrations, an effect enhanced by PIP-2 (Lin and Gilman, 1996). Very likely, the activation of dynamin GTPase by PIP-2 involves the inositol head group because D-IP-3 can activate the enzyme to the same extent as PIP-2. Interestingly, PtdIns(3,4,5)P3 was inactive in these assays, suggesting high specificity of the binding pocket (Salim et al., 1996). Salim et al. (1996) have further characterized the PIP-2 binding site in dynamin in a very elegant study. First, they could show that the GTPase activity of a truncated dynamin molecule lacking amino acids 541-618, corresponding to the PH domain, is not activatable by PIP-2 but can still be activated by SH3 domaincontaining proteins. They further studied direct binding of the isolated dynamin PH domain (amino acids 511-630) to phosphoinositides using a

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novel technique. The GST-fusion protein is captured via anti-GST antibodies on a biosensor surface. Large unilamellar liposomes containing 10% phosphoinositides and 90% PC are then added and changes in the refractive index close to the sensor chip surface are measured. Using this technique it could be shown that the dynamin PH domain interacts selectively with PIP-2 and PIP but not with PtdIns(3,4,5)P3, PI, and PC, which is in agreement with the data of Salim et al. (1996) on GTPase activation. The later findings are in contrast to those by Lemmon et al. (1995), who, using centrifugation and gel filtration assays, found no evidence for binding of the dynamin PH domain to PIP-2-containing vesicles. Possibly the assays used were not sensitive enough to detect interaction. Zheng et al. (1996) could detect only a selective interaction of PIP and PIP-2 with the dynamin PH domain using lipids solubilized in SDS. They measured quenching of intrinsic tryptophan fluorescence by lipids. Salim et al. (1996) further mutated several basic amino acids (lysines 535, 539, 554, 562, and 598) in the dynamin PH domain to methionines and found abolishment of phosphoinositide interaction for all these point mutations, defining the binding pocket located between loops 1 and 3 of the PH domain. It is not clear whether only polar interactions are involved. According to Zheng et al. (1996), PIP2 and PIP bind with considerably higher affinity to the dynamin PH domain than inositol (1,4,5)-phosphate, suggesting interactions involving fatty acid side chains. In summary, dynamin interaction with phospholipids may be best explained by the presence on dynamin of two distinct lipid binding sites-one located in the PH domain, specific for phosphoinositides, and one allowing interaction with acidic phospholipids, such as PS, located in the basic,qroline-rich C-terminal domain. The possible relevance of different dynamin domains in targeting the protein to plasma membranes and coated pits has been addressed by Shpetner er al. (1996). They found, using truncated mutants of dynamin expressed in COS-7 cells, that a C-terminal domain (amino acids 651-851), including the basic C terminal and especially amino acids 786-794, is essential for directing dynamin to clathrin-coated pits. The latter sequence is a typical SH3-binding domain. The PH domain is not involved in this process. In contrast, the PH domain as well as amino acids 733-746 and the GTPase region are involved in interaction with nonclathrin-coated regions of the plasma membrane. It remains to be shown whether phosphoinositides and other phospholipids participate in siru in the membrane interaction of dynamin.

VI. Interaction of Intermediate Filaments with Membrane Lipids Intermediate filaments clearly have the capacity to interact in vitro with acidic phopholipids, cholesterol, and triglycerides, using different binding

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sites for charged and neutral lipids. The question of whether intermediate filament-associated proteins such as plectin also share this property has not yet been addressed. Recently, a physiological relevance for these in vitro interactions has emerged. Intermediate filaments appear to be involved in intracellular transport of lipids and in stabilization of lipid droplets. Whether the capacity of PIP-2 to modulate intermediate filament assembly has a physiological role is as yet unknown.

A. Interaction of Intermediate Filament Proteins with Lipids in Vitro Traub and collaborators have analyzed this interaction in great detail, using a number of different techniques such as density gradient centrifugation, electron microscopy, hydrophobic photolabeling, gel filtration chromatography, circular dichroism measurements, and phospholipid vesicle leakage and aggregation. A starting point for these studies was the observation that the intermediate filament protein vimentin, isolated from mammalian cell lines, is highly contaminated with phospho- and neutral lipids (Traub et al., 1985). Vimentin (53 kDa) consists of a central rod domain of approximately 310 amino acids with a high propensity for a-helix formation, a Nterminal head domain rich in arginine, and a negatively charged tail domain (Shoeman and Traub, 1995). Using sucrose and KBr density gradient centrifugation, Traub et al. (1986) demonstrated an interaction of delipidated vimentin isolated from Ehrlich ascites tumor cells and of desmin isplated from porcine smooth muscle with phospholipid vesicles. They observed a selectivity for acidic phospholipids, especially PI and PS, when using sucrose gradients at low ionic strength. The proteins vimentin and desmin require an intact N terminus for interaction and, indeed, the purified N terminus of vimentin is able to interact with phospholipids to the same extent as the intact protein (Traub et al., 1986). The capacity to bind lipids appears to be a general property of intermediate filament proteins because similar results for vimentin were obtained for neurofilament protein and glial fibrillary protein (GFAP). The high content of arginines in the N-terminal domain of vimentin could contribute to binding. However, basic histones containing 13 or 14% arginines do not interact significantly in these assays, suggesting that a positive charge alone is not sufficient (Traub et af., 1986). Indeed, hydrophobic interactions also appear to be important, as demonstrated using hydrophobic photolabeling. Vimentin was shown to be labeled by a phosphatidylcholine derivative containing a carbene-generating probe at the end of a fatty acid, indicating insertion into the bilayer. Vimentin also reacted with 1-azidopyrene when associated with liposomes (Perides et al., 1987). 1-Azidopyrene has been shown to label membrane-associated

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domains of rhodopsin, and labeling is not significantly reduced by a watersoluble nitrene scavenger such as glutathione (Smith er al., 1981). Labeling of vimentin required the presence of acidic phospholipids (PS, PI, PG, and PA) and also occurs at physiological ionic strength. Cholesterol plays a synergistic role in this interaction. The authors moreover confirmed the presence of a lipid binding site in the N-terminal domain by hydrophobically labeling the intact protein, followed by proteolytic degradation and analysis of radioactivity bound to the fragments. Incorporation was only observed into the N-terminal domain (amino acids 1-96). Circular dichroism measurements indicate an increase of a-helical content to 30% upon conact of vimentin with PI-containing vesicles. Vimentin may thus, in a first step, interact electrostatically with the negatively charged phospholipid head groups, followed by a conformational change and insertion into the bilayer, similar to findings with the actin-binding protein vinculin (Niggli et al., 1986). The N terminus of vimentin also contains a relatively high amount of hydroxylated and hydrophobic amino acids that could mediate the interaction (Perides et al., 1987). An indication for insertion into the bilayer and ensuing disruption is also the finding that intact vimentin and the N terminus of vimentin induce at low concentrations rapid leakage of fluorescent molecules from the interior of liposomes containing PI, a process inhibited by 100 p M Ca2' (Horkovics-Kovats and Traub, 1990, 1991). Vimentin also appears to contain a second binding site for neutral lipids in its a-helical rod domain (Traub et al., 1987). Using gel filtration chromatography, sucrose density gradient centrifugation, and electron microscopy, an efficient association of delipidated vimentin with cholesterol, cholesterol fatty acid esters, and mono-, di-and triglycerides was observed. Not only does monomeric vimentin at low salt interacts with neutral lipids but also vimentin polymerized to filaments in 150 m M KCl. The purified N terminus is inactive, whereas the a-helical rod domain binds these lipids as efficiently as the intact protein (Traub et al., 1987). Traub and collaborators also analyzed functional effects of lipids on vimentin. They found, using electron microscopy, that liposomes containing 20% of acidic phospholipids interact closely with vimentin filaments. However, when added to monomeric vimentin, at the initiation of polymerization, acidic phospholipids totally block vimentin filament assembly. At low concentrations PIP-2 and PIP are most active followed by PG, PA, PI, and PS. PC, SM, and P E are inactive but appear to associate with filaments according to electron microscopy studies. The latter interaction appears to be of low affinity, however, because these lipids dissociate in sucrose density gradients. Moreover, association of vimentin filaments appears to depend on the fatty acid species present in PC: Saturated, but not unsaturated fatty acids supported an interaction. PIP-2 and PIP are also most active in inducing disruption of preformed filaments and in inducing labeling of

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vimentin filaments with the hydrophobic probe 1-azidopyrene. Because the N termini are essential for vimentin filament assembly, acidic phospholipids very likely block assembly by interacting with these domains (Perides et al., 1986a,b). Because 3 p M of phosphoinositides is sufficient to induce disassembly, they could also play a role in situ. However, no further information is available on this point. Leterrier et al. (1996) observed a modulatory effect of PIP-2 on neurofilaments purified from bovine spinal cord. PIP-2 (10 p M ) increases the elastic storage shear modulus of neurofilaments by 200%. Neurofilaments also break at larger stress values in the presence of this lipid. PIP is not effective. PIP-2 thus selectively increases flexibility of the filaments, possibly by affecting neurofilament cross-bridges.

6.Evidence for in Situ Interaction of Intermediate Filament Proteins with Lipids Two special types of in situ interactions of intermediate filaments with lipids have been documented: an association of intermediate filaments with lipid droplets containing cholesterol or triglycerides and colocalization of glycosphingolipids with intermediate filaments in intact cells. Franke ef al. (1987) first described that highly ordered, paracrystalline sheets of vimentin are formed around triglyceride lipid globules during adipocyte conversion in murine 3T3-L1 cells using immunofluorescence staining and electron microscopy. Practically all the cellular vimentin is associated with the lipid globules. In contrast, neither actin filaments nor tubulin showed any association. The reorganization of vimentin is not accompanied by a change in the level of polymerized vimentin. Vimentin filaments are also closely associated with lipid droplets containing cholesterol in bovine adrenal cells, as documented by immunogold electron microscopy and transmission electron microscopy (Almahbobi et d., 1992; Almahbobi, 1995).The filaments form an interconnecting network enveloping the entire droplet, although the array is not as regular as that described by Franke et al. (1987). A different type of interaction was observed by Gillard et al. (1991,1992). In human umbilical vein endothelial cells, a colocalization could be shown of vimentin filaments with glycosphingolipids using immunofluorescence staining and immunogold electron microscopy. Similar observations were made for fibroblasts, smooth muscle cells (desmin), keratinocytes (keratin), and glial cells (GFAP). The extent of colocalization appears to be rather variable in different cell types. It is most striking after treatment of cells with colcemid, which results in perinuclear intermediate filament bundles. The finding is not due to unspecific cross-reaction because the antiglycosphingolipid antibody does not cross-react with fibroblast proteins on

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nitrocellulose blots. Kotani et al. (1994) similarly observed colocalization of the ganglioside GM2with vimentin in fibroblasts using immunofluorescence. The previous observations do not allow to distinguish between a direct association of the lipids with the filaments and an association of membrane vesicles containing these lipids. In support of a direct association, Asch et al. (1990) found that keratins in the Triton X-100-insoluble cytoskeleton from mammary gland primary cell cultures still contain tripalmitin and possibly cholesteryl esters extractable with,organic solvents. Upon labeling of cells with [3H]palmitate, protein bands corresponding to keratins still have radioactivity bound that is extractable with organic solvents, even after SDS-PAGE. Kotani et al. (1994) found that vimentin isolated from the Triton X-100-insoluble cytoskeleton contains strongly bound ganglioside GM2,as detected using an antibody against this lipid. Upon separation by SDS-PAGE, without boiling this lipid is still bound to vimentin, but it dissociates after boiling. All these observations support a physiological relevance of the interactions of intermediate filament proteins with neutral and acidic lipids observed in vitro. Intermediate filaments may be involved in intracellular transport of lipids and in stabilization of lipid droplets. Sarria et al. (1992) provide evidence for the former notion using cell lines derived from a tumor of the adrenal cortex that either lack or contain vimentin as the only intermediate filament protein. They show that decreased esterification of cholesterol occurs in vimentin-free cells. Reexpression of vimentin led to increased cholesterol esterification, in correlation with the level of vimentin expression. This finding suggests that delivery of cholesterol to the site of esterification (ER) is dependent on vimentin filaments. Lieber and Evans (1996) provide evidence for a role of vimentin in stabilizing lipid droplets in 3T3-L1 cells. First, nocodazole leads to a collapse of intermediate filaments and to a significantly reduced production of triglyceride-containing lipid droplets. Second, microinjection of antivimentin antibodies leads to a 50% reduction in formation of lipid droplets. Third, expression of a truncated form of vimentin results in aggregation of filaments and significant inhibition of lipid droplet formation.

VII. Conclusions

Growing interest and significance has led to the accumulation of new data concerning the covalent and noncovalent modifications of cytoskeletal proteins by lipids. Cytoskeleton-lipid interactions seem to be involved in mediating the anchorage of the cytoskeleton in the membrane bilayer and also in defining the architecture of specific membrane areas. The application of

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biophysical techniques, such as neutron reflection, DSC, and CD spectroscopy, or hydrophobic photolabeling of membrane constituants now allow the analysis of the folding and insertion behavior of cytoskeletal proteins during their electrostatic and hydrophobic interactions with lipid molecules after reconstitution into planar lipid mono- and bilayers as well as into liposomes. The direct in v i m interaction of a number of cytoskeletal proteins with lipids frequently has been regarded skeptically as an in vitro artifact without having any physiological relevance. Solid evidence has now been provided that contradicts this view. The identification of a binding site in dynamin with a selectivity for PIP-2, which does not interact with PtIns(3,4,5)P3, argues against proteins acting simply as ion exchangers that bind to any acidic phospholipid through a basic protein domain. Obviously, highly specific lipid-binding pockets exist, and we consider it extremely unlikely that such domains have no physiological relevance. As summerized in Table I, examples for lipid binding sites in cytoskeletal proteins exhibit a precise spacing of basic residues but also of nonpolar amino acids such as leucine, valine, etc., which may be of importance. Basic residues are often flanked by clusters of hydrophobic residues as, for example, in vinculin (aa 913-940). The notion that many cytoskeleton-associated proteins may be regulated by lipids in a selective and specific manner particularly stresses the impact that protein-lipid interactions may have on structure-function relations and thus on signaling pathways originating at membrane interfaces.

Acknowledgments The authors’ work is supported by Grants Is 25/7-2 and SFB 266; C-5 from the Deutsche Forschungsgemeinschaft to G. I. and a grant from the Swiss National Science Foundation to V. N. V. N. greatly acknowledges the comments of Dr. E. Sigel on sections V and VI and continuous support by Prof. H. U. Keller. Dr. E. Sackmann has kindly provided Fig. 1. We thank Dr. S. Kaufmann for organizing the manuscript.

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Cell Biology of Autoimmune Diseases Johannes M. Van Noort* and Sandra Amort *Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden, The Netherlands; and ?Department of Immunology, The Rayne Institute, St. Thomas’ Hospital, London SE1 7EH, United Kingdom

Autoimmune diseases such as insulin-dependentdiabetes mellitus, rheumatoid arthritis, and multiple sclerosis are common in the western world and are often devastating diseases which pose serious health problems. The key feature of such diseases is the development and persistence of inflammatory processes in the apparent absence of pathogens, leading to chronic breakdown of selected tissues. To date, no comprehensive explanation can be given for the onset or persistence of autoimmunity. As a rule, the chronic activation of helper T lymphocytes reactive against self proteins appears to be crucial for fueling the destructive autoimmune process, but why this occurs remains to be established. In this review, we present an overview on the rules that govern activation of T lymphocytes and on the factors that control it. The contribution of both genetic and environmentalfactors are discussed, clarifying that most autoimmune disease are of multifactorial origin. Special emphasis is given to the contribution of infectious events and the role of stress proteins in the process. In attempts to dissect the mechanisms involved in autoimmunity and to develop ways of blocking disease, experimental animal models are widely employed. We describe the various experimental models that exist for the study of multiple sclerosis, diabetes, and other autoimmune diseases and on the experience that has been gained in such models with experimental therapies to block the activation of self-reactive T lymphocytes. The lessons that can be drawn from these studies provide hope that continued efforts will lead to the successful development of antigen-specific strategies which block the development of autoimmunity also in humans. KEY WORDS: Autoimmune diseases, Autoreactive T lymphocytes, Immune therapy, Stress proteins, Multiple sclerosis, Allergic encephalomyelitis.

1. Introduction

Inflammatory processes usually develop only when pathogens invade the body. Under those conditions, cells of the immune system recognize struclnrernarional Review of Cyrology. V d 178

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tures and molecular signals that are different from what the body is used to and an immune response ensues. Sometimes, however, inflammatory processes develop in the apparent absence of pathogens and these processes may become persistent. Different tissues may become affected or sometimes even completely destroyed by such events. Resulting disease, often chronic, may lead to lifelong impairment or even death. When this happens, and no pathogen can be held responsible for the onset of destructive immune processes, we are dealing with autoimmune diseases. Well-known examples of such disease are rheumatoid arthritis, insulin-dependent diabetes, and multiple sclerosis. The study of autoimmune diseases is an important research area in immunology, fueled by the hope that understanding the molecular basis of such disease will allow rational development of therapy. In this review, we address some questions that are pertinent to autoimmunity and review current ideas on the cell biological aspects of disease mechanisms. During the inflammatory process leading to tissue destruction, a variety of cells traffick and communicate, they secrete signaling and effector molecules, and respond to molecular signals in their environment. We will review important mechanisms and relationships in this respect and discuss factors that, together, determine whether the immune system may enter the pathway to autoimmunity. Also, we will deal with models for autoimmune disease and with attempts to block autoimmunity in that context. Lessons that can be drawn from those studies are encouraging and the perspectives they offer justify the hope that continued studies will ultimately allow the design of successful intervention.

A. The Spectrum of Autoimmune Diseases In some autoimmune diseases autoreactive T cells or antibodies are specific for antigens found only in the target organ, whereas in other autoimmune diseases there is involvement of many tissues and widespread distribution of the antigen(s). The former so-called “organ-specific’’ autoimmune diseases are best exemplified by Hashimoto’s disease in which autoantibodies react with the thymus tissue giving rise to highly localized lesions. In contrast are the “non-organ-specific” diseases in which many tissues are affected due either to the widespread nature of a single autoantigen or to the recognition of many autoantigens. Systemic lupus erythematosus (SLE) is such an example. These two diseases represent different ends of the spectrum of autoimmune diseases and although many autoimmune diseases may be easy to classify, others do not fall neatly into either category. For the purpose of this review we will focus only on some autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis (MG), SLE, and insulin-dependent diabetes mellitus (IDDM). These

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examples cover the spectrum of non-organ-specific as well as organ-specific diseases, although there may be controversies as to the category to which some diseases belong. Throughout the chapter, MS will be taken as an example to illustrate the points raised. This is not to imply that the issues dealt with in this review apply only to MS; it is simply the area of research that we are most familiar with. The precise role that B cells and antibody, T cells and other immune cells, and soluble factors of the immune response play in the initiation and perpetuation of autoimmune diseases in humans can only be speculated on. Likewise, the question of which self-antigens trigger disease in humans cannot be definitively answered as yet. Thus, it is useful to turn to experimental situations that attempt to mimic the human condition. In such a setting, well-defined self-antigens can be examined on their potential to induce pathogenic immune responses. In general, the association of a particular self-antigen-directed response to an autoimmune disease should fulfill the following criteria, although obviously other considerations may also be applicable (Rose and Bona, 1993): a. The autoantigen and immune responses against it should correlate with disease. b. Removal of the antigen-specific response cures the disease. c. Transfer of antigen-specific T cells or antibodies induces the disease in animal systems. d. Induction of the specific response re-creates the disease. Very few autoimmune diseases fulfill such criteria, although an excellent example of one that does is MG. Antibodies to the acetylcholine receptor (AChR) are thought to be the major autoreactive factor in this disease and several experiments have shown that transfer of anti-AChR antibodies from humans with MG leads to disease in mice. Furthermore, plasmapheresis of patients ameliorates disease due t o a reduction in levels of anti-AChR antibodies. Additionally, in several other antibody-mediated autoimmune diseases clinical signs may also be observed in the infants of affected mothers as a result of immunoglobulins crossing the placenta. Thus, antibodies to thryoid-stimulating hormone receptor in a pregnant patient with Grave’s disease will induce hyperthyroidism in her newborn, although this may be corrected by plasmapheresis. In other autoimmune diseases it is more difficult to identify the autoantigen, or, indeed, the autoreactive T cell or antibody responsible for disease. MS, for example, is an inflammatory and demyelinating disease of the central nervous system. Figure 1 shows a demyelinated region in the white matter of an MS patients, showing how inflammatory processes in MS can fully destroy the protective myelin sheath of axons in the central nervous system. Extensive studies have attempted to define the target antigens and the role of immune

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FIG. 1 Multiple sclerosis plaque showing lesions of demyelination in the CNS. As the final result of local activation of T cells, massively infiltrated and activated macrophages strip the protective myelin sheath off axons. The inflammatory degradative process occurs only in the white matter of brain and spinal cord. The pale area represents the region where demyelination has occurred.

factors in the pathogenesis of disease but to date these are elusive. It is probable that for this disease the cause is multifactorial and experimental models, both viral and autoimmune, have defined the importance of both autoreactive T cells and autoantibodies and other contributing factors such that the criteria listed previously may be very difficult to fulfill. In the

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experimental model of MS, i.e., experimental allergic (or autoimmune) encephalomyelitis (EAE) (see Section IV,A), disease can be clearly mediated by major histocompatibility complex (MHC) class 11-restricted CD4+T lymphocytes. These are lymphocytes that bear the CD4 receptor and recognize a pathogenic peptide in the context of MHC class I1 molecules on antigen presenting cells. As a rule, T cells recognize a complex of a peptide fragment bound to a MHC molecule. For CD4 lymphocytes, the appropriate context is provided by MHC class I1 molecules and for CD8 lymphocytes by MHC class I molecules. Furthermore, lymphocytes will only recognize the peptides if presented by a MHC molecule compatible with the lymphocyte. This concept of MHC restriction earned Zinkernagel and Doherty the Nobel prize for medicine in 1996 (reviewed by Zinkernagel and Doherty, 1997). However, although EAE can be mediated by CD4' lymphocytes, directing studies in MS to also concentrate on the T lymphocyte, antibodies do play an important role. This holds not only for EAE but most likely also for MS. It can be safely assumed that in other autoimmune diseases originally thought to be T cell mediated, disease will turn out to be T cell dependent with antibodies playing a synergistic role.

6.Organ Specificity in Autoimmunity The entry of leukocytes into an organ or tissue is determined by specific combinations of adhesion and signaling molecules expressed on both the endothelial cells of the organ and the migrating cell itself (see Section 11,A). However, the question of why particular pathogenic cells are effective only in a specific organ giving rise to localized damage is more difficult to clarify. To explain the exquisite specificity of lymphocyte trafficking it would be ideal to identify adhesion molecules specific for each organ, limiting migration to only those cells that recognize specific molecules within the target tissue. The most significant distinction in lymphocyte homing concerns the trafficking of naive and memory/effector populations, the latter of which display tissue-selective patterns of recirculation to sites where they are more likely to encounter, or indeed reencounter, their specific antigen. For some tissues a specific homing receptor is known. One example is cutaneous lymphocyte-associated antigen, which directs memory T cells to the skin and the mucosal addressin (Butcher and Picker, 1996). It remains to be seen whether CNS-specific adhesion molecules will be found in, e.g., MS, or synovial-specific adhesion molecules in RA. Aside from local production of cytokines within tissues, chemokines must also be considered as agents involved in imparting organ specificity because of their chemotactic specificity for functionally distinct lymphocyte subsets.

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By far the major factor that probably determines specificity is the expression and “correct” presentation of the autoantigen in the target organ enabling the local activation of potentially pathogenic effector cells. An obvious factor in antigen presentation to T cells that contributes to tissue specificity is tissue-restricted expression of the target antigen. However, with the advent of more sensitive detection systems for both protein and messenger RNA, previously presumed tissue restriction in the expression of various self-antigens has frequently turned out to be much less obvious than once believed. It is becoming increasingly likely that factors other than tissue-restricted expression also control the phenomenon of tissue selectivity in autoimmune diseases. In Section II,B, it will be discussed that the initiation of specific T cell responses in tissues requires more than just the presence of the correct antigen alone. It may well be those additional, costimulatory factors that play a role in determining organ specificity, with one type of tissue being able to provide them whereas another cannot. One mechanism by which entry of circulating lymphocytes into tissue may occur is via recognition of autoantigen in the context of class I1 antigens-together with the correct costimulatory molecules-on the endothelial cells of the target organ. That CNS endothelial cells expressing MHC class I1 are able to present a relevant CNS antigen, i.e., myelin basic protein (MBP), to T cells has been demonstrated to occur in EAE (Wilcox et af., 1989), although other cells, e.g., pericytes, associated with the perivascular region may also function as antigen presenting cells. Such presentation and recognition in the vascular region may allow activation of T lymphocytes and subsequent migration into the target organ where they exert their effect. However, antigen presentation is not necessarily a prerequisite for the transmigration of T cells through the blood-brain barrier as shown by the studies using bone marrow chimeras. Activated MBP-specific T cells from strain A mouse injected into irradiated strain B (which had been previously reconstituted with bone marrow from strain A) were able to migrate across the bloodbrain barrier and induce EAE in the absence of antigen presentation by strain B cells (Hickey et af., 1991). These elegant studies show that factors other than CNS-specific antigen recognition control the transmigration of the activated T cells. Once within the CNS, however, the antigen-specific cells meet their target antigen and initiate recruitment of nonspecific cells (Cross et af., 1990). Another way by which tissue-restricted damage may be controlled is via antibodies. Important degradative processes are mediated by macrophages. These cells carry surface receptors for the Fc portion of antibodies thus allowing such antibodies to target specifically activated macrophages to certain sites. In this way, degradation may be intensified at sites marked by antibodies, explaining the synergistic effect tissue-specific antibodies may have in autoimmunity.

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II. Immune Mechanisms in Autoimmune Diseases A. Lymphocyte Trafficking Rules that govern the movement of lymphocytes into tissues are complex and they are very much determined by the state of T and B cells themselves. Naive resting T and B cells have a selective homing pattern into, for example, lymph nodes via so-called high endothelial venules. Lymphoblasts and memory T and B cells may display selective migratory behavior to skin, mucosal epithelium, or other types of tissues. Finally, all T and B lymphocytes together with neutrophils and macrophages may transmigrate into inflamed tissues in response to localized signals that mediate their trafficking. In the context of this review, the extravasation of blood leukocytes into inflamed tissue will be discussed because it is obvious from histopathological studies that this process occurs in tissue-specific autoimmune diseases. Although it is likely that more specific events precede the massive infiltration of affected tissue by leukocytes, these events are currently unclear. Apart from being mediated by surface adhesion molecules, trafficking of leukocytes is also influenced by soluble mediators secreted by endothelial cells including chemokines of various types. These may selectively activate lymphocytes and favor transmigration of certain types of leukocytes over others (Del Pozo et al., 1996). In this review, however, we will limit ourselves to the most important surface-expressed adhesion molecules (Springer, 1990). Below, it will be clarified that T cell trafficking is influenced by its state of activation. Activated T cells migrate much easier than resting cells. On the other hand, even resting or naive T cells can be efficiently captured by endothelial cells and recruited into the tissue when the endothelial cells themselves provide the appropriate signals. It still remains to be established whether specifically activated T cells or, conversely, distressed tissues themselves provide the initiating signals for massive leukocyte trafficking as is seen in autoimmunity. Several steps in the process of leukocyte extravasation can be discerned, each involving cross-talk between surface molecules of leukocytes and those of endothelial cells. Figure 2 illustrates these steps and the molecules involved. The first step in the interaction between circulating leukocytes and endothelium is defined as capture: initial interactions slowing down leukocytes that rapidly flow by in the bloodstream. In this initial step, the selectin family of adhesion molecules on the surface of endothelial cells plays a crucial role. P-selectin in particular is important in the capture of leukocytes, causing them to slow down and roll over the vascular endothelium. P-selectin is constitutively found in secretory granules of endothelial cells and certain triggers such as cytokine signals may cause its rapid mobilization onto the endothelial plasma membrane.

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FIG. 2 Main molecular interactions during leukocyte extravasation. Transmigration of leukocytes is effected in several stages. First, leukocytes flowing by in the bloodstream are captured by endothelial cells through interactions that are primarily mediated by selectins. P- and Eselectins on endothelial cells and L-selectin on leukocytes are the main players in this process. These interactions result in the slowing down of leukocytes, causing them to roll over the endothelial surface to a final arrest. Second, additional interactions between pairs of integrins mediate opening of the endothelial surface and penetration of leukocytes into the tissue. Major integrins involves at this stage are VLA-4 and LFA-1 on the surface of leukocytes that can bind to VCAM-1 and ICAM-1, respectively, on the surface of endothelial cells.

The molecule recognized by P-selectin is P-selectin glycoprotein ligand-1 (PSGL-l), a dimeric mucin on the surface of leukocytes. Another player in the process, L-selectin, is constitutively present on the surface of most circulating leukocytes and it may recognize a glycoprotein of approximately 50 kDa that is universally present on vascular endothelium, but only if this molecule is appropriately glycosylated. Finally, additional recognition steps take place between E-selectin on the surface of endothelial cells and Eselectin ligands on leukocytes surfaces, probably including PSGL-l. These interactions will allow the slowing down of leukocytes and their final arrest. It is of interest to note that the molecules on the endothelial surface that play a role in these initial steps may be inducible, whereas those on the surface of leukocytes are generally present in a constitutive way. This implies that the vascular endothelium of tissues itself has a strong hand in regulating leukocyte arrest. After leukocytes have been captured and immobilized on the surface of vascular endothelium, additional interactions-mostly via integrins-may

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occur that will finally determine whether leukocytes will actually transmigrate. These interactions include recognition between pairs of adhesion molecules such as between lymphocyte function-associated molecule-1 (LFA-1, an a& integrin) on the surface of leukocytes and intercellular adhesion molecules-1 and -2 (ICAM-1 and -2) on the surface of endothelial cells and between very late antigen-4 (VLA-4, an a& integrin on leukocytes) and VCAM-1 (on endothelium). In these interactions, the state of activation of lymphocytes involved plays a more distinct role: Levels of expression of LFA-1 and VLA-4 on T cells, for example, are strongly elevated when these cells are in activated state and, thus, such cells are more likely to rapidly extravasate. Also, memory T cells have approximately two- or threefold higher expression of these molecules than naive T cells. Again, however, the state of activation of endothelial cells is important and their interaction with leukocytes at this stage is firmly augmented when, for example, proinflammatory cytokines are present that boost expression of endothelial adhesion molecules. Finally, the process of mutual recognition results in the transmigration of leukocytes through the vascular endothelium into the tissue: The extracellular matrix is partially removed by proteases and leukocytes are pulled through.

6. Lymphocyte Activation and the Importance of Costimulation

The activation of T lymphocytes depends on a number of signals that are required to initiate the cascade of intracellular second messengers leading to the transcription of genes that mediate immune effector functions. These include genes encoding surface molecules for trafficking and communication with other cells as well as genes encoding soluble factors such as cytokines that may have both autocrine and paracrine regulatory effects. Of prime importance for controlling this array of effector functions is the T cell receptor (TcR), a highly variable surface structure. T cell receptors are built in such a way that they recognize a combination of a short protein sequence entrapped in the so-called peptide-binding cleft of MHC molecules on the surface of other cells. Hundreds of millions of different variant TcRs are present, each on a different cell and each tailored to recognize another MHC-entrapped protein sequence. Coreceptors, however, are extremely important in determining the way the T cell responds to recognition of its target structure via the TcR. In this discussion, we will limit ourselves to helper T cells that are of prime importance in controlling immune responses by other effector cells such as macrophages and antibody-producing B cells.

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Helper T cells are designed to recognize protein sequences in the context of MHC class I1 molecules (Harding and Unanue, 1990;Brodsky and Guagliardi, 1991; Germain, 1994). As a rule these sequences are generated from extracellular material when this is ingested and partially degraded by proteolytic enzymes (see Section V,A). The T cells themselves do not perform this function but another cell is required, a so-called antigen presenting cell (APC). Dendritic cells, macrophages, and B cells can be effective in this respect because they not only perform the peptidegenerating function but also express the necessary MHC class I1 molecules, molecules that are not present on the surface of all cells of the body. In some cases, peptides presented by MHC class I1 molecules can be derived from intracellular proteins (Weiss and Bogen, 1991), but this is a minor route, probably only applicable to intracellular proteins that are long-lived in the cytosol. In search for their appropriate antigen-MHC target, TcRs may encounter peptide-MHC complexes that may not be entirely what the TcRs were looking for but are structurally sufficiently complementary to the receptor to allow at least some interaction. Over the past few years, it has become apparent that this degenerate or partial recognition, as well as the strength of the interaction, may have significant influences on how T cells will respond. In fact, it is the quality of the response by the T cell rather than its intensity that is affected by these events of sometimes partial recognition. There is a high level of complexity in the intracellular signaling pathway switched on by full engagement of the T cell receptor and partial engagement of the receptor may lead to activation of some elements in this pathway (for example, membrane-bound events or intracellular phosphorylation of selected second messengers) while failing to trigger the whole repertoire of signaling functions. Thus, some functions may be switched on while others will not and this will become visible by a qualitatively different response at the end. Dependent on the readout system one employs to measure T cell activation (for example, proliferation, production of certain cytokines, or cytotoxic potential) one will detect responses in some of the readout systems but not in others (Kumar et al., 1995). In some cases, partial engagement of the TcR may even lead to shutting down of the T cell, disabling it to properly respond to its authentic target structure later on (Sloan-Lancester et al., 1994). The underlying mechanism causing these effects is very subtle and difficult to grasp in simple rules because it is dependent on the exact way the TcR makes contact with its target. Therefore, the rules are different for every TcR. For defined TcR structures, peptide sequences can be identified that will trigger a grossly different response compared to the natural target of the T cell. Such experimentally altered peptide ligands (APLs) may be used for manipulating specific T cell responses in a predictable manner (Sloan-Lancester et al., 1993). This

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interesting option is further discussed in Section VI,B. The previous effect is also relevant in real life. It will probably play a role in organizing an appropriate immune response to abundant versus scarce antigens and it will most certainly influence the process of thymic selection of T cells. Recognition of peptide-MHC complexes is key to the activation of T cells as a first signal. On its own, however, it is only sufficient for the continued triggering of T cells that are already in an activated state. Primed, resting T cells as well as naive resting T cells require more than this. In fact, recognition of just peptide-MHC complexes by resting T cells may have quite the opposite effect and this first signal alone may induce such cells to become unresponsive or anergic (Mueller et al., 1989; Schwartz, 1992; Harding er al., 1992), and T cells may even die from such an encounter (Russell et al., 1991). In order to understand these effects it is important to appreciate the role of other interactions that take place between T cells and antigen presenting cells in addition to the recognition of peptide-MHC by TcR. Apart from the first signal a T cell receives by the MHC-peptide complex, a second signal can be given by so-called costimulatory molecules-sets of complementary molecules on the surfaces of T cells and APCs. So-called costimulatory molecules on the surface of the APC include B7-1 (CD80) and B7-2 (CD86) and these may interact with their ligand molecules on the surface of T cells designated CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4). At later stages, additional costimulatory interactions between T cells and APCs are mediated by CD40 on the surface of APCs and the CD40 ligand (CD40L) that appears on the surface of activated T cells. This interaction is of prime importance in the onset of antibody-mediated responses. Other molecular interactions between T cells and APCs take place but a discussion of these is beyond the scope of this review (the reader is kindly referred to an excellent review by Springer, 1990). Activation of resting T cells by peptide-MHC complexes will occur only when costimulation by B7-1 or B7-2 takes place simultaneously. This appears to be the case for all resting T cells, be it naive or memory T cells. It is important to note that the expression of costimulatory molecules, such as B7-1 and B7-2, that are therefore essential to start both primary and secondary T cell responses is not a constitutive property of antigen presenting cells but an inducible one, dependent on a variety of signals. As with T cell trafficking, T cell activation therefore has evolved to become dependent on inducible signals and it is regulated in this way. This phenomenon is extremely important for the maintenance of tolerance as will be discussed in Section II,E. Although CD28, the low-affinity ligand for B7-1 and B7-2, is constitutively expressed on all T cells, the high-affinity ligand CTLA-4 is inducible. On the other hand, B7-1 and B7-2 can be detected on most dendritic cells, B cells, and macrophages but the constitutive low levels may be insufficient

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for proper costimulation. A variety of triggers that can generally be referred to as stress (see Sections II,E and II1,C) may induce strong upregulation of B7-1 and B7-2 with some inducers leading to the expression of both molecules, whereas other triggers may induce just one of them. Apart from providing the essential costimulation of T cells that allows their productive activation, details of the interaction between B7-1 and B7-2 on APCs with CD28 and CTLA-4 on T cells influence the quality of the response that is initiated. By using animal models, including mice, that lack either one of these costimulation signals and by studying the effects of experimentally blocking B7-1 or B7-2, intriguing phenomena were recorded indicating a role of these interactions in steering T cell responses to either proinflammatory effector functions or regulatory functions and assisting antibody responses (Jenkins, 1994). For example, blocking CTLA-4 and B7-2 interactions leads to a blockade in the development of spontaneous diabetes in nonobese diabetic (NOD) mice (Lenschow et al., 1995). On the other hand, development of an experimentally induced form of autoimmunity, i.e., EAE, is blocked by antibodies against B7-1, whereas anti-B7-2 antibodies exacerbate disease (Kuchroo et al., 1995; Racke et al., 1995). Clear-cut rules on exactly how the interactions between CD28 and CTLA-4 on T cells with their counterparts B7-1 or B7-2 on APCs affect the outcome of immune responses cannot be defined as yet because some of the currently available data are apparently conflicting. Most likely, the signaling effects are equally subtle, as in the case of the TcR signaling pathway described previously, and strongly depend on slight differences in the structure of the complexes that can be formed. In this rapidly developing field, more data may soon be expected to provide perhaps a clearer view.

C. Cytokines and the Regulation of Lymphocyte Effector Functions Once T cells are activated, they may perform a number of effector and signaling functions. These include the activation of other effector cells in the immune system, such as cytotoxic T cells, macrophages, and B cells, via direct cell-to-cell contact as well as by secretion of a large array of signaling molecules including cytokines. However, not all T cells will go on to perform the same set of functions. As clarified in the previous section, engagement of the TcR as well as of costimulatory receptors on T cells will generally activate only a selection of the whole repertoire of effector functions. Selection of which functions are activated and which are not will depend on subtle parameters of the interaction of the T cell with a variety of surface structures on APCs including peptide-MHC and costimulatory molecules. Also, soluble factors are critically involved in determining which

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functions will be developed to an effector stage and which functions will remain silent. The most important soluble factors in this respect are cytokines. Apart from a few exceptions, cytokines usually serve local functions within a short time span. Although immunologists have long regarded cytokines to be their own pet signaling molecules, it is quite clear that cells other than those of the immune system are capable of producing them. Especially in the central nervous system, signaling pathways exist between resident CNS cells that make use of the very same cytokines that are produced and recognized by cells of the immune system. Therefore, especially in this environment, interesting possibilities exist for communication between the CNS and cells of the immune system. This is discussed further in Section II1,E. It is now well accepted that in both rodents and man, certain types of cytokines produced by activated T cells tend to cluster in two groups (Mossman and Coffman, 1989). This clustering of cytokine production during maturation of T cells is depicted in Fig. 3. The first group of cytokines is associated with a response that tends to promote inflammatory processes, cell-mediated immune responses, and less pronounced antibody formation. This type of response is referred to as a type 1 response and the T cells that sustain it are usually called type 1 helper cells (Thl cells). The major cytokines that belong to this group are interferon-y (IFN-y), tumor necrosis factor-a (TNF-a), and lymphotoxin and T cells that produce them also usually produce fair amounts of interleukin-2 (IL-2). The other group is associated with immune responses involving marked antibody production, including IgG2b, IgE, and IgA, with less inflammation-generally referred to as a type 2 response. Accordingly, T cells that are involved in the development of such a response are termed type 2 helper (Th2) cells. Prime cytokines belonging to this group are IL-4, IL-5, and IL-10. Although in most cases mature activated T cells produce cytokines that are representatives of just one of the two groups, this dichotomy is not absolute and hybrid production of both type 1 and type 2 cytokines by a single T cell is not an unusual event. Based on experiences in studies of mucosal immune responses, some have suggested the existence of a third type of T cell, tentatively designated Th3, that would be characterized by mainly secretion of transforming growth factor-p (TGF-P) (Weiner et al., 1994). This cytokine has peculiar, broad suppressive effects in a large array of immune effector systems. This proposal is, however, still controversial and requires further substantiation. A special cytokine is IL-12, which is not produced by T cells but rather by APCs of the monocytelmacrophage lineage. IL-12 appears to be of critical importance in initiating type 1 responses, primarily by triggering the production of IFN-y by T cells. In turn, IFN-y acts as a powerful positive

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humoral responses

IL-5

Y e l g G 2 , IgA, IgE

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FIG. 3 Polarization of cytokine-mediated T cell effector functions. Upon activation of naive T cells the integrated signals from peptide-MHC complexes and costimulatory molecules on the surface of antigen presenting cells codetermine whether T cells will go on to support cellular inflammatory responses (type 1 responses) or rather the production of antibodies (type 2 responses). Also, cytokines play an important role in this development. IL-12 secreted by antigen presenting cells will stimulate maturation of T cells into the direction of Thl cells that produce IFN-y, IL-2, TNF-a, or lymphotoxin (type 1cytokines). In turn, type 1 cytokines provide positive feedback signals on maturing T cells favoring their development into Thl cells. In the absence of IL-12, development may ensue of Th2 cells that mainly produce IL4, IL-5, and IL-10. These type 2 cytokines will provide the maturation signals for the development Th2 cells. Some controversy exists as to the question of whether or not a third type of T cell may develop. It has been claimed that such cells, tentatively designated as Th3, can originate in the intestinal mucosa and mainly produce the modulatory cytokine TGF-P.

feedback signal to APCs for the production of IL-12. However, IL-12 also exerts its proinflammatory function through other pathways, as was observed in mice deficient for the IFN--y receptor (Car er al., 1995). Apart from providing maturation and effector signals to other types of cells, T cell-secreted cytokines also affect T cells themselves. Most important, IL-2 is required for the proliferation of activated T cells themselves and the very same cells that produce it will consume it again for their own growth. In a more general sense, cytokines belonging to either type 1 or type 2 immune responses will favor the maturation and activation of other T cells with the same type of effector function, in this way amplifying the immune response while maintaining the same quality. It is quite likely that

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the previously described dichotomy in T cell effector functions has led to the suggestion for the existence of so-called “suppressor” T cells, i.e., cells clearly suppressing certain effector functions by other T cells. Most likely, the phenomena reported in this context can be attributed to certain populations of T cells with an extreme polarization in cytokine effector function and in the selection of readout systems that were particularly suitable to monitor regulatory or suppressive functions that were exerted by these cytokines. Thus, suppressor T cells are probably just normal T cells that happen to secrete cytokines with a distinct regulatory power in certain experimental readout systems.

D. Termination of a Response: T Lymphocyte Death and Apoptosis Once a specific immune response is initiated it is of obvious interest to the host to be able to terminate it again. As a rule, the signals that control the initiation of the response, i.e., the appearance of peptide-MHC complexes and costimulatory molecules, and the production of T cell-regulating cytokines will also probably be employed for shutting down the response. The most straightforward strategy to accomplish termination is the removal of these initiating signals. Once bacteria and viruses are cleared, antigen derived from these pathogens will no longer be available for presentation to T cells. Once tissue destruction has been resolved and repaired, the induction of nonspecific recruitment and activation factors will similarly cease. In this process, important contributions can be made by corticoid hormones that assist in downregulating costimulatory molecules and proinflammatory cytokines. However, apart from this simple solution it is also useful to actively limit effector functions of the immune system to a well-defined site while immediately terminating them when they threaten to extend into tissues or sites of the body where no responses are required. The fact that cytokines are short-lived and short-ranged mediators ensures that this source of activating signals is unlikely to extend into tissues where no response is needed. More or less the same holds for costimulatory molecules. They are membrane-bound molecules and expressed only on cells that are directly exposed to danger-associated signals, i.e., signals that justify a response. Important to note, however, is the fact that the absence of costimulation does not just fail to trigger T cells; it is also a major signal for active downregulation of the immune response by inducing T cell death via apoptosis. In general, cells may die either via nonspecific destruction or via a wellordered scheme of intracellular events. A major difference lies in the final result. When cells are destroyed nonspecifically they may lyse, fall apart,

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or in other ways disintegrate while releasing intracellular components into their environment. This is generally referred to as necrosis. Necrosis thus results in the exposure of surrounding tissue to the intracellular components of perished cells. As a.result of this exposure these surrounding cells are triggered to produce cytokines and costimulatory molecules. In other words, cell death via necrosis will provide the signals for the onset of immune responses. Getting rid of T cells in this way would therefore not be a very productive strategy for terminating immune responses. A more elegant way for cells to die is via apoptosis, a mechanism that may be specifically designed to prevent intracellular components of the perishing cell to be released. Apoptosis is a morphologically distinct form of programmed cell death (Vaux et af., 1994). Triggered by specific signals, catabolic enzymes degrade macromolecular structures of the cell within the near to intact boundaries of the plasma membrane (Enari et af., 1996). DNA is fragmented and chromatin condenses into characteristic granules. Finally, the cell will be engulfed by and destroyed within macrophages that probably recognize an apoptotic cell by abnormal plasma membrane features. This way, no intracellular components of the dying cell will be released into the environment. Apoptotic death can occur in virtually any cell and it is executed via several stages. First, potentially death-inducing signals are received; in some cases these may be specific death-signaling factors, whereas in other cases they may just be the lack of necessary survival factors. One example of a specific death-inducing signal is ligation between Fas and Fas ligand (or APO-1; Nagata and Goldstein, 1995). Also, receptors for TNF-a and its family members have the capacity to transmit specific death-inducing signals. In addition, more nonspecific mechanisms, such as the lack of nutrients, heat, irradiation, or other forms of metabolic stress, may induce apoptosis when they remain below a certain threshold. In the effector phase of apoptosis, these signals are intracellularly translated and evaluated and the decision to die can be made. Regulation of this stage is still possible and some of the regulatory molecules that play a role have been identified, a major one being the protein encoded by the bcf-2oncogene whose appearance usually (but not always) blocks the execution of apoptosis. Finally, the actual stage of degradation is initiated, and from that point on regulation or rescue are no longer possible. It is only at the last stage that the morphological characteristics of apoptosis become apparent, such as DNA fragmentation, nuclear condensation, and massive protein degradation. Evidence is rapidly accumulating that programmed cell death, and especially FasFasL-mediated apoptosis, is a very important way of controlling and terminating immune responses as well as of maintaining the immune privileged status of various tissues such as the central nervous system and the eyes (Enari et al., 1996; Ju et af., 1995; Ford et af., 1996; Griffith et

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al., 1995). As elaborated previously, the activation of T cells is critically dependent on multiple signals and their integrated effect on intracellular signaling pathways. Especially when T cells have been primed, encounter with antigen-MHC complexes induces a state of dependence on costimulatory signals. When these fail to be delivered, an apoptotic pathway may be entered. Also, engagement of TNF receptors and the Fas/FasL interaction may trigger apoptosis in such cells unless a salvage signal is received (Griffith et al., 1995). Because healthy, resting tissues cannot provide these rescuing signals, the T cells will readily die via apoptosis (Russell et al., 1991). For the central nervous system, for example, it has been shown that resident microglia fail to provide the necessary survival signals to T cells (Ford et al., 1996). At best, T cells may briefly secrete their cytokines as a partial response to local antigen, but it is their swan song: They rapidly die afterwards. In this way, even primed T cells are limited in their action to those sites that really require their activity. Outside these areas, these T cells may survive for as long as they fail to encounter their antigen, but when they do encounter it in the context of a healthy environment they will die.

E. Peripheral Immunological Tolerance: The Danger Model For a long time, it was believed that the adult human T cell repertoire was devoid of T cells capable of recognizing the body’s own proteins. This was supposed to be the result of thymic selection: Emerging T cells from the bone marrow pass the thymus and, at that stage, all cells potentially responsive to “self” targets would die, leaving only T cells fit to recognize “nonself” structures. Because thymocytes (immature T cells) are unable to receive any form of costimulation, their encounter with thymic antigens in the context of MHC molecules will lead to their death if the antigen is recognized with sufficient avidity. This view on removal of potentially selfreactive T cells from the mature repertoire, however, has turned out to be far from complete. Even healthy individuals have large numbers of T cells in their circulation that can recognize protein sequences derived from selfproteins. This is particularly likely to be the case for self-proteins that are not expressed in the thymus and for self-proteins that are inducible, i.e., only expressed at certain developmental stages or expressed as the result of disease or other forms of stress. The question then arises how the immune system deals with these T cells. How are potentially self-reactive T cells prevented from starting autoimmune responses after having escaped thymic deletion? Key to answering this question is the notion that it takes more than anigen-MHC complexes to trigger cellular immune responses. This notion

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professional APC + peptide-MHC no costimulation

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FIG. 4 Emerging rules for T cell activation and tolerance. When a T cell meets its appropriate peptide-MHC complex, it will respond in different ways, dependent on the T cell's state of activation as well as on the status of the antigen presenting cell (APC). Naive T cells will only be activated when the right peptide-MHC complex is presented by full professional APCs such as interdigitating dendritic cells in lymph nodes; all other APCs will cause naive T cells to die. Resting, memory T cells may become reactivated by both professional and amateur APCs, but only if these are activated and provide the appropriate costimulatory signals. Otherwise, memory T cells will die. Already activated effector T cells require only an encounter with peptide-MHC complexes, irrespective of costimulation. After having been triggered, effector T cells will either return to a resting state fairly quickly or they may die, dependent on the APC (reviewed by Matzinger, 1994). A special case is formed by thymocytes, immature T cells in the thymus. These immature T cells are most likely unable to recognize any type of costimulation. As a consequence, the encounter of a thymocyte with its peptideMHC target will always cause the thymocyte to die if the interaction has sufficient avidity. This inability of thymocytes to receive costimulation is probably a major mechanism underlying the phenomenon of clonal deletion.

and its implications have been summarized by Matzinger (1994); Figure 4 depicts the rules for T cell activation that are now becoming clear. As already described in detail, naive as well as resting, primed T cells fail to respond to their peptide-MHC target if they do not receive the appropriate costimulatory signals at the same time. Instead, they may even die from such an encounter. Because a number of self-antigens and even MHC molecules may be constitutively expressed at some sites, of critical importance to the maintenance of peripheral tolerance is the system that regulates the expression of costimulatory signals, notably of B7-1 and B7-2.

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One of the signals that induces these costimulatory molecules are macromolecular structures that d o not occur in mammalian cells and tissues and, therefore, may conveniently be taken as a sign of pathogen entry. A classic example of such nonmammalian structures are lipopolysaccharides (LPS) derived from bacteria. Ample evidence indicates LPS to be capable of activating resting APCs, thereby inducing high expression of B7-1 and B72. Most likely, molecules that exclusively belong to the intracellular space of mammalian cells may have similar effects, explaining the proinflammatory effect of necrosis whereby such molecules are released. In other instances, specific viral sequences have been shown to bind to the surface of APCs, resulting in their activation and the appearance of costimulatory molecules. The list of possible signals for activating APCs, however, is probably frustratingly long and includes reactive metabolites, mediators of oxidative stress, heavy metals, proinflammatory cytokines, certain hormones, and amino acid homologs (Janeway, 1992; Ibrahim et al., 1995). In fact, this list is remarkably similar to the list of circumstances generally captured as “stress.” Immunologists now frequently use the term “danger” to refer to the similar array of signals that are required for the induction of costimulatory potential in APCs: Signals that can be regarded as unmistakable signs of destructive infection, cellular damage, or metabolic malfunction-signs that something is very wrong. This appears to make perfect sense. By limiting the activation of T cells to those sites that express costimulatory molecules, the body has chosen a system in which an immune response is triggered only when some damage has already occurred. Harmless pathogens and ubiquitous self-antigens are thus ignored even though the T cell repertoire harbors the appropriate specificities to attack these. However, why should an immune response be mounted if no damage is recorded? Second, a distinction is made between full professional APCs, capable of priming naive T cells, and partially competent APCs that only support effector functions. Resident tissue APCs, such as Langerhans cells in the skin and microglia in the central nervous system, as well as circulating APCs, such as macrophages and B cells, are generally not full professional APCs as are, for example, interdigitating dendritic cells in lymph nodes. Danger signals may well boost APC potential, but even then full APC competence is not achieved. As a consequence, these “amateur” APCs can sustain memory T cell effector functions, including the secretion of cytokines in response to the danger signal, quite well but they do not trigger T cell proliferation or priming of naive T cells that may have infiltrated tissue during its inflamed state (Croft et a/., 1994). In this way, the system is designed to allow effector functions but avoids largescale activation of naive T cells with an autoreactive potential as the unintended result of localized immune responses that will release such autoantigens in the context of danger. This ensures that, as a rule, a

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local inflammatory response will be self-limiting and will disappear when the danger signal disappears. Only the persistent combination of antigen and danger will lead to a persistent response, but even then priming of new specificities in the system will remain the primary task of lymphoid organs, such as the lymph node and spleen, that harbor or recruit sufficient professional APCs, routinely interdigitating dendritic cells, that are essential for this purpose. One well-known example of recruitment of professional APCs into the lymph nodes is the migratory behavior of Langerhans cells residing in the skin. While resident, Langerhans cells are only amateur APCs, even in the presence of danger signals. When these cells receive a danger signal, they will become activated and migrate to the nearest lymph node carrying a molecular sample of their original environment in the context of MHC class I1 molecules. While migrating (but only then) the Langerhans cells develop into full professional APCs that, on arrival in the lymph nodes, are able to activate even naive, resting T cells with the appropriate specificity. These activated lymph node T cells then migrate into the skin to locally exert appropriate effector functions when they reencounter their antigen. For this final step, they require only local amateur APCs (resident Langerhans cells) and these can trigger effector function, but only if the danger signal is still there. When the danger signal disappears, even their ability to support the effector function ceases. Thus, by regulating the levels of costimulation on potential APCs the immune system confines priming of new specificities to lymphoid organs while allowing local amateur APCs to sustain immune effector functions, but only when these effector functions are called for. In conclusion, the long-held view that the immune system is programmed to distinguish self from non-self structures is probably only partially true. In addition, the immune system is also designed to distinguish stressed or endangered cells from healthy cells and comes into action only when stress has occurred. It may even be hypothesized that the immune system is primarily regulated by this latter mechanism and that it really does not care about self or non-self structures as such (Matzinger, 1994). An interesting implication of these emerging views is that self-antigens that may play a role in tissue-specific autoimmune responses may well be expressed elsewhere in the body without causing inflammatory responses. If their expression at other sites is not accompanied by local signs of distress or danger, the immune system will simply ignore them. Another interesting implication is that the presence of stress-inducible molecules, notably heat shock proteins (hsp) or stress proteins, may not be limited to secondary stages of inflammation. Rather, stress-inducible molecules such as hsp form part of the environment that T cell activation is critically dependent on and they may therefore play

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a role in regulating immune responses from the very beginning. This is further discussed in Section 1II.C.

F. Effector Mechanisms in Tissue Destruction: Metalloproteinases Immune-mediated mechanisms for the actual destruction of tissue probably involve the contribution of mainly macrophages. In some cases, specific cytotoxic T cells may also contribute to the lysis of cells that contain the target antigen by secretion of perforins and other degradative enzymes, but immunopathological studies strongly suggest that contributions from this source are probably only minor. Most likely, direct ingestion and destruction of tissue-derived material by activated macrophages and secretion of degradative and oxidative substances by these cells are the main factors that control degradative processes. An important example of a harmful substance produced by activated macrophages is nitric oxide (NO), which is discussed in Section VI,F. The main degradative enzymes include metalloproteinases, which can be secreted by activated macrophages as well as by other local cells that receive the instruction to do so during inflammatory processes. Matrix metalloproteinases (MMPs) are the prime effectors of degradation of extracellular matrices and, apart from playing a role in autoimmune disease, MMPs are also strongly implicated in tumor formation and tumor outgrowth. Under normal conditions, MMPs fulfill a variety of useful functions in embryological development, angiogenesis, and during bone turnover. One particularly interesting function of one of the MMPs, designated TNF convertase, is the cleavage of membrane-bound TNF-a, releasing it as an active cytokine that is very much involved in the amplification of proinflammatory processes (Gearing et al., 1994; Crowe et al., 1995). MMPs comprise a family of potent enzymes and they share structural homologies and a number of common properties. As a rule, MMPs are secreted as inactive proenzymes and are only activated on partial proteolysis (or sometimes by mercural compounds). They are critically dependent on the presence of a zinc atom in their active site and, as a rule, they require calcium. Thus, MMPs are quite sensitive to chelating agents. The MMP family can be divided into at least four classes: the stromelysins, the collagenases, the gelatinases, and the membrane-type MMPs. MMP representatives have now been given numbers to facilitate nomenclature. Especially because MMPs are such potent enzymes, their expression and activity are tightly regulated at different levels. Growth factors and cytokines control their synthesis, enzymes control the conversion of inactive pro-MMPs into active ones, and active inhibition of mature MMPs is effected by so-called tissue

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inhibitors of metalloproteinases (TIMPs). For example, proinflammatory cytokines, such as IL-lP and TNF-a, and factors such as epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor, may stimulate MMP production. Conversely, factors such as TGF-P, IL-4, corticoid hormones, retinoic acid, insulin-like growth factors, and surprisingly perhaps, IFN-y downregulate MMP synthesis (Cawston, 1996). In addition, some of these factors also act by stimulating synthesis of TIMPs, blocking the MMP activity via this route. The conversion of inactive pro-MMPs into active versions of the enzymes usually require proteolysis at a sequence that is conserved among MMPs : PRCGVPD. The cysteine residue in this sequence is bound to a zinc atom and disruption of this interaction by proteolysis initiates activation of MMPs (Van Wart and Birkedal-Hansen, 1990). Complete activation ensures removal of the propeptide either by other proteinases or via an autocatalytic mechanism. The zinc atom remains in the active site of the enzyme and catalyzes the cleavage of peptide bonds in the substrate upon binding. Propeptide-removing proteinases can also be MMPs. For example, stromelysin can activate procollagenase (He et al., 1989), but other proteinases such as plasmin and mast cell-derived proteinases can perform similar functions in activating inactive pro-MMPs. Some MMPs may be activated within the cell that they are produced in and, thus, be secreted in an active form. For example, stromelysin-3 contains a sequence that can be cleaved by a Golgi-associated serine protease (Pei and Weiss, 1995). Inhibition of MMPs is usually effected by special TIMPs. TIMP-1 and TIMP-2 are similar 28-kDa glycoproteins that can bind to MMPs in a 1:1 ratio, thereby inactivating the MMP. Exactly how this binding takes place is not fully known. Another TIMP, TIMP-3, has been identified as a protein bound to the connective tissue matrix. It appears likely that other variant TIMPs will be identified in the future. Also, the expression of TIMPs is under the influence of inducing factors including, for example, TGF-P, but much remains to be established on this issue. Obvious interest is now directed at understanding how MMPs can be therapeutically inhibited in order to prevent MMP-mediated tissue damage and productive processing of TNF-a (Cawston, 1996). One approach is to choose a zinc-chelating substance and attach it to a peptide that mimics an MMP target sequence. Alternatively, the crystal structure of relevant MMPs can be used to design a synthetic structure that would fit into the active site and block its interaction with natural substrates. Also, optimizing the naturally inhibiting effect some antibiotics have on MMPs, including tetracyclin, for example, is an option. Current efforts involve the testing of a large variety of strategies to selectively target MMPs and their potential applicability in RA, MS, and IDDM; encouraging data from animal models still catalyze these efforts.

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111. Factors That Contribute to the Development of Autoimmune Diseases A. Genetic Factors The strongest evidence for the existence of genetic factors that contribute to susceptibility to autoimmune disease emerges from family studies. By comparing the relative frequency of a given autoimmune disorder in the general population to that found for monozygotic or dizygotic twins, parent-children relationships, or other familial relationships, it is evident that genetic factors must be involved. For multiple sclerosis, for example, disease concordance is found in 25-30% of monozygotic twins and lifetime risk of MS in siblings of affected individuals is approximately 30-fold higher than that for others (Sadovnik and Ebers, 1993). Also, epidemiological and migration studies are consistent with genetic factors in autoimmunity, although in these cases serious confounding factors exist such as the uneven geographical distribution of infectious pressure or dietary peculiarities that similarly influence susceptibility. The issue is to link these patterns of inheritance of disease to those of individual genes in order to identify those genes that may have a specific role in pathogenesis. The search for susceptibility genes in IDDM is perhaps the most advanced of ongoing studies on genetic factors in autoimmunity (Davies et al., 1994;Todd, 1995). For MS, significant progress has recently been made. It is clear that both IDDM and MS have a complex polygenic pattern of inheritance, but for both the main susceptibility locus in the genome is the region that encodes MHC molecules, i.e., the human leukocyte antigen (HLA) locus. In addition, inheritance of the insulin locus on chromosome 11 is also linked to IDDM (Hashimoto et al., 1994). Other loci have been reported but these are not always found and, as a rule, they display a weaker linkage to disease than MHC-encoding loci. For MS, candidate loci for susceptibility markers other than the HLA locus have been suggested to comprise polymorphisms in the regions that encode the T cell receptor, myelin basic protein, TNF-a, or the IL-1 receptor antagonist. A recent scanning of the genome with microsatellite markers to identify loci linked to MS has yielded data suggestive of the presence of perhaps as many as a dozen genes that may be involved (Ebers et al., 1996; Sawcer et al., 1996; Multiple Sclerosis Genetics Group, 1996). It is clear that in none of the major autoimmune disease (IDDM, RA, and MS) does a single gene exist that confers susceptibility on its own. An interesting approach to identify genes that confer susceptibility for autoimmune disease is the study of animal models. Using animals from inbred strains, genetic factors in disease may be dissected much more easily

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than in humans. The NOD mouse provides perhaps the best animal model for IDDM. NOD mice develop spontaneous diabetes with a disease penetrance of approximately 80% in females, although this can vary around the world. The NOD mouse harbors an unusual MHC class I1 molecule designated H-2g7, which bears some similarity to the human HLADQBl"0302 that is common in diabetes patients as well as in a subgroup of MS patients. The disease process in NOD mice involves insulitis, destruction of beta cells and, finally, diabetes. In this inflammatory process, which appears to be preceded by enlargement of some islets of Langerhans, cellular responses to a number of autoantigens including heat shock proteins, the enzyme glutamic acid decarboxylase (GAD), and insulin itself have been recorded. Like in humans, disease inheritance in the NOD mouse is polygenic, as can be inferred from cross-breeding the NOD mouse with other strains of mice. Identification of genes involved in the development of disease in NOD mice can clearly contribute to the search for genes that determine human autoimmunity (Serreze and Leiter, 1994). Also, in the animal model for MS, EAE, the search for susceptibility genes is ongoing (Baker et al., 1995; Sundvall et af., 1995), although in this case its relevance to the human situation is somewhat obscured by the fact that E A E is not a spontaneous model but must be induced by immunization with autoantigens. The exact nature of the experimental antigen may well affect the range of genes involved in the disease process. This may perhaps be less so in models of E A E that make use of virus-induced demyelinating disease (Bureau et al., 1993). Again, the MHC region turns out to be the dominant susceptibility locus in EAE but results also highlight some of the important problems in identifying susceptibility genes. Even when using large populations of genetically homogeneous animals, identification of susceptibility genes in autoimmunity is difficult and evidence for linkage in some populations may not always be reproducible in other populations. The epistatic effects of genes coinfluencing the disease process may especially cause such problems. These considerations emphasize that identification of the full range of genes that contribute to the development of human autoimmune diseases remains an elusive goal.

B. Infection and Autoimmunity The susceptibilty to autoimmunity and the mechanisms leading to tissue damage in autoimmune diseases are probably multifactorial but autoimmune diseases are ultimately a result of the breakdown in the regulation of tolerance or unresponsiveness to self-antigens. Although the exact mechanisms by which this event may occur are uknown and it is suggested that genetic predisposition has a role in deciding susceptibility, this is not the

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whole story. Monozygotic twins are not fully concordant for any chosen autoimmune disease. Thus, other risk factors must also be involved and the prime candidates implicated in initiating the disease are environmental factors. Epidemiological studies have identified several risk factors for the development of IDDM. These include maternal child blood group incompatibility, fetal viral infections, and high exposure to nitrosamines. For several autoimmune diseases, including R A and MS, certain infection patterns during the early years in life (up to early childhood) including, e.g., conditions of delayed primary infections, clearly contribute to susceptibility. Such factors may be important in the initiation of disease, whereas other risk factors may perpetuate an ongoing autoimmune process. Risk factors that may promote ongoing damage may include microbial infections, various forms of stress, dietary factors, and neuroendocrine influences. These different factors will be dealt with separately. Activation of autoreactive T cells is the prerequisite for the induction of autoimmunity. Resting autoreactive cells become activated when the TcR becomes engaged with the pathogenic epitope of an autoantigen in context with the appropriate MHC molecule. Infectious agents may induce activation and clonal expansion of these autoreactive cells by acting as superantigens that trigger autoreactive T cells bearing particular TcRs. Alternatively, peptide sequences of viruses and bacteria may be sufficiently similar to a pathogenic sequence of an autoantigen so as to trigger these cells in the periphery. That the onset of many autoimmune diseases is preceded by exposure to infectious agents suggests that infectious agents play a role in the initiation and progression of such diseases. Viral infections frequently precede autoimmune myocarditis and IDDM and, although central nervous system diseases in which demyelination is observed are rare, they are a significant phenomenon following infection with measles, mumps, or rubella. Indeed, myelin-reactive T cells known to be encephalitogenic in experimental animals are found in the blood and CNS of patients presenting with rubella, measles encephalitis, and postinfectious encephalitis (Hafler et af., 1987). Although the primary purpose of the immune response to microbial infections is to remove infectious agents, in doing so the immune response has the opportunity to become activated to host components and initiate an autoimmune response. Several of these mechanisms are discussed in the following section. 1. Release of Host Antigens

A major factor in viral replication and spreading is cytolysis, which enables the release of newly formed virions for reinfection of other host cells. However, the mere act of cytolysis causes the release of host cellular components. It is conceivable that host cell antigens (i.e., potential autoantigens)

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released in this way become available for degradation and subsequent presentation to the immune system. Alternatively, as in the case of enveloped viruses, viruses may “bud” from the surface of the host cell. The mechanism of budding from the cell may lead to incorporation of host cell antigens, for example, proteins or glycolipids, into the viral envelope. Generally, host cell proteins are displaced by the viral proteins during budding but this may not always be the case. Potter (1980) has reported the presence of host cell proteins in vesicular stomatis virions. Although one would expect the host to be tolerant to these self-proteins, possibilities do exist for such self-proteins to trigger autoimmunity. In contrast to protein, host cell glycolipids are routinely incorporated into the viral envelope. There is controversy over whether these host cell glycolipids can initiate an autoimmune response in their own right. However, glycolipids can clearly be immunogenic and antibodies as well as T cell responses have been detected against them in disease. Glycolipids are known to enhance MBPinduced EAE and antibodies directed to galactocerebroside (a major host cell glycolipid) induce damage to myelinated cultures (Dubois-Dalcq et af., 1970). Thus, during viral infection the immune system will recognize and attempt to clear viral particles, but in doing so it may well become sensitized to host cell antigens as well, which could possibly evoke a pathogenic autoimmune response. That enveloped viruses may induce such autoimmune responses has been shown for Semliki Forest virus, a Togavirus (toga = cloak/envelope) and for Langat virus, a tick-borne virus of the Ffaviviridae (Amor and Webb, 1988). Incorporation of host cell components into viral envelopes has been suggested to be a mechanism by which autoreactivity occurs in MS (Dalgleish et af.,1987) and during HIV infections. In simian immunodeficiency virus infection macaque monkeys are protected against a viral challenge if they are previously immunized with uninfected host cells. Also, HIV has been shown to incorporate MHC molecules into the envelope during replication and the protection afforded appears to correlate with anti-HLA titers (Dalgleish, 1995). It is pertinent that many of the viruses implicated as possible etiological agents of MS, such as measles and HTLV1 (Russell, 1997), are indeed enveloped viruses. It is possible that incorporation of CNS tissue into the viral envelope may precipitate autoreactivity to myelin leading to myelin damage. That viruses are able to trigger experimental autoimmune disease has been demonstrated by Liebert and coworkers (Liebert et al., 1988), who showed that rats infected with the JHM strain of mouse hepatitis virus were able to mount a T cell proliferative response to MBP, a well-known encephalitogen, and subsequently developed EAE.

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2. Bystander Damage It is well known now that productive infection of a host cell may result in the cytotoxic action of the immune response to remove the infected cell. Recognition of pathogen-derived peptides in the context of MHC class I molecules by cytotoxic T cells causes the release of perforin 1, which creates a hole in the infected cell membrane. Release of matrix metalloproteinases and granzymes, free oxygen radicals, and, in some cases, IFN-y and TNF-a accompany this process. For example, TNF-a is released from astrocytes infected with the neurotropic virus Newcastle disease virus and it is detectable in the brain following Semliki Forest virus infection (Morris et al., 1997). The cytotoxic effect of this cytokine has been shown in culture in which myelin and oligodendrocytes are damaged. Activation of the immune response, particularly of macrophages, is known to induce the release of free oxygen radicals, which have been implicated in tissue damage. Thus, killing of a virally infected cell is accompanied by the release of an array of degradative and cytotoxic products that may induce damage to nearby, otherwise innocent cells. This phenomenon is known as bystander damage, a term first used by Cammer and co-workers (1978) to describe damage to myelin. Bystander mechanisms have also been suggested to be the cause of the demyelination as observed in E A E (Cross et al., 1990). In these studies, it was found that after inoculation of naive mice with radiolabeled MBPspecific T cells only 1-4% of the infiltrated cells in the brain were MBPspecific T cells, suggesting that large numbers of other nonspecific cells may indirectly contribute to local damage. However, these results may also be explained by the phenomenon of “determinant spreading” to other autoantigenic peptides (discussed in Section V,B).

3. Molecular Mimicry That infectious pathogens have structural similarities with self-antigens may be coincidental in nature. To the immune system, however, it may be confusing to be confronted with a pathogen-derived sequence that is identical to a portion of a self-protein. Given the right circumstances, T cells or antibodies may not be able to discriminate between different sources of a given amino acid sequence. In such cases, we are dealing with molecular mimicry. It is quite conceivable that molecular mimicry may incidentally lead to autoreactivity as the result of microbial infection. Epitopes shared between coxsackie B3 virus and heart tissue are thought to contribute to virus-induced myocarditis and mimicry between poliovirus and the AChR has been suggested to be involved in the

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pathogenesis of MG. The availability of databases containing myriad peptide and protein sequences has led to many reports identifying sequence similarities between pathogens and self-antigens although few studies have conclusively shown that peptides sequences of pathogens can induce experimental autoimmune disease. An exception is MBP, a major protein found in myelin and commonly used to induced EAE. A short peptide sequence of MBP contains six amino acids identical to a sequence contained within the polymerase of hepatitis B virus. A study by Fujinami and Oldstone (1985) revealed that this sequence was able to induce EAE in rabbits when injected together with adjuvant. Thus, this observation demonstrated that viral sequences can in principle elicit an autoimmune response when they are sufficiently homologous to self-proteins. Additional sequence similarities between MBP and a number of pathogens, including Epstein-Barr virus and measles virus, have been identified (Wucherpfennig and Strominger, 1995). Other examples include sequence similarities between glutamic acid decarboxylase65 (GAD-65, a possible autoantigen in IDDM) and a noncapsid protein of coxsackievirus B and similarities between thyroid-stimulating hormone receptor, the primary antigenic target of autoimmune hyperthyroidism, and the HIV-1 nef protein. With respect to homologies between HIV and self-antigens, extensive searches have produced an impressive list of options including sequence similarities with HLA molecules (Dalgleish, 1995). A more complicated hypothesis has been proposed for the role of molecular mimicry in rheumatoid arthritis (Albani and Carson, 1996) and for IDDM (Baum et al., 1996). In these cases a pathogenic link is suggested between sequences of MHC molecules on the one hand and human pathogens or peptides known to induce disease in experimental models on the other hand. Although certainly interesting, an in-depth discussion of this topic is beyond the scope of this review. Other wellknown examples of homologies between pathogens and human selfproteins include stress proteins (Kotb, 1995), but these are dealt with in Section II1,C. Many intriguing examples have been described of molecular mimicry between pathogens and potential autoantigens in human disease or experimental autoantigens. However, the role of this phenomenon in induction of disease in humans remains to be determined. 4. Superantigens As a general rule, activation of T cells is dependent on binding of the T cell receptor to the complex of target antigen and MHC molecule. However, an intriguing alternative exists. So-called “superantigens” (SAgs) produced by bacteria and viruses are able to polyclonally activate T cells through the Vp element of the T cell receptor. SAg-mediated activation of T cells

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bypasses the normal constraints of MHC restriction by operating outside of the peptide-binding groove of the MHC molecule. In some cases, the peptide itself may also comprise part of the binding site (Woodland et al., 1997) but this is not necessarily so. In this way, SAgs may engage up to 30% of T cells rather than 0.01-0.0001% for conventional recognition of antigens. Two classes of SAgs have been described: soluble proteins secreted by bacteria and endogenous viral proteins. Examples of the former group are staphylococcal enterotoxins (e.g., staphylococcal enterotoxin B or SEB) or the toxic shock syndrome toxin, and an example of the latter group are the antigens encoded by murine mammary tumor virus (Held et al., 1994). The possible consequences of SAg-mediated activation of T cells include the activation of potentially autoreactive T cells or, conversely, the induction of immunosuppression and tolerance. The exact effects are believed to be codetermined by the presence of costimulatory signals and concentration of SAg. Of importance is the possibility that activation of T cells by SAgs could also lead to the elimination of regulatory T cells that otherwise control autoreactive T cells. Thus, exposure to SAgs could increase or decrease the activity of self-reactive T cells. Furthermore, by interacting with MHC molecules, SAgs can activate B cells, triggering them to produce antibodies including potentially autoreactive ones. Also, macrophages can be activated, leading to the production of excessive amounts of cytokines, nitric oxide, matrix metalloproteinases, adhesion molecules, costimulatory molecules, and other factors that may perpetuate an inflammatory process. SAgs also act as adjuvants enhancing T cells t o respond to cross-reactive epitopes and expand beyond the threshold level required to induce autoimmunity as has been shown for streptococcal M protein SAgs (Kotb, 1995). The SAgs harbor sufficient possibilities to activate autoimmune responses. In IDDM, it has been suggested that a SAg associated with pancreatic islets may be involved in the pathogenesis of disease (Conrad et al., 1994). Likewise, activation of autoreactive T cells by SAgs has been proposed as a mechanism giving rise to R A (Paliard et al., 1991). Experimentally, several studies have shown that the SAg SEB is able to activate autoreactive T cells, which subsequently are able to induce autoimmune disease in animals. This has been shown in EAE (Racke et al., 1994), in which T cells expressing distinct Vp receptor sequences were shown to be activatd by SAg and able to transfer disease. Several autoimmune diseases are associated with prior infection of viruses and bacteria and the auotimmune disease may be assumed to be triggered by infection. Infection may modulate the immune response by a variety of complex mechanisms that may not be mutually exclusive. However, the exact role of these factors in the pathogenesis of postinfectious autoimmune diseases requires further evaluation.

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C. Role of Stress Proteins in Autoimmune Diseases From the simplest prokaryote to mammals, all living cells contain stress proteins whose primary function is to manage unfolded polypeptide structures. A constitutive role is played by stress proteins during transport of proteins across membranes such as the mitochondria1 membrane. Also, stress proteins are key to the proper maintenance of routine protein biosynthesis and protein turnover that generate partially unfolded proteins in preventing these from unwanted interactions with other structures. This particular function has also been designated as chaperone function and it is an essential property of stress proteins. However, stress proteins are better known for their inducibility by circumstances that strongly increase the requirement for chaperoning or protective potential within the cell such as heat shock, exposure to reactive chemical substances, cellular transformation, oxidative stress, and so on. These conditions usually increase the likelihood for damaged and unfolded proteins to be generated and, therefore, the cell has to make a particular effort to maintain homeostasis. It does so by producing much higher levels of stress proteins than under normal conditions. Under those conditions, stress proteins can be rapidly recruited from intracellular storage sites and transcription of their genes is dramatically increased. Also, the patterns change depending on which stress proteins are postranslationally modified, but currently it is not clear what exactly is affected by this change. Thus, stress proteins belong to the normal physiology of the cell as well as to changing conditions as the result of stress. Initially, the inducibility of stress proteins was often examined following exposure of cells to high temperatures. The proteins appearing were accordingly termed hsps, but currently the term stress proteins is more commonly used. The different families of stress proteins are usually classified on the basis of their molecular weight and the best studied hsps are hsp60 and hsp70. Bacteria generally express just one, or at most two, members of these families, whereas mammalian cells usually have multiple homologs that may be localized in different subcellular compartments. One particular family of stress proteins, the so-called small hsp (or hsp25), appears to be unique to eukaryotic cells and, although humans have two representatives of this family, hsp27 and aB-crystallin, no proteins equivalent to these exist in bacteria. Members of the same family of stress proteins usually share significant structural homologies with each other as well as from one species to another. The sequence of the human hsp60, for example, is almost 50% identical to that of the bacterial homolog. The conditions under which stress proteins can be induced are generally referred to as stress but it is important to appreciate the fact that not all individual types of stress will lead to induction of the same range of stress

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proteins. The intracellular second signaling mechanisms for stress-induced transcription of hsp genes include so-called heat shock factors (HSF) and HSF-binding sequences that accompany and regulate the transcription of hsp genes. HSF may be intracellularly complexed to a variety of structures rendering them inactive as gene regulators. When signals are recorded at the cell surface or when certain substances enter the cell, HSF may be released from their complexes and become available for switching on genes by binding to HSF-binding sequences in the regulatory DNA domain. It has now been well established that a multitude of different HSF-binding sequences exist, each related to particular types of stress, and that each type of stress-provoking condition will trigger the transcription of its own given set of stress proteins and related genes. Different types of cells and tissues, however, and even individual cells, may respond differently to any given type of stress. In a recent study of MS lesions, it was demonstrated not only that the expression of stress proteins in inflammatory lesions was different for two selected stress proteins, i.e., aB-crystallin and hsp60, but also that different types of CNS cells were very different from each other in their expression pattern of these stress proteins (Bajramovic et al., 1997). Although oligodendrocytes in early developing lesions, for example, produced high levels of aB-crystallin, adjacent astrocytes, microglia, or neurons failed to do so. In contrast, astrocytic expression of the stress protein was prominent at later stages of lesional development but no expression could be detected in oligodendrocytes, microglia, or neurons again. This emphasizes that complex rules govern the expression of stress proteins and that each individual stress protein has its own rules that may be different in the context of different cells. Apart from their physiological importance, several stress proteins are quite interesting to immunologists. For reasons not fully understood, several stress proteins stand out as potent immunogens and a significant proportion of the immune repertoire of both T and B cells appears to be directed at stress proteins, even by birth (Fisher et al., 1992). Obviously, thymic deletion is not very effective in removing potentially hsp-directed T cells from the repertoire. In particular, the marked structural homologies between bacterial hsp and mammalian hsp have led many to investigate the anti-hspdirected responses in the context of autoimmune diseases. Anti-hsp immune responses could be relevant to autoimmune mechanisms in several ways. The structural homologies between bacterial and mammalian hsp could well form the basis for molecular mimicry in which an initial response to a bacterial hsp cross-reacts with a self-hsp and triggers autoimmunity. Many studies have been devoted to this issue, focusing on anti-hsp60 responses in the context of rheumatoid arthritis and diabetes (Van Eden, 1991). An observation that has triggered much of this interest was that, in Lewis rats immunized with heat-killed mycobacteria in mineral oil, an experimental

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form of arthritis developed that could be transferred to healthy animals by T cells directed at the bacterial hsp60. Intriguingly, these T cells were found to be cross-reactive to preparations of rat cartilage. However, subsequent investigations revealed that the explanation for what was observed could not be simple molecular mimcry because disease-preventing T cells were isolated that reacted to the very same determinant that was recognized by the pathogenic T cells. Also, cross-reactivity to the rat hsp60 homolog could not explain the findings entirely because it was not the homologous stretches in the hsp60 sequences that triggered disease (Anderton and Van Eden, 1996). Clearly, more complex processes are involved in the relationship between anti-hsp responses and pathogenic autoimmunity. Confusing to the potential pathogenic role of anti-hsp responses is the fact that they are so prominent in healthy individuals and, therefore, are difficult to consider dangerous on their own. This is not only true for the hsps that are common between mammals and bacteria but also for strictly self-hsps such as the small hsp. For example, a recent study has revealed strong human T cell responses to the small hsp aB-crystallin in all subjects included in the study-multiple sclerosis patients and healthy individuals (Van Noort et al., 1995). In many other studies, both cellular responses and humoral responses to hsps, most notably hsp60, have been documented in diseased and in healthy individuals. Clearly, anti-hsp-directed immune responses form part of the normal immune repertoire and they may even perform a routine surveillance task for the signaling of stressed or infected cells. In this way, anti-hsp immunity could possibly serve as a generalized boosting mechanism for the onset of protective immunity against antigens encountered in the context of a stressed or damaged environment. This makes particular sense given the emerging evidence that stress or danger provides other initiating signals for the immune system to come into action, most notably by inducing the appearance of costimulatory signals for T cells. As such, the contribution of anti-hsp immunity to the development of autoimmunity would perhaps be determined by the strength and quality of such a boosting mechanism and its control rather than the mere fact that hsps are recognized. Increased anti-hsp responses have been recorded in autoimmune diseases of a variety of tissues including the joint, the pancreas, the eyes, the blood vessels, the nervous system, the skin, and probably many others (Cohen, 1996). In parallel, many experimentally provoked anti-hsp responses may precipitate organ-specific diseases in laboratory animals including experimental forms of arthritis and diabetes. At the same time, however, increased anti-hsp responses have been associated with downregulation of disease. This has been observed, for example, in experimental arthritis in Lewis rats (Van den Broek et al., 1989; Van Noort et al., 1994) and in juvenile chronic arthritis in man (De Graeff-Meeder et al., 1991). Thus, there is an apparent paradox in anti-hsp responses and

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they are associated with both the induction and the control of disease. This renders it quite likely that the quality of the response and its control will be key to understanding how anti-hsp responses play a role in autoimmune diseases. Because infectious events are associated with enhanced appearance of hsps and their presentation to the immune system, it is conceivable that the way the immune system is programmed to respond to hsps is strongly affected by the way the individual must deal with primary infections. Thus, the strength and quality of anti-hsp memory responses are likely to parallel those against important pathogens that are the first to induce presentation of hsps to the immune system. It is well known that the quality of protective immune responses to a given pathogen may differ markedly when primary infection occurs at different ages. For example, the first encounter with Epstein-Barr virus may go unnoticed when it occurs early in life, but the very same event may cause massive, pathogenic cellular responses when it occurs in early adolescence, giving rise to infectious mononucleosis. In this way, different patterns of infections, especially during the early years in life, may have a profound impact on the quality of antimicrobial responses as well as on accompanying responses such as those against hsps. Recent evidence has shown that the stress protein aB-crystallin is expressed at elevated levels not only in the white matter of MS patients but also in virally infected peripheral blood B cells, where it ends up being presented via the MHC class I1 pathway (van Sechel et al., 1997). A tentative scheme depicting a role of a given hsp in T cell responses involved in both infectious events and tissue-specific responses is given in Fig. 5. The relationship suggested in Fig. 5 would be consistent with the widely supported view that certain childhood infections, and especially delayed primary viral infections, contribute to the susceptibility to autoimmune diseases (Waksman, 1995; Gianani and Sarvetnik, 1996).

D. Dietary Influences Diet and the nutritional status of the host are known to have significant influences on immune function, on resistance to infection, and on susceptibility to autoimmune diseases (Harbige, 1996). Nutrients encompass a wide range of compounds, each influencing the effect of others, and the study of specific nutrients in particular diseases is therefore difficult. Impaired immunity and susceptibility to infectious diseases are strongly linked to nutritional deficiencies such as protein-energy malnutrition and vitamin A deficiency. In measles infection low vitamin A levels are known to be associated with severe clinical disease and low anti-measles antibody titers. That, in turn, measles is associated with autoimmune diseases, such as

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FIG.5 Possible role of hsp-directed cellular responses in the development of autoimmunity. In this simplified scheme, a link is suggested between the involvement of hsp-directed responses in both infectious events and the development of autoimmunity. When microbial infections occur, a resting immune system will be challenged by presentation of both microbial antigens and self-derived hsp sequences that emerge as the direct result of cellular infection. Presentation of some hsps may specifically be triggered by only some groups of pathogens. Within regional lymph nodes, the T cell repertoire will thus be primed against both sets of antigens, and after infection has been resolved, the immune repertoire will be enriched for memory T cells directed not only against microbial determinants but also to self hsp sequences. The quality of both repertoires may well be affected by the temporal pattern of first microbial contacts during the early years of life. At a second stage later on in life, some forms of local stress or “danger” within a tissue (even in the absence of pathogens) will cause reappearance of these same hsps while at the same it provides the signals for nonspecific recruitment of the immune system. Under these conditions, hsp determinants will be presented to nonspecifically infiltrated memory T cells by stress-activated local APC, which is sufficient to reactivate these memory T cells. In the absence of any pathogen, the quality and strength of the anti-hsp memory T cell response may become of prime importance in determining whether pathogenic autoimmune responses will develop.

subacute sclerosis panencephalitis and postviral encephalitis, in which autoimmune respones to CNS tissue may be implicated in the disease suggests that vitamin A supplementation may be useful as a treatment or as part of a combined therapy. Vitamin A is known to affect cytokine production and complement activity and macrophage cytotoxic as well as phagocytic function. The precursor of the vitamin A, /3-carotene, is known to exert antioxidant effects although the exact mechanism of the vitamin is not fully understood. Another antioxidant, vitamin E, which includes several naturally occurring tocopherols, has been reported to have profound effects

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on infections of animals. Supplementation with vitamin E can enhance antibody responses and phagocytic function of PML and cocktails of antioxidants including vitamin E potentiate T cell activity and can augment CD4+ T cell numbers. Their effects in autoimmune diseases, however, still require evaluation. Other vitamins known to influence immunological status are pyridoxine and folic acid, although the effects of these on the development of autoimmune diseases are unknown. Minerals and trace elements likewise influence susceptibility to infectious agents and autoimmune disease. Many studies have demonstrated the importance of iron, which is a prerequisite for many essential biological processes and a central player in the control of immune function. Iron levels influence T and B cells levels and may even control the regulation of T h l and Th2 subsets (Weiss et al., 1995), thereby modulating the susceptibility to autoimmune disease and its regulation. Also, many of the biological effects of nitric oxide, a potent cytotoxic component of macrophages and implicated in many inflammatory diseases including autoimmune conditions, are dependent on the interactions with iron. Zinc deficiency in the NZB mouse, a spontaneous model of the human autoimmune disease SLE, caused a delay in the onset and increased longevity associated with lower antibody titers. In apparent contrast, however, human studies on supplementation of malnutritioned children with zinc revealed that this treatment improved delayed-type hypersensitivity (DTH) responses, pointing to augmented T h l function, which generally worsens major autoimmune diseases. Likewise, zinc supplementation in the elderly enhanced DTH responses to several recall antigens. Recent studies have addressed the effect that fats, both saturated and unsaturated, may have on the development of immune-mediated disease. Diets low in fats or deficient in essential fatty acids increase the survival rate during experimental autoimmune SLE. In contrast, highly saturated fat leads to an increase in deposition of immune complexes, increased titers of anti-DNA antibodies, and decreased T cell mitogenic activity. Thus, modulating dietary fat composition may be a useful therapeutic regimen in order to manipulate autoimmune diseases. More specific studies have dissected immunomodulatory effects of unsaturated fatty acids. These include monounsaturated fatty acids (e.g., olive oil) and polyunsaturated fatty acids (PUFAs), which encompass the omega3 (e.g., fish oils) and omega-6 PUFAs (e.g., safflower and borage oils). The effects of omega-3 PUFAs on autoimmune diseases, both in experimental animal models and in human disease, are summarized in Table I. Supplementation of the diet with omega-6 PUFAs in these studies has shown modulation of the immune response by attenuation of leukotrieneB4 (LTB,) production and of IL-1, IL-6, TNF-a, and IL-2 production suggesting that less proinflammatory activity occurs. This was most clearly observed in R A patients who were supplemented with fish oil. In these

JOHANNES M. VAN NOORT AND SANDRA AMOR

162 TABLE I Effects of Omega-3 PUFAs on Autoimmune Diseases Disease SLE NZB X NZW F1 mouse MRLllpr mouse Human Human

PUFA Fish oil rich in omega-3 Omega-3 Omega-3

Effect Delayed onset Increased longevity Equivocal Clinical benefit

Arthritis Protects Collagen-induced arthritis Fish oil (mouse) Fish oil Augments Rat High polyunsaturated/low saturated Beneficial Human fat + 1.8 g EPA' MS EAE in rats EAE in mice a

Omega-6 Omega-6

Decreased incidence of disease

EPA, eicosapentaenoic acid.

patients, a significant reduction in clinical signs correlated with a decrease in LTB4production. The other class of PUFA known to influence autoimmune conditions are the omega-6 PUFAs, which include primrose, safflower, and borage oils and which differ by their proportion of linoleic (LA) to ylinolenic acid (GLA) ratios. Whereas the administration of LA-rich oil augmented experimental SLE or was ineffectual, supplementation with safflower oil (LA + GLA) led to a greater survival rate. Comparable effects have been observed in EAE. Recently, other sources of omega-3 PUFAs have been shown to be more beneficial in EAE, possibly related to their potential to induce production of the immunoregulatory cytokine TGF-0. With respect to human studies, such as MS, epidemiological studies suggest that diet plays a distinct role in codetermining susceptibility. This role may be particularly determined by the proportion of saturated versus unsaturated fats in the diet (Swank et af., 1952; Agranoff and Goldberg, 1974; Alter et af., 1974). Such an association is strongly suggested by the high incidence of MS in the farming communities in Norway compared to the low incidence of MS among the fish farmers on the coastal regions (Swank et af., 1952). Figure 6 illustrates these epidemiological findings. Such correlations can also be recorded for RA: The strikingly low incidence of both RA and MS in Eskimos could well be attributable to the high levels of fish oils and low levels of saturated fats in their diet.

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- 2.5 - 5.0 - 7.0 00 000 per year

FIG. 6 Diagrammatic representation of the geographical distribution of MS in Norway. Selected regions in Norway may differ strongly in incidence of MS. In the farming communities (inland regions), MS occurs quite frequently, whereas in the fishing communities (coastal regions) the incidence of MS is much lower. It has been hypothesized that this is codetermined by differences in diet between these regions.

E. Neuroendocrine Influences Susceptibility to infections and onset of disease as a result of biological and psychological stress is well recognized and provides compelling evidence for interaction between the immune and neuroendocrine systems that is relevant to autoimmune processes. Cognitive stimuli, such as chemical, emotional conditions, or stress, are registered and conveyed to the nervous system via hormones and peptides providing a biochemical rationale for this interaction. Further substantiation stems from observations that lymphocytes produce stress-associated peptides and express many receptors for neuroendocrine peptides and, conversely, that neuroendocrine cells express receptors for cytokines. Thus, the phenomenon of bidirectional communication between the endocrine and nervous system is now well recognized (Blalock, 1994; Savino and Dardenne, 1995). In response to stress, be it infectious stress or some other form, a cascade of events take place, some of which modulate the immune responses. This cascade is commonly referred to as the hypothalamic-pituitary-adrenal

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FIG. 7 A simplified diagram showing the major interactions between the hypothalamicpituitary axis, the adrenal gland, and immune system. As a result of stress, the hypothalamus is stimulated to produce CRH, which in turn activates the pituitary gland into producing ACTH. This hormone reaches the adrenals and triggers these to secrete glucocorticoids. Glucocorticoids are strongly implicated in regulating immune functions and they can do so by a large variety of different mechanisms. Overall, glucocorticoids favor the development of type 2 immune regulatory responses while downregulating type 1, proinflammatory processes. Such a change in balance between these types of immune responses can make an important contribution to the control of autoimmune diseases.

axis (HPA axis). Figure 7 highlights the key features of the complex network that exists between neuroendocrine hormones of the HPA axis and immunological activity. As an initial stress response, corticotrophin-releasing hormone (CRH) is produced by the hypothalamus. CRH stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary, which in turn acts on the adrenal causing the release of glucocorticoids. These have a wide variety of effects including alterations in metabolism and suppression of immune function. CRH levels in the synovial fluid of RA patients correlate with mononuclear infiltrates in the joints and administration of anti-CRH antibodies suppresses streptococcal cell wall-induced arthritis. In experimental animals, failure to release significant levels of CRH in response to stress correlates with susceptibility to autoimmune diseases.

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The susceptibility of Lewis rats to adjuvant-induced arthritis and E A E may be due to a defect in the hypothalamic release of CRH during inflammation. This results in a lack of production of ACTH, glucocorticoid levels fail to increase and, thus, emerging autoimmune responses fail to be regulated. Resistant strains of rats may be made susceptible to autoimmune disease by lowering glucocorticoids levels. Following induction of MBP-induced EAE, Lewis rats recover spontaneously and such a recovery is clearly associated with release of corticosterone. Administration of glucocorticoids prevents the onset and progression of E A E in the Lewis rat, thereby demonstrating a direct involvement of the endocrine system in regulating autoimmune disease. The effects of glucocorticoids are most likely attributable to their influence on cytokine production by macrophages. T cells, monocytes, and natural killer cells (Cupps and Fauci, 1982). Although potent inhibitors of the immune response, glucocorticoids also induce atrophy of the adrenals due to the inhibition of ACTH production by the pituitary. Unfortunately, this effect renders them unsuitable for application in long-term therapy of autoimmune disease. Other hormones released by the pituitary are also associated with autoimmune diseases. In both experimental SLE and in the human disease itself, clinical symptoms are associated with hyperprolactinemia. An inhibitor of the hormone prolactin, bromocriptine, affects the development of T cells in the thymus and spleen. Administration of bromocriptine increases the longevity of NZB X NZW F1 mice, which otherwise show typical clinical parameters of SLE. a-Melanin-stimulating hormone (a-MSH), derived from ACTH, occurs in the pituitary among other organs and it is increased at sites of inflammation. Although it has no major influence on glucocorticoid secretion, a-MSH counteracts proinflammatory cytokines, such as TNF-a, nitric oxide, and prostaglandins, and it increases the production of IL-10. In experimental adjuvant arthritis, a-MSH inhibited the development of chronic inflammation. These data suggest that therapeutic administration of a-MSH may have beneficial effects in autoimmune diseases requiring further study. The effects of cytokines on the release of hormones are shown in Table I1 (top). The influence of the hormones on the outcome of the immune response and autoimmune disease is shown in Table I1 (bottom). This is not a comprehensive list and for more detailed information readers are referred to another review (Blalock, 1994). Together, the available data provide clear evidence for a role of neuroendocrine hormones in the shaping of T cell repertoire, in modulating T cell responses in the periphery and, hence, in influencing the course of autoimmune responses. Their influence on cytokine networks requires further investigation in order to provide a more complete understanding of the complex interactions that apparently exist.

TABLE II Hormonaland Cytokine Influences in Autoimmune Diseases

Influence on neuroendocrine system ~~

~~

~~

Cytokines IL-1 IL-2 IL-6 TNF

Causes release of ACTH, CRH, acts on adrenals, synthesized by TSH-containingcells, rIL-1 decreases CRH, IL-lfl injected intracerebrally inhibits secretion of LH and ovulation Regulates pituitary cell proliferation, influences secretion of GH, PRL, ACTH, and CRH Causes release of ACTH, synthesized by TSH-containing cells, increases corticosteroid levels Increases corticosteroid levels Influence on neuroendocrine and/or immune response

Influence on Autoimmune Disease

Elicits release of ACTH, increases NK cell activity, induces IL-1 production by macrophages, produced intrathymically

Present in arthritic joints, high levels in synovium of arthritic joints, administration to SCW-induced arthritis decreases inflammation

ACTH GH TSH PRL

Causes production of glucocorticoid by adrenals, produced intrathymically Augments antibody synthesis, increases neutrophil levels Increases antibody production If depleted, IL-2 proliferation is inhibited

Inhibits antoimmune diabetes

a-MSH

Increases IL-10 production, inhibits prostaglandin synthesis

Inhibition of PRL increases survival in murine SLE Inhibits development of chronic inflammation in arthritis models

Blocks macrophage IL-1 production

Suppresses EAE

Hormone Hypothalamus CRH Pituitary

Adrenals Glucocorticoids

Abbreviations used: IL, interleukin; TNF, tumor necrosis factor: ACTH, adrenalcorticotrophic hormone; CRH, corticotrophin releasing hormone: GH, growth hormone; TSH, thyroid stimulating hormone; PRL, prolactin; a-MSH, a-melanin stimulating hormone.

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IV. Models of Autoimmune Diseases Ideally, models of autoimmune diseases should re-create the wide array of aspects predisposing disease, the clinical, physiological, histopathological features of disease, and allow the investigation of therapeutic strategies. In addition, the ideal experimental animals should be cheap to breed and easy to maintain, and the physiological and biochemical system of the animals should sufficiently resemble that of humans to allow interpretation of experimental data in terms of the human immune system. Also, specific reagents such as antibodies to monitor disease mechanisms and to allow in v i m experimenting should be available. Rodents, in particular rats and mice, fulfill many of these criteria and they are therefore widely used as experimental animals. Models of autoimmune diseases may be classified as those that occur naturally or spontaneously and those that require experimental induction. Both infectious models and autoimmune models exist in each of these categories (Table 111). Tissue damage typical of that seen in several autoimmune diseases, such as MS and IDDM, may also be recreated with toxic chemicals. Furthermore, with the development of molecular genetics, transgenic mice expressing tentative autoantigens [e.g., MBP and proteolipid protein (PLP) in the model for MS], class I1 molecules (e.g., H-F7expressing I-E), and T cell receptor molecules (e.g., TcR specific for MBP) have allowed the development of new models of autoimmune disease. Some of the more popular models are reviewed and compared to the corresponding human autoimmune disease. Specific attention is devoted to models for MS, whereas other models are discussed more briefly. Several excellent reviews cover some of the individual models in greater detail (Fazakerely et af., 1997; Pender, 1995; Boitard and Bach, 1991).

A. Models of Multiple Sclerosis Multiple sclerosis is an inflammatory and demyelinating disease of the central nervous system. Epidemiological studies have shown that there are high-, medium-, and low-risk areas and the disease affects 5-300 per 100,000 people in Western societies. The etiology of the disease is unknown, although many viruses have been implicated in the onset and progression of the disease (Russell, 1997). However, once initiated the disease is probably autoimmune mediated. The notion that immune mechanisms play a major role in MS is strongly supported by studies on the lesions in the CNS white matter and by examination of the T and B cell responses against selected myelin antigens such as MBP, PLP, myelin oligodendrocyte glycoprotein

TABLE 111 Models of Autoimmune Diseases

Type of disease Natural Infectious

Autoimmune (spontaneous)

Induced Infectious

Autoimmune

Chemical induction

Model

Species

Human disease

Visna" Canine distempef Mice transgenic for TCR specific to Mspb HLA-B27 mouseb BB rats Nonobese diabetic mice MRL/lpr, gld mutations; (NZB X NZW)FI B 10D2 mice DB N1 rat

Sheep Dog Mice Mice Rat Mouse Mouse Mice Rat

Multiple sclerosis Multiple sclerosis Multiple sclerosis Ankylosing spondylitis Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Systemic lupus erythematosus Scleroderma Arthritis

Semliki Forest virus Theiler's murine encephalomyelitis virus Mouse hepatitis virus Eubacreria-induced arthritis Encephalomyocarditis virus Coxsackie B virus Rubella infection Experimental allergic encephalomyelitis Collagen type 11-induced arthritis Adjuvant-induced arthritis Experimental autoimmune myasthenia gravis, transfer of anti-AChR antibody Expenmental allergic neuritis Experimental allergic uveoretinitis Neonatally thymectamized BALBlC mice Low-dose streptozotocin Cuprizone Ethidium bromide Lysolecithin

Mouse Mouse Mouse Rat Mouse Mouse Syrian golden hamster Mouse, rat, primate Mouse Mouse, rat, rabbit

Multiple sclerosis Multiple sclerosis Multiple sclerosis Rheumatoid arthritis Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Insulin-dependent diabetes mellitus Multiple sclerosis Rheumatoid arthritis Rheumatoid arthritis Myasthenia gravis

Mouse, rat Mouse, rat Mouse Mouse Rat Rat Rat

Guillain-BarrC syndrome Uveoretinitis Autoimmune gastritis Insulin-dependent diabetes mellitus Multiple sclerosis Multiple sclerosis Multiple sclerosis

"Can also be induced experimentally. 'Do not express disease in germ-free conditions.

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(MOG), myelin associated glycoprotein (MAG), and other antigens, e.g., the heat shock protein aB-crystallin, present in the CNS of MS patients (Van Noort et al., 1995). The role for autoimmunity is also supported by data from the experimental autoimmune model EAE, which may be induced with CNS antigens in rodents and primates. Generally, immunization of animals with spinal cord homogenate (SCH) is quite effective in inducing EAE but many studies make use of individual myelin proteins or peptides derived thereof to induce disease. These experimental antigens include MBP, PLP, MOG, and MAG. Active induction of disease occurs following immunization of susceptible animals with antigen in adjuvants such as complete Freund’s adjuvant (CFA). The type of EAE induced is dependent on the immunization protocol, animal strain, and antigen used and although some protocols result in a single acute episode of EAE, others induce a chronic relapsing disease. In this latter type of EAE, acute disease initially resolves, but in time further neurological phases of clinical relapses develop. Chronic relapsing E A E is reminiscent of MS because animals develop accumulating neurological deficits following each disease phase. In mice the onset of disease is observed as a weight loss following increasing neurological deficit (Amor et al., 1994). Figure 8 illustrates the clinical signs of EAE as induced by different individual myelin proteins; Figure 9 shows some of the histopathological features of the induced disease. EAE may also be induced by transfer of lymphocytes preactivated with myelin antigens into naive animals; this form of disease induction is referred to as passive EAE. In mice, different MHC haplotypes typically associate with different regions (epitopes) of experimental antigens being effective in disease induction. The major encephalitogenic epitopes for several mouse strains are listed below (Table IV). Note that some epitopes are effective in the induction of EAE in a number of strains despite haplotype differences. Such broadly effective epitopes are generally referred to as “promiscuous” epitopes; the region 89-101 of MBP is an excellent example of this type. Characterization of encephalitogenic epitopes of myelin antigens has enabled studies at the molecular level of peptide-MHC and peptide-MHC-T cell receptor interactions that are relevant to disease. In these studies, even individual amino acid residues have been identified that are critically involved in T cell activation and disease induction, thus forming the basis for the development of specific peptide therapy. This interesting development is discussed further in Section VI,B. Recently, the Biozzi ABH mouse has been shown to exhibit reproducible relapsing remitting disease following injection with SCH (Baker et al., 1990) and the major epitopes of MBP, PLP, and MOG have been described in detail (Amor et al., 1993,1994,1996a). Figure 8 represents the typical course of E A E as observed in Biozzi ABH mice following immunization with

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1st relapse

acute

MBP 4~ 3

10

14

18

22

26

30

34

38

42

Days post sensitization FIG. 8 Clinical course of relapsing EAE in Biozzi ABH mouse induced with different CNS encephalitogenic antigens. Antigens used for EAE induction are spinal cord homogenate (SCH), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), and myelin basic protein (MBP). Note that induction of EAE with MBP does not result in the development of relapses. Immunization with MOG results in a clinical course that is different from that seen after treatment with SCH and PLP.

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FIG. 9 Mononuclear cell infiltrates in the subpial areas of the spinal cord of a mouse with EAE induced with PLP. After PLP immunization in mice, perivascular lesions develop in the CNS but no infiltrates can be seen in the peripheral nerve.

different myelin proteins. Disease in this mouse strain may also be induced with MAG, glial fibrillary acidic protein, and the heat shock protein a Bcrystallin (S. Amor, unpublished data). Although pathogenic autoreactivity in EAE may well be initially restricted to responses against a single experimentally introduced antigen, there is clear evidence that in the course of disease T and B cell responses diversify to other antigens and epitopes. This phenomenon of so-called determinant spreading is an important concept in autoimmune disease. Especially when developing strategies for antigenspecific intervention, the possibility of determinant spreading must be taken into account. This is further discussed in (Section V,B). The fact that for a single mouse strain, i.e., Biozzi ABH mice, a significant number of different epitopes derived from different myelin antigens have now been identified

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TABLE IV Maior lmmuncdominant Epitopes for Induction of Disease in Mice

Encephalitogenic myelin epitope Mouse strain SJL PLIJ Biozzi ABH NOD SWR C3H/HeJ BALB/c BlO.Rl11

Haplotype

H-2A

S

S

U

U

dql g7 q k d r

g7

MBP

PLP

MOG

89-101 Acl-11 12-24

139-151 43-64 56-70 56-70 103-116 215-232 56-70

92-106 35-55 8-22 8-22

g7

q k d

r

89-101 Acl-11 12-24 89-101

8-22

provides an excellent starting point for the exploration of this concept and its application in antigen-specific therapies. Also, in other experimental models of autoimmune disease and the human disease itself, determinant spreading requires further studies. Extensive studies in EAE have shown that disease may readily be induced by the activity of myelin-reactive MHC class 11-restricted CD4+ T cells. Especially the passive form of EAE underscores this notion. For this reason, it has been assumed that the T cell plays center stage in MS. Although this may well be the case, the role of anti-myelin antibodies certainly deserves consideration. MBP-induced disease in the Lewis rat results in inflammatory lesions of mononuclear cells in the CNS but very little if any demyelination. This type of pathology is typical of acute EAE. In the relapsing models, however, demyelination-which is the hallmark of MS-is more prominent in the relapse phase in which anti-myelin antibodies are detected. Injection of anti-MOG antibodies during MBP-induced disease in the Lewis rat (Linington et af.,1992), or in SCH- and MOG-induced EAE in Biozzi ABH mice, results in augmentation of disease and lesions of demyelination (M. M. Morris, unpublished data). Thus, although acute EAE is useful to study the inflammatory process, the relapsing model is vital to study aspects of myelin damage and myelin repair, the later being explored as a potential therapy in MS. No spontaneous models of EAE have been described, although mice transgenic for the T cell receptor specific for MBP exhibit disease when housed in a “dirty” environment, whereas those kept under germ-free conditions do not (Goverman et af., 1993), suggesting a role for infection in coprecipitating disease. Alternative and contrasting models of MS are the virus-induced models, the two studied being Semliki Forest virus (SFV) infection and Theiler’s murine encephalomyelitisvirus (TMEV) infection. These models have been

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extensively reviewed (Fazakerley et al., 1997; von Herrath and Oldstone, 1995) and only a brief outline of the diseases will be given here. TMEV is a natural pathogen of mice and only in rare cases gives rise to spontaneous CNS disease. To induce reproducible disease the virus must be inoculated intracerebrally, unfortunately precluding examination of factors such as blood-brain barrier disturbance in this experimental model. The course of the disease is virus strain dependent and mouse strain dependent. The virus strains of use are GDVII, DA, and BeAn and susceptibility to disease has been linked to the H2D locus, with mice of the s, f, p, r, and q being susceptible and those of b and d resistant to chronic disease. In the BALB/c mouse (d haplotype), for example, a monophasic disease course is observed following injection with TMEV, whereas SJL mice (s haplotype) exhibit chronic disease. Elegant studies by Miller and colleagues (1995) have shown that Theiler’s virus infection of SJL mice gives rise to T cell-mediated demyelination in the CNS. T cell response to viral antigens occurs 7-10 days postinfection and T cell proliferative responses to PLP 139-151 and MOG 92-106 (these are encephalitogenic epitopes in SJL mice) are detected at later times following infection. These findings clearly demonstrate that at least in mice neurotropic viral infections are able to induce autoreactivity to myelin proteins. The SFV infection of mice gives rise to focal lesions of demyelination in the CNS of immunocompetent, but not athymic ndnu or severe combined immunodeficient (SCID) mice, clearly suggesting that the myelin damage is immune mediated. Figure 10 shows a region of the murine CNS following SFV-triggered inflammation and demyelination. Adoptive transfer of splenocytes or populations of cells enriched for T lymphocytes as well as depletion studies demonstrate that demyelination in this case is T cell dependent and reliant on CD8+lymphocytes (Fazakerley et al., 1997; Suback-Sharpe et al., 1993;Amor et al., 1996b). SFV infection induces demyelination following intraperitoneal inoculation and therefore allows the study of early inflammatory events and changes in the blood-brain barrier. In contrast to EAE, in which inflammation in the CNS is mainly characterized by type I cytokines (Baker et al., 1990), cytokine profiles in the CNS following SFV infection are less clear-cut and both type 1 and type 2 cytokines are detected (Morris et al., 1997). It is of significant interest to note that in MS brains, cytokine profiles within the lesions consist of both type 1 and type 2 cytokines (Cannella and Raine, 1995). There are obvious advantages to using the autoimmune model of chronic relapsing EAE. Most important, these models resemble MS much better with regard to pathological and clinical features. Of interest is the notion that virus models of EAE clearly illustrate how common viruses may give rise to CNS lesions and autoimmunity, underscoring that such mechanisms be seriously considered in attempts to explain the pathogenesis of MS.

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FIG. 10 Demyelination within the cerebellum of a Biozzi ABH mouse infected with Semliki Forest virus. Following infection with SFV,Biozzi ABH mice develop an inflammatory demyehating disease. After 18 days of infection, cellular infiltrates are present within white matter lesions.

B. Models for Insulin-Dependent Diabetes Mellitus

Destruction of the insulin-secreting pancreatic j3 cells leads to IDDM. During IDDM, hyperglycemia is observed months or years after the immune response against the j3 cells has been initiated. IDDM occurs in association with other autoimmune disorders such as Hashimoto’s disease and it correlates to HLA serotypes DR3 and DR4. Because the damage

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is restricted to the pancreatic p cells, IDDM is considered an excellent example of an organ-specific autoimmune disease. In IDDM patients, autoantibodies can be detected to a variety of structures including islet cell autoantigens, insulin itself, GAD, and the insulin receptor (Song et al., 1996). Despite the known involvement of T cells in disease, which of the putative autoantigens may play a key role is still a matter of debate. Support for an autoimmune contribution to IDDM comes from experimental animal models that include spontaneous, infective, and chemical methods of induction of experimental IDDM. Spontaneous disease is observed in the biobreeding (BB) rat and the NOD mouse. In the BB rat, diabetes is observed at 90-120 days and all clinical forms of the human disease are exhibited, i.e., from subclinical to overt diabetes. The islet infiltrates consist of macrophages, B cells, and both CD4' and CD8+ lymphocytes. Diseased rats also present circulating autoantibodies and complement-fixing antibodies. Unlike the human or NOD mice, BB rats are characterized by a recessive inherited lymphopenia. Genetic studies have revealed associations of disease with the lyp gene (also called iddm 1) on chromosome 4 which is necessary for disease. Other loci have also been described and contribute to disease. One of these is linked to the MHC (Theofilopoulos, 1995). That the mechanism of disease induction in BB rats is immune mediated is supported by adoptive transfer studies. Mitogen-activated lymphocytes are able to transfer disease to naive recipients and cyclosporin A, anti-asialo GM1, silica blockage of macrophages, and neonatal thymectomy abrogate disease. In the NOD mouse, IDDM occurs from 6 weeks to 6 months. The disease predominates in females and is observed in up to 80% of the females and in 20% of the males. Many lines of NOD mice have been developed throughout the world and the variability in the incidence of disease in the different colonies may be due to environmental factors. The cellular infiltrate in the islets is composed of mainly CD4'T cells, whereas CD8' and B cells are only observed at later stages of disease. Anti-insulin autoantibodies and anti-islet cell antibodies are detected in diseased animals but an important role is played by T cells as evidenced by adoptive transfer experiments. Splenocytes from diabetic NOD mice transfer disease to nondiabetic NOD recipients. Early diabetes can be induced following administration of cyclophosphamide or by thymectomy at 3 weeks of age, whereas neonatal thymectomy prevents disease. Like in the BB rat, microsatellite analysis in NOD mice aimed at identifying susceptibility loci has revealed the presence of multiple candidate loci (Todd, 1995). An important candidate locus determining IDDM in the NOD mouse is the MHC locus. The MHC class I1 of NOD mice does not contain an I-E molecule, whereas the I-A chain is composed of Aad and Apg7. The presence of serine at position 57 may be involved in susceptibility because transgenic NOD mice with aspartic

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acid at this position are resistant to disease. Furthermore, transgenic mice that express I-E molecules do not succumb to disease. IDDM may also be induced with low-dose streptozotocin, a selective p cell cytotoxin. In this T cell-mediated model male mice are somehow more susceptible. The role of viruses in induction of IDDM is illustrated by three virus models of disease. Encephalomyocarditis virus induces disease with p cell destruction in both immunocompetent and Swiss nude mice but not C57BL16 nude mice. The exact mechanism of tissue damage in this model is, however, still controversial. In contrast, reovirus infection induces transient diabetes in which islet cell autoantibodies are detected as is the case with diabetes following rubella virus infection of Golden Syrian hamster. In the former model, disease is prevented with cyclophosphamide and antithymocyte serum, suggesting that in these cases disease is immune mediated.

C. Other Models of Autoimmunity RA is a heterogeneous disease that ranges from mild arthritis to severe disease involving the internal organs. It is primarily a chronic inflammatory disease affecting synovial tissue in which destruction to the joint results in crippling disease. The rheumatoid factor (RF), an autoantibody to the Fc portion of IgG, is produced in the affected synovium and is a serological marker of disease. Activated T cells are also present in the joint and anti-CD4 antibodies markedly improved disease outcome. The antigens implicated in disease include articular cartilage because RA patients are sensitive to chondrocyte antigens. Furthermore, 50% of RA patients have plasma cells specific to type I1 collagen. Even more pertinent to RA than to perhaps other autoimmune diseases is the supposed role of microbial infection in the pathogenesis. Such a role has been suggested, for example, by studies that revealed the presence of parvovirus B19 in the synovium of RA patients. However, a multitude of different viruses and bacteria have been implicated in the development as has been the role of stress proteins, most notably hsp60. As in other autoimmune diseases, however, genetic, environmental, and hormonal factors clearly affect disease. Adjuvant arthritis (AA) may be induced in Lewis rats following immunization with CFA. T cell lines derived from these immunized rats are able to transfer disease into irradiated naive recipient rats. In contrast, resistance is obtained in nonirradiated animals. The stress protein hsp60 has been identified as the critical antigen in the AA model but its role in the induction of AA is still poorly understood, particularly because T cells reactive against the very same hsp60 determinants that act as arthritogens can prevent disease induction. Other models of disease include collagen type 11-induced arthritis in mice and streptococcal cell wall-induced arthritis. Arthritis is

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also a feature in Brown Norway rats treated with gold salts and mice transfected with the HTLV-1 tax gene develop arthritis-like signs. Guillian-BarrC syndrome (GBS) is an acute demyelinating polyneuropathy of unknown etiology and pathogenesis. A possible autoimmune involvement in the disease is suggested by studies in patients that have revealed the presence of antibodies and T cells directed at myelin components of the peripheral nervous system. The clinical course of GBS correlates with titers of antibodies to peripheral nerve myelin and antibodies with complement-fixing capacities. In rodents, clinical signs reminiscent of GBS may be induced with peripheral myelin and individual protein components derived from it. The experimental model disease is referred to as experimental allergic neuritis. The major neuritogenic component of peripheral myelin is the P2 protein, which induces disease in Lewis rats and SJL mice (Saida et al., 1983). In Lewis rats, the P2-derived sequence 53-78 is neuritigenic on its own. Endoneurial injection of antisera to the myelin lipid galactocerebroside and the myelin protein PO suggests that antibodies are quite important for induction of the demyelination (Taylor and Hughes, 1985). The prevalence of myasthenia gravis (MG) is approximately 5 - 7 3 100,000. The disease is observed as muscle weakness that first becomes apparent in the extraocular and bulbar muscles but also affects muscles in the extremities. The disease most likely results from the presence of antibodies directed against the AChR. Neonatal MG can result from passage of AChR antibodies across the placenta of an affected mother; the disease in these infants, however, is transient. The antibodies are thought to mediate intracellular degradation of AChR but patients with MG frequently also have thymus abnormalities including tumor formation, hyperplasia, or atrophy. Genetic studies have shown that both susceptibility and resistance to MG are influenced by HLA type (McCombe, 1995) and there is also an association with immunoglobulin isotypes. Histologically, the disease manifests itself by degradation of the postsynaptic regions of the motor endplate. Although there are no signs of inflammation in these regions, perivascular infiltrates are observed in the muscle. Antibody complexes and complement can be demonstrated at the endplates and patients have high AChR antibodies in the serum. Also, T cells specific for AChR can be isolated from the thymus of MG patients and they are present in the blood but their relevance is currently unclear. Experimental allergic MG (EAMG) can be induced in rodents following the passive transfer of anti-AChR antibodies, following immunization with recombinant AChR subunit administered in adjuvants or by passive transfer of AChR-sensitized lymphocytes. Also, passive transfer of antibody from patients with MG or from EAMG animals inoculated with AChR induces disease in naive recipient animals. Disease is also observed in SCID mice in which tissue from human MG thymuses have been implanted. Such mice

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have been shown to produce significant levels of anti-AChR antibodies. Decomplementation with cobra venom factor or removal of antibody abrogates disease in rats. Furthermore, complement-deficient mice do not succumb to disease. Although anti-AChR plays a major role in disease, susceptibility does not correlate with the ability to produce antibody but instead is linked to the MHC. This strongly suggests that at least in this case, T cells are somehow involved. Lewis rats immunized with either the (Y subunit of AChR or the peptide sequence 100-116 exhibit disease. The disease manifested depends on the mode of inoculation and antibody or antigen used and, although the physiology is similar to human MG, the histology of the experimental disease varies and in no case is there involvement of the thymus. SLE manifests itself in a wide variety of clinical and immunological features and, as with many of the autoimmune diseases, the etiology is currently unknown. Genetic, hormonal, and environmental factors contribute to a heterogeneic clinical picture that affects up to 1/2000people depending on the ethnic population studied. Much higher numbers of females are affected compared to male (13 : 1 in adults). Although rare in children, 25% of SLE cases occur in the first two decades of life. SLE is diagnosed if 4 of 11 criteria are met: erythema; erythematosus; photosensitivity; oral ulcers; arthritis; serositis, renal, neurological, and hematological disorders; and laboratory diagnosis of immunological disorders and abnormal anti-nuclear antibody (ANA) titers. The most effective therapy available to date includes administration of corticosteroids and cytotoxic drugs. Apart from primates, only dogs and mice succumb to SLE and, in both, spontaneous models are available. The canine model is characterized by polyarthritis, dermatitis, myalgia, and glomerulonephritis. Like in the human disease, ANA levels are elevated and an association between MHC class I and SLE has been reported from German shepherd dogs. Murine models of SLE are probably more useful for the study of pathogenesis and therapy of disease because most of the immunological abnormalities of human SLE occur in the mouse model. F1 generations of NZW (H-2") X NZB (H-2d) mice develop spontaneous lupus nephritis and hemolytic disease. The short life span of the female in the F1 mice has been utilized to determine efficacy of therapeutic strategies. The other murine models of SLE are the MRL and BXSB mouse strains. Further inbreeding of the MRL mice has led to one strain developing lymphoproliferative disorder (lpr). The lpr gene was transferred to other strains, which led to production of diverse autoantibodies and development of autoimmune disease. Other mutations that give rise to generalized lymphoproliferative disease (gld) occurred in the C3HIHe mouse. Further cross-breeding of this mouse gave rise to another SLE mouse, BXSB. The various mouse strains are also useful to Sjogrens syndrome, posterior uveitis, and muscle inflammation.

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Some of the histological abnormalities of SLE are common to all strains as are the serological abnormalities, whereas others are not. The immunology of the various murine models of SLE has been reviewed (Theofilopoulos, 1992). In conclusion, the genetics of the disease in both humans and experimental animals is complex and a large variety of studies have demonstrated the presence of various predisposing factors, including autoimmune factors.

V. T Cell Recognition of Autoantigens A. T Cell Recognition of Dominant and Cryptic Epitopes in Autoantigens

A likely basis for the development of inflammatory responses in tissue in the apparent absence of pathogens is recognition of self-proteins, or autoantigens. It is obvious that inflammatory responses to autoantigens are not exactly a healthy condition. In order to avoid them, the immune system is programmed by thymic selection in such a way that T cells that are responsive to self-proteins are deleted but, as previously explained, this selection process is far from complete. In all humans, proinflammatory T cells can be found that are responsive to self-proteins, especially to those proteins that have a restricted pattern of expression outside the thymus or to those that are only expressed as the result of aging, disease, or stress. However, cellular responses can be mounted even to proteins that are present in the thymus if the responses are directed to autoantigen-derived determinants that are not normally presented to T cells. In order to understand how this may come about, some features of antigen processing and presentation by APCs must be appreciated. When an extracellular protein antigen is captured by an APC, it is directed toward an intracellular network of vesicles with relatively low intraluminal pH. These also contain proteolytic enzymes such as cathepsin D and B. Exposure of endocytosed protein antigen to these conditions results in partial proteolysis and selected protein sequences become available for interaction with MHC molecules. Newly formed MHC molecules or MHC molecules recycled from the surface have an intracellular trafficking route that intersects the degradation pathway of endocytosed proteins. When MHC molecules and partially degraded antigen meet, some antigen fragments end up in the peptide-binding groove of MHC molecules and these are transported to the plasma membrane for presentation to T cells (Harding and Unanue, 1990; Brodksy and Guagliardi, 1991; Germain, 1994).

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FIG. 11 Processing of antigens for presentation in the MHC class I1 pathway. MHC class I1

molecules routinely present protein fragments derived from extracellular antigens. When such antigens are taken up by the antigen presenting cell (APC) they are directed to the endosomal/ lysosomal degradation pathway. In early and late endosomes, the intravesicular pH decreases, reducing conditions are created, and capthepsins will catalyze partial cleavage of antigenderived proteins. At this stage, endosomal vesicles with antigen fuse with vesicles that contain newly formed MHC molecules. These have been assembled in the endoplasmic reticulum by the concerted actions of calnexin and heat shock proteins, and MHC class I1 molecules become glycosylated in the Golgi network. In some APCs, including B cells, storage vesicles exist for newly synthesized MHC class I1 molecules. During assembly MHC class I1 molecules associate with a so-called invariant chain that directs their intracellular routing and at the same time covers the peptide-binding groove. When MHC molecules enter the endosomal degradation pathway, the invariant chain is removed by proteolysis and the peptide-binding groove becomes accessible for antigen-derived peptides. Peptide-MHC complexes are then transported to the plasma membrane while peptide trimming continues.

Figure 11 depicts the main events required for presentation of exogenous protein antigens to T cells. An individual MHC molecule, however, cannot bind just any peptide it encounters because the fine structure of its peptidebinding groove imposes restrictions on the structure of the peptide that

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will have to be met. Thus, each MHC molecule has its own set of rules-the peptide-binding motif-for selecting peptides from the myriad fragments they encounter intracellularly. In this way, the selection of antigen-derived sequences that are finallypresented is determined by their pattern of release during partial antigen degradation, their relative stability in the environment, and their ability to bind to the peptide-binding groove of a given MHC molecule. As a result of this selection certain sequences from a given protein antigen will be available for recognition by T cells, whereas other sequences will not become available. The sequences that can effectively be recognized are usually referred to as dominant epitopes. The previous scenario applies to a situation in which intact protein antigen is taken up by APCs for presentation to T cells. However, it may not always be the intact antigen that is present. Especially in the context of inflammatory processes, proteolytic enzymes may operate in tissues and they may cause the appearance of fragmented proteins in the environment. When this happens, APCs may suddenly be confronted with protein structures that are already partially degraded in a way that is different from what their own endocytic catabolism would have generated. As a possible consequence, protein fragments may end up in the peptide-binding groove of MHC molecules that would never have made it in the context of their parental structure. T cells may find themselves confronted with protein sequences different from those generated under normal conditions and they may respond to them. Epitopes that are presented only when available as isolated sequences but never as part of a whole protein molecule are generally referred to as cryptic (or hidden) epitopes. It has been hypothesized that such cryptic epitopes may have a special role in initiating or perpetuating autoimmune responses (Sercarz et al., 1993). Because they cannot be generated under normal conditions, such as during thymic selection of T cells, the T cell repertoire may very well not be tolerant to cryptic epitopes. It is of interest to note that a special category of cryptic epitopes may be formed by protein determinants that may become covalently modified by, e.g., phosphorylation or glycosylation under certain conditions. It is well known that patterns of such modification may change during aging and as the result of disease or stress. If posttranslational modification of a protein sequence that represents a major determinant occurs and if this does not prevent its binding to MHC molecules, a neodeterminant may be presented to T cells for which they may, again, not be tolerant. An interesting example of this phenomenon was recently recorded in the murine T cell response to aB-crystallin. One of the major epitopes of this stress protein turned out to be a site of phosphorylation and murine T cells were grossly affected in their response to the protein by direct recognition of a phosphorylated sequence presented via MHC class I1 (Van Stipdonk et al.,

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1997). Thus, when considering crypticity it is of importance to also include this possibility.

6.Chronicity and the Phenomenon of Determinant Spreading Cryptic epitopes may be especially relevant to the perpetuation of autoimmune disease by determinant spreading. In this process it is assumed that additional T cell specificities are recruited in the course of a chronic disease by the release and emerging presentation of new autoantigens that become available as the result of continued breakdown of tissue. In chronic animal models for MS, the emergence of new T cell specificities as the result of an ongoing destructive inflammatory response in the CNS has been demonstrated to include T cells that can be pathogenic on their own (Lehmann et al., 1992;McRae et al., 1995). The activity of these new CNS-specific T cells in animals suffering from E A E has been recorded by assessing T cell specificities in the spleen or by measuring delayed-type hypersensitivity responses in the skin. In this way, activated T cells were detected against other sequences of an experimental autoantigen than the one used to trigger the disease and even against other autoantigens from the affected tissue. In some cases, this pattern of newly detectable T cell specificities may take a predictable course during disease (Yu et al., 1996). It should be noted, however, that it still remains a matter of debate whether such new T cell specificities are actually generated in the local inflammatory lesions and subsequently recirculate or whether, in fact, the T cells involved are activated in peripheral lymphoid organs that have recruited professional APCs carrying antigen samples from inflammatory regions back into the lymph nodes and spleen. Although the phenomenon of antigen spreading during autoimmunity has been convincingly documented in animal models, it remains to be established whether the same occurs in human autoimmune diseases. Currently, the availability of an animal, i.e., the Biozzi ABH mouse, for which major encephalitogenic epitopes derived from a number of different myelin antigens have been fully delineated provides a unique system to test the impact of determinant spreading on strategies for antigen-specific intervention in chronic relapsing EAE (Amor et al., 1993, 1994, 1996a). Continued studies on this issue will provide invaluable information on how to apply such strategies in humans.

C. About Autoantigens in Human Auotimmune Disease The definition of relevant autoantigens in human autoimmune disease has remained an elusive goal. Several reviews have been devoted to this

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issue (Weiner et al., 1994) and the information available on the subject could well be repeated again here but perhaps more informative is some reflection on exactly how autoantigens should be defined. From what has been explained previously, some rules can be defined that describe T cell activation by proteins. When referring to autoantigenic stimulation, we mean to say that T cell activation occurs to protein sequences that belong to the body itself. At the same time, however, lack of understanding exactly how autoimmune diseases develop also implies that we cannot fully appreciate exactly what the status of autoantigens is o r should be. Must autoantigens be tissue-specific antigens when we are dealing with tissue-specific autoimmune diseases or may they be just as well expressed elsewhere in the body when the absence of costimulation then renders them invisible to the immune system? Are autoantigens “cryptic” structures for which the immune system may not have been tolerized or, rather, are they more likely to be immunodominant structures for which regulation fails? Are autoantigens stable entities throughout the development of autoimmunity over years or do autoantigens change over time? Are autoantigens the same from one patient to another, especially if clinical symptoms and histopathology vary? Must immune responses to autoantigen be detectable only in patients or may they be part of normal immune repertoires? Must the immune system be primed for autoantigens or may primary responses also play a role? Must autoantigens for human diseases meet the criterion of pathogenicity in healthy laboratory animals or is their autoantigenic status codefined by an altered state of the tissue in which they reside? Currently, we have to acknowledge that we do not have the answers to these questions and thus, that conclusive statements as to which antigens are actual autoantigens in autoimmune disease cannot be made at this point. Perhaps the only exception to this is the acetylcholine receptor as target antigen for autoreactive immunoglobulins in myasthenia gravis. Most proteins that have been suggested as autoantigens in tissuespecific autoimmune diseases are tissue-specific proteins against which marked cellular proliferative responses can be detected in patients. Sometimes, T cell responses in patients may be stronger or qualitatively different from those in healthy control subjects, but given the complexity of what a T cell response entails, final statements on this issue cannot be made. Second, many claims on certain proteins being autoantigens in human disease have been substantiated by the observation that transfer into healthy laboratory animals of autoantigen-reactive T cells or antibodies results in clinical and pathological symptoms that are similar to those of the human disease. At the least, this establishes the pathogenetic potential of those specific responses. These two criteria are usually considered to be the most important. Especially in the case of

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cellular responses to MBP, many studies have been performed to test the initial expectations of what an autoantigen should be. At first, MBP appeared to be a good example of an autoantigen, being a more or less tissue-specific protein and being capable of inducing T cell-mediated encephalomyelitis in laboratory animals (Zamvil and Steinman, 1990; Martin et al., 1992). Instead of confirming differences between MS patients and healthy controls, however, the study on MBP has clarified that autoreactive, potentially pathogenic cellular responses, even to hidden, tissue-specific proteins, are a normal feature of the adult human T cell repertoire. No quality or feature of the MBP-directed T cell repertoire or its status in MS patients has so far been identified that is any different from what is seen in control subjects. If anything, these studies have demonstrated that we should be cautious in employing the criteria of tissue specificity and pathogenicity in laboratory animals as criteria sufficient for the identification of autoantigens. Recently, a different approach was taken to identify potential autoantigens in multiple sclerosis (Van Noort et al., 1995). In this study, the possibility was considered that not only could deviations in the immune repertoire cause autoimmunity to develop but also differences in the molecular makeup of the target tissue of disease itself. Thus, central nervous system myelin preparations were examined for their potential to trigger human T cell responses and the targets of these responses were delineated using high-resolution separation of all proteins contained in the tissue, using myelin from either MS patients or from control subjects. In this study, it was noted that both MS patients and healthy controls mounted cellular responses to virtually every protein in the preparation but that a single strong response was recorded when using MS brains as a source of antigen but not when using control material. The protein that was the target of this response was identified as the small stress protein aB-crystallin. This study demonstrated that human T cell responses can be generated to virtually every myelin protein, at least in vitro. In addition, the fact that the strongest T cell trigger in MS-affected tissue was a stress protein may be just a little too much of a coincidence. Previously, the idea has been put forward that the pathogenic response developing within the white matter of MS brains could perhaps be attributed more to dysregulation of a local stress response than to intrinsic aberrancies of the T cell repertoire (Van Noort, 1996). More data are required to examine the possibility that in human tissue-specific autoimmune disease, it is the target tissue that provokes disease just as much as the immune system attacks it. If this were true, autoantigens could also be molecules that are only expressed or strongly upregulated in diseased tissue and the criterion of pathogenicity in healthy laboratory animals could become a problematic one.

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VI. Intervention in Autoimmune Diseases A. Reestablishment of Immunological Tolerance to Autoantigens Recently, much interest has been focused on strategies for reestablishment of immunological tolerance fueling the hope that identification of relevant autoantigens in autoimmunity could then be translated into novel therapeutic approaches. Basically, two aims may be defined for reestablishing immunological tolerance to a given antigen. The first is the manipulation of the quality of an antigen-specific response and turning it from a proinflammatory response into a regulatory response. In this context, tolerance does not actually mean lack of responsiveness but rather a lack of pathogenic responsiveness. The second aim is the deletion or inactivation of antigenspecific T cells, ways to actually kill off autoreactive T cells by the induction of apoptosis. Mechanisms that influence the quality of the response (see Sections II,B and I1,C) probably also underly the phenomenon of oral tolerance: the well-known lack of classical proinflammatory responses to any protein ingested via the oral route (Weiner et al., 1994). Somehow, the way the immune system deals with antigens that enter the body via the nasal or oral mucosa or via the gastrointestinal tract is different from what happens during classical immunization. Instead of activating T cells capable of secreting IFN-7, lymphotoxin, or TNf-a (type 1 responses), oral administration of defined protein antigens appears to more readily activate T cells that secrete large amounts of TGF-P, a modulatory cytokine (Miller et al., 1991; Khoury et al., 1992). Such T cells, tentatively designated Th3 cells, may downregulate inflammatory responses and, at least in experimental models, they ameliorate autoimmune responses (Higgins and Weiner, 1988;Fukaura et al., 1996). Prevention of experimental autoimmunity by oral or intranasal administration of the trigger antigen has been well established in several different model systems. Clearly, it appears to be an attractive option to try and it beneficially influences autoimmune disease by feeding patients the antigen that elicits the response. In fact, ongoing clinical trials in, for example, multiple sclerosis and rheumatoid arthritis are based on the expectation that by feeding the putative autoantigen(s), one may shift the specific immune response to one with a less inflammatory quality (Weiner et al., 1993; Trentham et al., 1993). However, this approach may not be as straightforward as one would hope. First, steering the autoantigen-directed response in patients suffering from, e.g., multiple sclerosis to type 2 responses may not be helpful when antibodies play a pathogenic role. In multiple sclerosis as well as in rheumatoid arthritis, evidence is available

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that this is indeed the case. Second, oral administration of antigen may very well lead to activation of some pathogenic T cells, including cytotoxic T cells, as was recently found in marmosets as well as in mice (McFarland, 1996). Thus, many questions still exist as to the exact way the immune systems deals with orally or intranasally administered antigen and the final outcome of this strategy. The possibility to control inflammatory responses in this way is sufficiently provocative and useful to justify ongoing efforts to try and find the appropriate answers. A different mechanism involved in the reestablishment of tolerance is costimulation or, rather, the lack of costimulation. As explained previously, when resting memory T cells encounter their antigen in the context of MHC molecules but in the absence of the proper costimulatory molecules, they will die, usually by apoptosis. It is the natural way of controlling established T cell responses, but at the same time it provides an interesting option to researchers for the specific inactivation of certain populations of T cells that are suspected of causing autoimmunity. What one would basically aim for is administration of antigen in such a way that T cells may encounter it in the context of MHC molecules but in the absence of costimulation. Experimental approaches to achieve this include oral administration of antigen (Chen et al., 1995; Weiner et al., 1993; Trentham et af., 1993), intravenous administration of antigen (Gaur et af.,1992; Racke et al., 1996), intraocular administration (Wilbanks and Streilein, 1991), intraperitoneal administration in incomplete adjuvant (Swierkosz and Swanborg, 1977), or administration of chemically prepared antigen-MHC complexes (Miller et al., 1979). In all cases, costimulation does not occur because the way the antigen is introduced in the body is sufficiently stress free. As a result, T cells reactive to the antigen introduced will undergo apoptotic death. In experimental models for autoimmunity, this approach has been quite successful both with proteins and with peptides representing the trigger antigens in such models. Key to application of antigen-specific tolerance induction in human autoimmune disease is of course the definition of the appropriate autoantigens and it is mainly for this reason that efforts to achieve this definition should continue. An alternative way of preventing the occurrence of costimulatory interactions between APCs and T cells is administration of antibodies directed against the costimulatory molecules involved. An excellent example of such a treatment and its effect on the development of autoimmune disease has been reported for antibodies against the CD40 ligand (Laman et al., 1996) and current efforts are devoted to application in human diseases. Even after onset of autoimmunity in experimental models, specific anti-CD40 ligand antibodies were found to be able to ameliorate disease (Gerritse et al., 1996).

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B. Antagonist Modulation of an Autoimmune Response

T cell activation occurs when the TcR engages with the MHC molecule, in which a specific peptide is bound. This is important not only in generating a protective response against invading microbes but also in the generation of autoimmune responses. Identification of autoantigenic peptides, such as the encephalitogenic peptides of MOG, PLP, and MBP, for the induction of E A E in mice such as the Biozzi ABH mouse (Amor et al., 1993, 1994, 1996a), and studies on how they interact with MHC class I1 molecules on the one hand and with TcR on the other hand, has allowed the development of altered peptide ligands (APLs). Subtle changes in the sequence of APL compared to the authentic peptide target may alter the affinity of binding to the MHC. Alternatively, such changes may occur in the regions recognized by the TcR. In each case the altered peptide may result in a different T cell response compared to that against the authentic peptide. Thus, altered peptides have been shown to inhibit (antagonize) or augment (superagonize) the normal T cell response, whereas others are able to deviate (partially agonize) the response completely, e.g., switching a type 1 response to a type 2 response. In the E A E model in the PLJ mouse the MBP peptide Acl-11 is the immunodominant epitope for induction of disease. The amino acids important in MHC binding and those thought to be the major T cell contact points have been identified. Computer modeling using the crystal structure of an MHC molecule can be employed to evaluate possible ways a short peptide sequence may interact with the peptide-binding cleft of the MHC molecule. An example of such a modeling study to identify amino acid side chains available for contacting the MHC and TcR, respectively, is given in Fig. 12. In the case of the MBP sequence binding to the particular class I1 molecule of the PLJ mouse, i.e., I-A", substitution at position 4 resulted in peptides with different affinities for the MHC. It is striking that the original encephalitogenic epitope binds with only low affinity. This lowaffinity binding has been suggested to be a major reason why potentially responsive T cells may escape negative selection in the thymus (Fairchild and Wraith, 1996). In another model of EAE in the SJL mouse the major immunodominant epitope is the PLP sequence 139-151. Adoptive transfer of T h l CD4'lymphocyte clones against this sequence induce disease in recipient mice. The use of peptide analogs demonstrated that tryptophan at position 144 is critical for TcR contact and activation. Substitution of tryptophan for glutamine abolishes the ability of the peptide to induce disease suggesting that peptide analogs may exert their activity via competition with the wild type due to the higher binding affinity of the analogs for MHC. However, because antagonist peptide analogs of PLP 139-151 (except A144) do not bind to MHC at any higher affinity

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FIG. 12 Model structure of the MHC molecule H-2Ag’ with a proteolipid protein (PLP)derived sequence that induces EAE in mice. Amino acid residues are given in three letter symbols. Leu, Ile, Asn, Val, Ile, His, Ala, Phe, Gln, Tyr, Val. A top view is represented showing the peptide (in a ball-and-stick representation) entrapped in the binding cleft of the MHC molecule (represented as a ribbon). Modeling studies such as these allow predictions to be made of which amino acids will be important to MHC binding and which may directly contact T cell receptors. Thus, a rational approach can be taken to the design of altered peptide ligands for possible therapeutic intervention.

than the wild-type peptide, it was concluded that mechanisms other than MHC blockage were at work. Further studies demonstrated that T cells induced by the APLs produced a predominantly type 2 response (IL-10 and IL-4) demonstrating that APLs were able to skew the cytokine profile. Furthermore, coimmunization of the authentic peptide with the glutamine APL protects mice from EAE. Striking examples have been reported of a single APL being able to suppress a multifaceted autoimmune response in vivo (Brocke et al., 1993). However, the mechanism by which APLs exert their effect has not been fully elucidated. It is probable that T cell manipulation by APLs is based on the differential activation of selected intracellular signaling pathways.

C. Cytokine Modulation of an Autoimmune Response Activation, amplification, and modulation of the effector phase in immunemediated responses occur via cytokines that can be either inhibitory or augmentary. Because many of the cytokines through to play a pathogenic

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role in autoimmune disease are type 1cytokines, including IFN-y, TNF-a, and IL-2, methods to counteract these have been the prime interest in efforts to direct therapy at the level of the cytokine network. Alternatively, because the type 2 cytokines IL-4, IL-10, and IL-13 inhibit the proliferation of T h l cells, therapies in which Th2 cells are augmented may also be beneficial. However, in autoimmune diseases in which autoantibodies predominate, such as in SLE, this strategy may be counterproductive because in those cases augmenting type 2 responses and inhibiting type 1 responses may be worsening disease rather than ameliorating it. In this section, we will concentrate on cytbkine modulation of autoimmune diseases thought to be mediated by CD4+ T h l cells. Although T h l and Th2 phenotypes have been well defined in mice, the distinction can sometimes be applied to human T cells with some difficulty. However, in humans IFN-y activates monocytes and macrophages to produce TNF-a, IL-1, and IL-6, which in turn induce an array of proinflammatory products including chemokines. IFN-y also upregulates human MHC molecules providing cells with the ability to present autoantigens. It should be noted that in several well-established animal models of autoimmunity manipulations through the cytokine network may result in surprising and sometimes even confusing data. The majority of the cells present within recently infiltrated islets of NOD mice secrete high levels of IFN-y. Administration of an antibody to IFN-y reduced the incidence of disease including insulitis. In other diseases the effects are less clearcut. IFN-y-secreting cells are present in the lesions and in the CSF of MS patients. In mice, systemic or intraventricular administration of IFN-y has been shown to inhibit E A E and neutralization of IFN-.)I exacerbates EAE. However, a different result is observed in the human disease. Administration of IFN-y in this case was shown to exacerbate relapse in MS demonstrating a fundamental difference between the animal model and the human situation. In the non-organ-specific disease SLE, IFN-.)I correlates with disease and, like in MS, administration of IFN-y and IFN-a can augment ongoing SLE in man and animals. Apart from their antiviral actions IFNa and IFN-p are also immunoregulatory. The effects of both IFN-a and IFN-p have been assessed in MS. Results from a number of trials with IFN-a have been inconclusive, whereas intrathecal IFN-p has been shown to both reduce the number of relapses in MS and improve the degree of disability. The mode of action of IFN-0 is thought to be via the inhibition of monocyte and T cell activation. In vitro studies suggest that IFN-p may exert its effect by modulating MHC class I1 expression. However, whether this also occurs in vivo is currently unknown. The other major T h l cytokine that has been targeted in therapies is TNF-a. TNF-a is released from activated macrophages and is known to induce expression of MHC class I and I1 antigens and upregulate adhesions

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molecules. TNF-a has been shown to be cytotoxic for myelinated oligodendrocytes in culture and intravitreal injection of TNF-a in mice results in lesions of demyelination similar to those observed following SFV infection. Although no effects have been reported so far on modulation of TNF-a in the virus model for MS, in the active EAE model systemic administration of anti-TNF-a antibodies ameliorates clinical and histological disease. A more dramatic effect is observed when the antibody is given intracerebrally (Baker et al., 1994), emphasizing the importance of locally produced cytokines and the possible pitfalls of such therapies in human disease. E A E can also be modulated using soluble TNF receptors. Chimeric human/ mouse neutralizing monoclonal antibodies to TNF-a have been shown to induce beneficial effects in R A and many trials directed at the action of TNF-a are now in progress. Repeated administration of anti-TNF-a antibodies decreases the pain score and morning stiffness, and increases grip strength (Elliot and Maini, 1996). Other TNF-a blocking therapies include recombinant human soluble receptor Ig : Fc fusion protein (effective in RA), chemicals such as pentoxifyllin (in E A E and experimental uveoretinitis), and therapies aimed at blocking the matrix metalloproteinases required for the maturation and release of TNF-a. IL-1 shares many of its biological functions with TNF-a and it is present in inflammatory infiltrates in many autoimmune diseases. Repeated injection of IL-1 in rats gives rise to tissue damage in the joint while, conversely, the IL-1 receptor (IL-lr) is effective in ameliorating disease. Clinical trials have also shown that IL-lr can be effective, although side effects such as flu-like symptoms were common. Inhibition of other proinflammatory cytokines have also been shown to be beneficial in autoimmune disease. The action of IL-6 in promoting humoral immunity suggests its therapeutic potential in some autoimmune diseases. Anti-IL-6 improves joint stiffness in R A and suppresses disease in lupus mice, as does blockage of IL-6 receptors. Recently, attention has turned to the use of anti-IL-12 antibodies to modulate autoimmune disease. This approach is beneficial in E A E and inflammatory bowel disease. IL-12 injections accelerate diabetes in NOD mice and therapeutic strategies inhibiting IL-12 in humans are now being considered (Trinchieri and Scott, 1994; Lamont and Adorini, 1996). Cytokines such as IL-4, IL-10, and TGF-0 inhibit inflammatory cytokine synthesis and may provide an alternative mechanism for treating autoimmune disease. IL-4 has been shown to induce immune suppressive effects in several models of autoimmunity (Rocken et al., 1996). In experimental autoimmune diabetes in which mice were engineered to produce high levels of IL-4 and IFN-7, diabetes no longer develops. Likewise, when IL-4 is given early after inoculation of MBP-specific T h l clones, E A E no longer develops in mice. In this system, IL-4 treatment appeared to coincide with the generation of MBP-specific Th2 cells. Mice receiving IL-4 at later time

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points did not produce MBP-specific Th2 cells and were not protected. IL10 was found effective in reducing collagen-induced arthritis. However, controversial studies have been described in EAE. In some studies IL-10 administration was reported to exacerbates disease, whereas in others, improvement of clinical signs was recorded. The most consistent downregulation of Thl responses and suppression of autoimmune disease has been experienced with TGF-P. This cytokine is upregulated in recovery from clinical disease and remission and administration in EAE has been shown to inhibit disease. Furthermore, the immunomodulatory action of omega6 PUFA has been shown to correlate with TGF-/3 production as has the beneficial effects of oral tolerization of rats with MBP (Weiner et af., 1994). However, the lessons that can be drawn from the previous studies have demonstrated that the effects of cytokine administration or cytokine blocking are highly dependent on the time and site of intervention and on the presence of other potentially synergistic factors.

D. Therapy Directed at the T Cell Receptor Over the past decade, tremendous efforts have been dedicated to the study of T cell receptors in autoimmunity. Two factors greatly contributed to this. The first was the discovery of so-called superantigens: viral or bacterial proteins capable of activating a significant proportion of the mammalian T cell repertoire by a peculiar mechanism (Scherer et af., 1993). Superantigens bind firmly to the MHC class I1 molecule but outside of the peptidebinding groove. At the same time, they can interact with certain conserved structural features of T cell receptors that exist in the partially variable amino acid sequence of its p chain. This results in a situation in which T cells endowed with the proper Vp sequence can engage in a superantigenmediated interaction with APCs even though the peptide in the MHCbinding groove may not fit the receptor at all. The superantigen simply overrules this requirement and causes the activation of the T cell based on its Vp structure. It is obvious that such an event may lead to the activation of a significant proportion of the complete T cell repertoire regardless of antigen specificity,including the activation of autoreactive T cells. However, if this happens, it is easily recognized by the fact that all activated T cells must then carry the same type of Vp structure, which, in turn, would provide a way for therapeutically attacking them again. The question arose as to whether superantigen-driven responses played a role in autoimmune diseases. At about the same time, it was found that in both rat and mouse models for MS the pathogenic (encephalitogenic) T cells were highly restricted in their Vp structures, all sharing more or less the same structure (Zamvil et

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af., 1988; Urban et af., 1988). Especially in Lewis rats, the restriction to the use of one particular family of Vp T cell receptor structures was striking. This observation was particularly challenging because the encephalitogenic rodent T cells sharing the same Vp did in fact recognize different antigenic sequences in the context of different MHC molecules. Although no simple explanation could be given for the restricted Vp usage of pathogenic T cells in rodent models of MS, this condition did allow therapeutic intervention (Acha-Orbea et al., 1988; Vandenbark et af., 1989). Therapies to block autoimmunity were designed to intervene at the level of T cell receptors. These included the administration of VP-specific antibodies, immunization with peptides representing the Vp sequence in order to induce regulatory immunity against pathogenic T cells, and whole T cell vaccination (BenNun et af., 1981). Many of these therapies were quite successful in blocking the development of EAE. Again, the question arose as to whether a similar approach was possible in human autoimmune diseases. Consequently, an impressive number of studies were undertaken to examine the Vp repertoire of autoreactive T cells in patients suffering from autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and diabetes. There have been a large number of reports on recognition of myelin basic protein, a major component of central nervous system myelin, by T cells from multiple sclerosis patients. Initially, several reports appeared that described a markedly biased V-gene repertoire in populations of patients compared to controls. Research evolved to also include studies on Va sequences and the CDR3 junctional region of T cell receptor sequences. Confusing,however, were the relatively small number of subjects frequently involved in those studies, the lack of appropriate control subjects, and the fact that reported biases were dissimilar from one research report to another. Also, the fact that still no answers are available to the question of which T cell specificities actually must be studied as being relevant to human autoimmune disease greatly adds to the continued uncertainties (Martin and McFarland, 1995). The issue of restricted Vp, Va, or CDR3 sequences has not been fully resolved but collective evidence suggests that it is very unlikely that in any disease, a universal V-gene usage skews the repertoire of patients compared to controls. The best that can be hoped for appears to be the possibility that in certain individuals, a given restricted V-gene usage of T cells reactive to relevant antigens is stable over time and may be used as a target for intervention. Immunization with peptides representing parts of a T cell receptor sequence (Vandenbark et al., 1996),vaccination with DNA encoding such sequences (Waisman et af., 1996), or T cell vaccination (Zhang et al., 1993) are approaches that could perhaps be used in this context but to date little evidence supports the idea that these approaches will be useful in the therapy of human autoimmune diseases.

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E. Gene Therapy Diseases for which specific gene defects are directly responsible, e.g., severe combined immunodeficiency, may be controlled by inserting the normal copy of the defective gene. The approach of gene therapy, in which cells from the patient are removed, the normal gene is inserted, and the corrected genes are transfused into the patient, bypasses problems of graft versus host disease and thus problems of rejection. In autoimmune diseases, although the proinflammatory type l cytokines have been implicated in tissue damage, the regulatory type 2 cytokines have been shown to control disease. Thus, situations in which the cytokine profiles may be modified may be beneficial as a form of therapy (see above). Transfusion of cells secreting type 2 cytokines or, more specifically, of cells into which a gene encoding a specific cytokine, e.g., IL-10 or TGF-P, is transfected are methods of selective immune modulation. However, in autoimmune diseases it is probable that cytokine production is limited to certain types of cells and tissues and to only certain episodes. Counteracting or modulating this is an effective, but controlled manner may therefore not be simple. Most of the approaches for cytokine gene therapy have come from studies on tumors because cytokines may directly modify the tumor cell and modification of lymphocytes is particularly attractive because of their ability to proliferate. Ideally, the cell of choice should be easy to transfect, selectively target the organ of choice, and be able to proliferate well in vitro in a controlled manner. It would be unwise to allow the transfected cell to secrete any quantity of cytokine wherever it chooses. Delivery of the cytokine by other types of cells such as genetically modified endothelial cells and fibroblasts is under investigation. Such an approach would allow the implantation of the transfected cell into the exact organ of choice. Alternatively, transfection of cells with the gene under a particular inducible promoter would allow direct regulation of the gene and, hence, cytokine production. In attempts to stop tumor growth (Lotze et al., 1994), fibroblasts have been transfected with the IL-4 gene to induce an immune response to tumor cells. In experimental diseases, transfected T cell hybridoma cells expressing IL-4 have been shown to control EAE and mice transgenic for IL-4 are protected against IDDM in the NOD mouse suggesting that, in the future, gene therapy may be beneficial in autoimmune disease.

F. Antioxidants in Therapy To understand the role of antioxidants as a therapeutic strategy in autoimmune disease it is first necessary to discuss some basic biochemistry to understand the importance of antioxidants in regulating free radicals (for

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a more comprehensive review, see Halliwell and Gutteridge 1984; Betz, 1993). A free radical is defined as an atom or molecule having an unpaired electron in its outer orbit. They are often derived from molecular oxygen because of its ability to readily accept electrons forming the superoxide anion radical (02-). This radical can undergo dismutation to form H202 either spontaneously or via the enzyme superoxide dismutase (SOD). The control of H202is regulated in turn by catalase and glutathione peroxidase. This is quite necessary because powerful oxidative hydroxyl radicals can be formed in the presence of, for example, iron salts. On its own, H 2 0 2is not toxic but it does induce oxidative stress. As a method of controlling the actions of H202, cells produce the nonspecific antioxidants ascorbic acid, a-tocopherol, glucose, and uric acid. Thus, by controlling the enzymes SOD and catalase and by regulating levels of glutathione, nonspecific antioxidants and iron control may be gained over the levels of free radicals and thus over the likelihood of tissue damage to develop. NO is also produced by macrophages. NO is an effective vasodilator and, together with oxygen radicals, an extremely effective antibacterial agent. However, the production of NO, like that of free radicals, may also have detrimental effects on the host tissue. Excessive NO activity has been implicated in tissue damage during degenerative disorders and in several autoimmune diseases. Of all the autoimmune diseases it would seem that damage by free radicals and NO may be particularly relevant in MS because myelin, the primary target of destruction, contains high levels of unsaturated fatty acids as well as of iron. Lipid peroxidation, which occurs when an hydroxyl iron is extracted from an unsaturated fatty acid, generates new species of free radicals and, hence, may cause more damage (Levine, 1992), particularly in the presence of iron. In MS macrophages may play a dual role, first, by releasing proteases and lipases that may cause myelin damage and thus release iron. Second, macrophages supply the necessary substrates for hydroxyl radical formation. Increased generation of superoxide radicals can be detected in the blood of MS patients (Glabinski et af., 1993) and lipid peroxidation products are detected in the CSF. The iron chelator deferoxamine is able to reduce both clinical and histological signs of EAE. On this basis, patients with advanced MS were also treated with deferoxamine and 4 of 12 patients improved (Levine, 1992). Experimental studies have also shown that the levels of uric acid and atocopherol are increased, whereas levels of glutathione are decreased in the CNS during EAE. Therapy of EAN, the experimental model of Guillain-Barre syndrome, with the oxygen radical scavengers superoxide dismutase and catalase has been demonstrated and daily administration of the free radical scavenger N-acetyl-L-cysteine protects against EAE. Treatment of EAE with SOD or catalase has so far produced rather confusing data that are difficult to interpret. Detection of free radicals is difficult due to

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their instability and short life. However, many studies have reported increased levels of free radicals and decreased levels of antioxidants in both human disease and experimental models of autoimmunity. Furthermore, therapies that reduce or inhibit free radical activity are effective in the inhibition of experimental conditions and may thus be a target for therapeutic intervention in humans.

VII. Perspectives on Future Therapies in Autoimmune Diseases

In this review, we have attempted to present an overview of immune mechanisms that operate in autoimmune diseases as well as of the factors that control these mechanisms. Also, we have discussed experimental models for autoimmune diseases that not only allow us to examine some molecular and cellular interrelationships in greater detail but also provide the tools to evaluate possible ways to control disease. Currently, what is clear is the fact that autoimmune diseases have a multifactorial basis and are effected by an impressive number of different mechanisms. We have not discussed repair and regeneration mechanisms, which are obviously of great interest as well. Their study is as relevant as the study on mechanisms that lead to destruction and, indeed, one may even wonder whether autoimmune diseaes may be determined by a failing repair system as much as by overactive degradative systems. Also, it appears to be quite likely that metabolic failure in certain tissues will contribute to autoimmune responses to become targeted at that particular tissue. Correction of such metabolic failure, therefore, may become a possibility to future therapy as well. Hopeful developments in the field of tissue and cell transplantation and gene therapy fuel the expectation that replacing diseased cells and tissue with healthy ones may become an option in the future. The question of whether this will be sufficient, however, cannot be answered as yet, and for the time being it appears to be a good idea to continue efforts to block destructive immune mechanisms. We have also discussed the possible contribution by microbial infection to the development of autoimmunity. Various patterns of infection with either bacteria or viruses most likely codetermine susceptibility to disease and its course of progression. However, no specific pathogens have been identified to explain the onset of major autoimmune diseases and it appears unlikely that this will happen in the future. Collectively,data on the relationships between microbial infection and autoimmunity suggest that immune processes triggered by infection are somehow relevant to disease but that several viruses and bacteria may have similar effects. Prevention of autoim-

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munity by selective vaccination, therefore, does not appear to be a realistic option. In fact, the reverse may even be considered. Data suggest that delayed primary infection with common pathogens such as herpes viruses increases the risk for the development of autoimmunity later on in life. This phenomenon has been used to explain, for example, the apparent differences in the incidence of autoimmunity between Western societies, in which excellent infant health care delays primary infections, versus less developed areas in which most children experience many types of microbial infection at an early age. Taking this into account, controlled contamination rather than vaccination may be a more productive strategy. Once triggered, many of the immune-mediated mechanisms that are involved in the development of human autoimmune diseases are essentially normal features of the human immune system, vital to long-term survival. This implies that most of these mechanisms cannot be completely switched off without causing serious general immune dysfunctioning inevitably leading to problems that may become more acute than the disease we want to cure. However, currently existing therapies, such as treatment with corticosteroids or cytotoxic compounds, as well as cytokine-directed therapies such as those against TNF-a usually operate at this general level. Many more forms of such generic therapies are currently being developed. To date, they include therapies aimed at favorably manipulating the cytokine network, the stress response, leukocyte trafficking pathways, T cell costimulation pathways, or oxidative and degradative processes such as those involving metalloproteinases. Although improved ways of intervention may result from these developments, fundamental difficulties will remain. First of all, there is a trade-off that will always have to be made between efficacy of a treatment and its side effects. High-level glucocorticoid treatment, for example, may be quite effective in supressing immune functioning; its severe side effects currently preclude continued application. When we aim at lesser side effects of a generic therapy, we will have to be prepared to pay a price in terms of efficacy. Interferon+ treatment of MS patients, for example, may be well tolerated for a prolonged time; the effect the treatment has on disease is only barely sufficient to justify doing it at all. Second, generic therapies intervene in mechanisms that usually influence a multitude of different biological functions and, therefore, final net effects are very difficult to predict and they may vary from one individual to another. Again, this is best exemplified by glucocorticoids that frequently ameliorate autoimmunity, even if only temporarily, but may very well worsen disease in some patients. Also, treatment of MS patients with interferon+ shows that although some delay in disease progression can be recorded in large groups of patients, a significant number of patients do not respond to treatment at all. Third, the mere importance of the pathways targeted by generic therapies to proper biological functioning will no doubt have been recog-

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nized by evolution, which will have incorporated the appropriate redundancy in the system. The cytokine network is a good example of such a system in which essential functions mediated by individual cytokines can also be performed by other cytokines when the first mediators are blocked. More than once, immunologists have been highly surprised to find perfectly healthy animals after disruption of supposedly vital genes such as those encoding major cytokines, MHC class I1 or class I molecules, and CD4 or CD8 molecules. Nature is resourceful and the more important a biological pathway is the more bypasses will exist to ensure its execution, even if crucial steps in the authentic pathway are blocked. These considerations suggest that generic approaches may well turn out to hold a balance between efficacy and acceptability, even when combined. In our view, the most challenging strategy to ameliorate autoimmune disease is the selective blocking of antigen-specific responses by the immune system. Although in essence we do not know what causes autoimmune diseases, we do know that in the major diseases, such as diabetes, rheumatoid arthritis, and multiple sclerosis, the activation of specific populations of helper T lymphocytes must be instrumental in disease development. This can now safely be concluded based on detailed immunohistochemical studies on inflammatory tissue lesions, the clear association of autoimmune diseases with genes that encode HLA-DR molecules, and experiences with animal models that provide us with the best clinical and pathological similarities to human diseases. Also, fundamental concepts in immunology tell us that helper T lymphocytes are key in controlling immune mechanisms, even if these are ultimately affected by other leukocytes. Clearly, the perspective of blocking the activation of only a small percentage of all T cells, i.e., only the ones involved in disease development, is a very attractive one. If we were to succeed in such a strategy, efficacy may be expected to be optimal and side effects minimal. The disadvantage of this approach, however, is that therapeutic approaches would have to be tailored according to antigenspecific responses that control each type of autoimmune disorder. Also, we do not know how many different antigens are involved as relevant T cell targets in the various autoimmune diseases. Although some may be pessimistic about the number of antigens that may be involved, there is also reason for optimism. Although a large variety of T cell responses may well be elicited against the array of tissue-derived antigens, some of these may trigger quite dominant responses. An example of this was recently reported for MS-affected myelin, in which it was demonstrated that in the multitude of different myelin proteins only one protein triggered dominant responses. It appears to be an attractive option to try and identify other such dominant T cell triggers in various tissues. Another reservation may be that antigenspecific responses may not be stable over time and perhaps different from one individal to another. Although this is yet to be proven in humans,

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experimental animal models provide an excellent option to examine how to deal with this problem should it occur. In recent years, several strategies to antigen-specific intervention have been developed up to the point that, at least in animal models, autoimmune processes can be fully blocked, even when intervention is applied after autoimmunity has already been initiated. We have discussed these strategies in Sections VI,A and VI,B. Oral tolerization and administration of antigen or antigen-derived structures in such a way as to prevent costimulation are important examples of such strategies. Their successful application in human autoimmunity obviously depends on the progress in identifying antigens that are relevant in this context. We hope we have convinced the reader of this review that continuing such efforts is worthwhile.

Acknowledgments We thank our colleagues for contributing to the studies cited in this review and sharing unpublished data with us. Our work is supported by the Foundation for the Support of Multiple Sclerosis Research in the Netherlands, theNetherlands Prevention Fund, the Multiple Sclerosis Society of Great Britain and Northern Ireland, and the European Commission via the concerted action’s program T-cell Autoimmunity in Multiple Sclerosis.

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Multiple Forms of Tubulin: Different Gene Products and Covalent Modifications Richard F. Ludueiia Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

Tubulin, the subunit protein of microtubules, is an d/3heterodimer. In many organisms, both ct and /3 exist in numerous isotypic forms encoded by different genes. In addition, both (Y and /3 undergo a variety of posttranslationalcovalent modifications, including acetylation, phosphorylation, detyrosylation, polyglutamylation,and polyglycylation. In this review the distribution and possible functional significance of the various forms of tubulin are discussed. In analyzing the differences among tubulin isotypes encoded by different genes, some appear to have no functional significance, some increase the overall adaptability of the organism to environmental challenges, and some appear to perform specific functions including formation of particular organelles and interactions with specific proteins. Purified isotypes also display different properties in vitro. Although the significance of all the covalent modifications of tubulin is not fully understood, some of them may influence the stability of modified microtubules in vivo as well as interactions with certain proteins and may help to determine the functional role of microtubules in the cell. The review also discusses isotypes of y-tubulin and puts various forms of tubulin in an evolutionary context. KEY WORDS: Tubulins, Tubulin isotypes, Posttranslationalmodifications, Microtubules.

1. Introduction Microtubules (MTs) are cylindrical organelles found in almost all cell types in all eukaryotes. They are involved in a great variety of cellular processes including mitosis, ciliary and flagellar motility, and intracellular transport Inlernorional Review of Cyrohgy, Vo/. 178

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of vesicles and organelles; they may also play purely cytoskeletal roles in determining the morphology of certain cells (Hyams and Lloyd, 1994). The structural subunit of MTs is the 100-kDa protein tubulin. The tubulin molecule has long been known to be a heterodimer of two polypeptide chains designated a and P (Bryan and Wilson, 1971; Ludueiia et al., 1977). Because of the variety of numerous functions in which MTs are engaged, it has always been tempting to hypothesize that different functions may require the existence of different forms of a and P. This idea was first proposed by Behnke and Forer (1967) on the basis of the observation that different subpopulations of axonemal MTs differed greatly from each other and from nonaxonemal MTs in their stability to temperature extremes and proteolytic enzymes. This idea was subsequently elaborated by Fulton and Simpson (1976) as the “multitubulin hypothesis.” Meanwhile, evidence appeared that tubulin could undergo posttranslational modification (Eipper, 1972); this would certainly be one mechanism for creating multiple species of tubulin. Since then, various other posttranslational modifications have been described; these will be discussed later. In 1981,the first complete sequences for both a- and P-tubulin were published (Krauhs et al., 1981; Ponstingl et al., 1981); this work was performed by the classical approach of preparing peptides and sequencing them by the Edman process. The results showed that in pig brain tubulin, there were at least four forms of a and two of P. This work suggested that variety among tubulins could arise from multiple forms of a and p. The term “isotype” has been used in the literature in three ways. First, the term is used to describe forms of a- or P-tubulin encoded by different genes and that differ in their amino acid sequences. Second, the term is used to describe different forms of tubulin that arise by posttranslational modification.Third, the term is used empirically to designate multiple forms of a- or P-tubulin that appear on isoelectric focusing or two-dimensional gels; in such cases, the multiplicity could reflect either multiple genes or modifications (Picquot and Lambert, 1988; Bughio et al., 1991). In this review, I shall discuss isotypes arising from both multiple genes and posttranslational modifications. I will discuss their phylogeny and their distributions in cells and tissues. The possible functions of isotypes will be addressed including the question of whether the difference is incidental to or necessary for the function.

II. Multiple Genes for

(r

and PTubulin

The structure, distribution, and function of tubulin isotypes arising from different genes have been reviewed several times (Cowan and Dudley,

MULTIPLE FORMS OF TUBULIN

209

1983; Cleveland and Sullivan, 1985; Cleveland, 1987; Sullivan, 1988; Little and Seehaus, 1988; Joshi and Cleveland, 1990; Luduefia, 1993; Gaertig et al., 1993; Raff, 1994). Some of these reviews have focused on multiple isotypes in plants (Fosket et al., 1993; Goddard ef al., 1994). There are many cases of multiple genes for either a-or P-tubulin in which the encoded proteins are identical (Youngblom ef al., 1984). These will not be discussed here. In this section, I shall focus on tubulin gene products in which the amino acid sequences are known to be different. In most cases, these have been deduced from the nucleic acid sequence. The modes of expression of the isotypes, namely, their temporal, tissue, and subcellular distributions, have generally been determined either by isotype-specific RNA probes or by antibodies. Tables 1-111 summarize current knowledge about the occurrence of isotypes of a- or P-tubulin in different organisms. No effort has been made to include every organism whose tubulin sequences have been determined. Those organisms that, according to the table, do not have isotypes are included because efforts to find more isotypes have failed. These organisms, therefore, constitute a useful comparison to organisms known to have isotypes. There are many organisms in which only one a- or 6-tubulin sequence has been determined, but it is not known if there are other isotypes; these organisms are not included in Tables 1-111. From these tables it is clear that multiple isotypes of a-or 0-tubulin are widespread among eukaryotes, occurring in many animals, plants, fungi, and protists. It is also clear that there is a wide range of variability among isotypes. Sometimes two isotypes in the same organism may differ from each other at only one position, as is the case with a from the green algae Polytomella and Volvox. At the other extreme, the two /3 isotypes of the ameba Reticulomyxa differ in 41% of their sequences. Some organisms have only one form of a but more than one form of /3 and others have the reverse. The actual number of isotypes is also variable, mammals appear to have six forms of a and seven of /3, whereas the mouse-ear cress Arabidopsis has six a’s and nine P’s.

A. Regulation of lsotype Expression

1. Protists Protists are single-celled organisms. Not surprisingly, many protists do not have multiple isotypes of tubulin; several of those that have isotypes do not regulate them differently (Table 111). It is interesting, however, that some protists can express isotypes differentially. The malarial parasite, Plasmodium falciparum, has a single P gene that is expressed in both the

TABLE I lsolypes of a- and 0-Tubulin: Animals" Number of isotypes and maximum divergence (70) Species Homo (human)

P

a

Chordate Chordate Chordate Chordate Chordate

2 3 (6) 24 ND* ND 6 (3.8)

24

Yes Yes Yes

22 7 (21.7)

Yes

Chordate Chordate Chordate

22 ND 5 (17)

24

Yes

Cricetulus (Chinese hamster) Gallus (chicken)

2 3 (27.7) 7 (17.3)

Yes

Xenopus (frog)

Chordate

22

22

Yes

Notothenia (fish) Oncorhynchus (chum salmon) Chaenocephalus (ice fish) Torpedo (electric eel) Salmo (rainbow trout) lctalurus (catfish) Mustelus (dogfish) Paracentrotus (sea urchin) Lytechinus (sea urchin) Strongylocentrotus (sea urchin) Homarus (lobster)

Chordate Chordate Chordate Chordate Chordate Chord ate Chordate Echinoderm Echinoderm Echinoderm Arthropod

22

22 ND 22 ND ND 22 22 3 2 (20.2)

Yes

sus (Pig) Bos (cow) Odocoileus (white-tailed deer) Mus (mouse)

Y Ranus (rat)

0

Phyluddivision

Differences in expression

4 (21.6)

ND 22 =2 ND ND 4 (24.2)

1 ND >Id

2 4 (8.8) 22

2 1 C

>Id

Yes

Yes Yes

Reference Little and Seehaus, 1988 Lewis et al., 1985a Krauhs et al., 1981; Ponstingl et al., 1981 Banerjee et al., 1988 Ludueiia et al., 1982 Little and Seehaus, 1988; Villasante et al., 1986; Hecht et al., 1988; Wang er al., 1986 F. D. Miller et al., 1987; Ludueiia et al., 1982 Lewis and Cowan, 1990, Ahmad et al., 1991 Monteiro and Cleveland, 1988; Pratt and Cleveland, 1988; Sullivan et al., 1986a,b Good et al., 1989; Bieker and Yazdani-Buicky, 1992; Wu and Morgan, 1994 Detrich et al., 1987, 1992; Detrich and Parker, 1993 Coe et al., 1992 Detrich et al., 1987 Canals et al., 1995 Garber et al., 1991 Detrich et nl., 1987 Detrich et al., 1987 Gianguzza et al., 1989, 1990, 1995; Casano et al., 1996 Alexandraki and Ruderman, 1983 Harlow and Nemer, 1987a,b Demers et al., 1996

Drosophila (fly) Heliothis (moth) Doryteuthis (squid) octopus Trichostrongylus Caenorhabditis

Arthropod Arthropod Mollusc Mollusc Nematode Nematode

3 (12.5) 22 ND 2‘ ND 22f ND 22 ND 2 2 (2.9) 3 (10.7)

Brugia Haemonchus Schistosoma

2 2 (21.5) ND Nematode 21 4 (3.3) Nematode Plat yhelminthes 2 (14.2)g ND

4 (32)

Yes Yes Yes Yes Yes Yes Yes

Theurkauf et al., 1986; Rudolph et al., 1987 Davis and Miller, 1988 Arai and Matsumoto, 1988 Tomarev et al., 1993 Grant and Mascord, 1996 Driscoll et al., 1989; Geary et al., 1992; Fukushige et al., 1993, 1995 GuCnette et al., 1992 Geary et al., 1992; Klein et al., 1992; Kwa et al., 1993 Duvaux-Miret et al., 1991; Webster et al., 1992

a The table gives either the actual known number of isotypes or states that there are at least a certain number. The symbol “2,” such as in “23,” means that at least three isotypes have been sequenced or identified, but that there is a reasonable probability of the existence of others, often based on the fact that closely related organisms are known to have more than three. Maximum divergence is calculated from comparing the sequences of the known isotypes. In some cases, only part of the sequence of an isotype is known. The maximum divergence is then calculated by dividing the number of known variant positions by the length of the total sequence and assuming that the actual divergence must be at least that large. For several mammals, when they are likely to have a very divergent gene that has not been sequenced, and very little sequence information is available for more than one isotype, the maximum divergence has not been calculated. Differences in expression (tissue, developmental, and subcellular) are indicated only when known. Phylogenetic classifications used here are based on those of Levine et al., (1980), Margulis and Schwartz (1988), and Hawksworth et al., (1983). * ND, not determined. There are at least 4 expressed P genes out of a total of 9-12 p genes. It is not clear if and to what extent the expressed genes differ in their amino acid sequence. In Homarus, there are four or five a genes and three to five P genes, but only one a and one /3 have been sequenced. It is therefore possible, but not certain, that there are multiple isotypes. The two isotypes were shown to differ by peptide mapping, not by sequencing; it is therefore conceivable that the isotypes arise from different posttranslational modifications rather than from genes encoding different proteins. f Only one a and one P gene were sequenced. The total number of a and p genes was not determined. However, the expression of P-tubulin mRNA appeared to be restricted to the lens, implying that other P genes must be expressed elsewhere. It is not clear if these other P genes encode polypeptides of different sequence. In contrast, a is widely expressed. g It is conceivable that these represent alleles rather than isotypes.

TABLE II

lsolypes of a-and &Tubulin: Plants and Fungi" Number of isotypes and maximum divergence (%)

Y R3 Species

Phyluddivision

Plants Arabidopsis (mouse-ear cress) Pisum (pea) Glycine (soybean) Solanum (potato) Nicotiana (tobacco) Eucalyptus Zinnia Prunus (plum) Lupinus (lupin) Zea (corn) Oryza rice

Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (dicot) Angiosperm (monocot) Angiosperm (monocot)

P

-4-5

8 (9.1) 3 (9.3) 3 (26.3) 2 2 (0.2) -3

>1'

ND

4 (11.3)

ND ND ND

ND

2 3 (9.3)

>1'

ND

ND

2 2 (1.3)

2 6 (12)

8 (10)

ND

3 (6)

Differences in expression Yes Yes Yes Yes Yes Yes Yes

Reference Kopczak et al., 1992; Snustad et al., 1992 Liaud et al., 1992 Guiltinan et al., 1987; Jongewaard et al., 1994 Taylor et al., 1994 S o m et al., 1996* Diaz et al., 1996 Yoshimura et al., 1996 Stocker et al., 1993 Vassilevskaia et al., 1996 Villemur et al., 1994; Rogers et al., 1993 Breviario er al., 1995; Koga-Ban et al., 1995

Fungi Histoplasma Aspergillus Colleiotrichium Candida Neurospora Erysiphe (grass mildew) Epichloe Saccharomyces Schizosaccharomyces Pneumocysiis Trichoderma Geotrichum

5

Deuteromycota Deuteromycota Deuteromycota Deuteromycota Ascomycot a Ascomycota Ascomycota Ascomycota Ascomycota Ascomycta? Hyphomycetes Hyphomycetes

1 2 (28) ND ND ND ND ND 2 (10.3) 2 (13.8) ND ND

ND

1 2 (17.4) 2 (21) 1 1 1 1 1 1 1 2 (13.7) 2 (36)

Yes Yes

No No

Hams et al., 1989 May et al., 1987; Doshi et al., 1991 Panaccione and Hanau, 1990; Buhr and Dickman, 1994 Smith et al., 1988 Orbach et al., 1986 Shenvood and Somerville, 1990 Byrd et al., 1990 Schatz et al., 1986a; Hiraoka et al., 1984 Toda et al., 19W, Hiraoka et al., 1984 Dyer ei al., 1992 Goldman et al., 1993 Gold et al., (1991)

See footnotes for Table I. In this work, the isotypes were characterized by gel electrophoresis. Based on analogous results obtained with other plants, it is likely that the electrophoretically defined isotypes correspond to polypeptides of different sequence. Only one (Y gene was sequenced; because it has only a localized distribution, it is highly probable that there are other expressed a genes, although it is not certain that they differ in amino acid sequence.

TABLE Ill lsotypes of a- and &Tubulin: Protists"

Number of isotypes and maximum divergence (%) Species

Phyluddivision

Encephalitozoon Physarum (slime mold) Dictyosielium Ectocarpus Polyiomella Volvox Chlamydomonas Chondrus Teirahymena Stylonichia Paramecium Achlya Toxoplasma Babesia Plasmodium Eimeria Crypiosporidium Leishmania Naegleria Trichomonas Reiiculomyxa

Microsporidia Myxomycota Acrasiomycota Phaeophta Chlorophyta Chlorophyta Chlorophyta Rhodophyta Ciliophora Ciliophora Ciliophora Oomycota Apicomplexa Apicomplexa Apicomplexa Apicomplexa Apicomplexa Zoomastigina Zoomastigina Zoomastigina Rhizopoda

a

See footnotes for Table I.

P

(Y

ND 3 (10) 1 ND ND ND 2 (0.4) ND 1 2 (>7.7) 2 (0.2) ND 1 ND 2 (5.8) ND ND ND ND ND 2 (2.2)

Differences in expression

1

4 (17) 1 2 (1.3) 2 (0.2) 2 (0.2) 1 2 2 (0.5) 1 ND 1 1 1 1 1 1 2 2 (2.3) >1 2 3 (2.7) 2 (41.2)

Yes

No

Yes Yes No

Reference Li et al., 1996 Walden ei ai., 1989a.b; Burland ei al., 1988; Paul et al., 1992 Triviaos-Lagos et al., 1993 MacKay and Gallant, 1991 Conner et aL, 1989 Harper and Mages, 1988 Silflow et al., 1985; Youngblom ei al., 1984 Liaud ei al., 1995 Barahona et al., 1988 Conzelmann and Helftenbein, 1987; Dupuis-Williams et al., 1996 Dupuis-Williams ei al., 1996 Cameron er ai., 1990 Nagel and Boothroyd, 1988 Casu, 1993 Wesseling ei al., 1989; Holloway er al., 1990; Rawlings et al., 1992 Zhu and Keithly, 1996 Nelson er al., 1991 Coulson ei al., 1996 Lai ei al., 1994 Katiyar and Edlind, 1994 Linder et al., 1997

MULTIPLE FORMS OF TUBULIN

215

sexual and asexual stages of the organism, but there are 2 a genes, 1 of which is constitutively expressed, whereas the other is expressed only in the male gametocyte (Wesseling et al., 1989; Holloway et al., 1990;Rawlings et al., 1992). Leishmania mexicana, although it appears to have only a single a gene, expresses at least 2 /3 isotypes, whose relative expressed levels differ between the mastigote and the amastigote stages (Fong et al., 1984; Coulson et al., 1996). The slime mold Physarum polycephalum, which has a complex life cycle, has 3 a and 4 P isotypes. Of the a’s, 1 is found in all three stages of the life cycle-ameba, plasmodia, and flagellate-but is particularly high in the flagellate and particularly low in the plasmodia. Of the P’s, 1 is also expressed in all three stages, the second /3 is expressed strongly in the flagellate and not in plasmodia, and a third is largely expressed in plasmodia. In short, one gene is mainly expressed in flagellates, one in ameba, and one in plasmodia (Paul et al., 1992; Cunningham et al., 1993). Naegleria gruberi, which is not formally known to have isotypes (and therefore is not listed in Table 111) has 8 a and 8-10 /3 genes. Only 1 of the P genes has been sequenced; it is expressed largely in the flagellate stage (Lai et al., 1994).Thus, if Naegleria has isotypes, they are likely to differ in their mode of expression. In short, certain protists exhibit a temporal or developmental difference in the expression of tubulin isotypes. 2. Fungi

As with the protists, certain fungi appear not to have isotypes. Some of those that do, such as the yeasts Saccharomyces and Schizosaccharomyces, do not appear to exhibit any differences in their mode of expression. However, the deuteromycote Colletotrichum gloeosporiodes has two P genes. One gene is expressed in all stages of the fungal life cycle: at high levels in vegetative mycelia and at low levels in conidia. In contrast, the other gene, a much more divergent one, is expressed only in conidiating mycelia (Buhr and Dickman, 1994). The related fungus Aspergillus nidalans has two a’s and two 0’s. One a is involved in nuclear division and the other in determining cell morphology; one P isotype is expressed in all life cycle stages and the other mostly in sporulation (May et al., 1987; Doshi et al., 1991). In the related fungus Fusarium moniliforme only one P-tubulin has been identified; its expression is apparently restricted to the vegetative growth and conidial germination phases. The P gene is not expressed during conidiation; presumably there is another P-tubulin expressed during conidiation, implying that this organism has two P genes and that they are differentially regulated (Yan and Dickman, 1996). 3. Plants

In contrast to protists and fungi, plants lacking multiple tubulin isotypes have not been identified. Also, in every case in which the question has

216

RICHARD F. LUDUENA

been asked, the isotypes appear to differ in their tissue distribution. In Arabidopsis one a isotype is expressed in all tissues, one only in pollen, and the others vary among the different tissues (Carpenter et al., 1992; Kopczak et al., 1992). Of the P isotypes, TUBZ is expressed most strongly in the root; TUB2, TUB3, TUB4, TUB7, and TUB9 mostly in the flower; and TUB5 in the leaves. TUB6 and TUB8 are more evenly distributed (Snustad et al., 1992). In addition, some 6 genes (TUB2, TUB3, TUB6, and TUB8) are expressed at lower levels at low temperatures and one (TUB9) is expressed at higher levels. This is consistent with the hypothesis that one reason for the existence of isotypes is to increase the range of adaptation to environmental conditions (Chu et al., 1993). In the soybean Glycine, expression of the P isotypes differs between enlarging leaves, mature leaves, and etiolated internodes (Fosket et al., 1993). One highly divergent soybean P isotype has the following distribution: It is expressed at low levels in the cotyledon and at times in the seedling root and at high levels only in the hypocotyl when the plant is grown in the dark. Thus, this isotype is subject to a distinct regulatory mechanism (Jongewaard et al., 1994). In maize there are at least five a isotypes. The tuaZ and tua4 isotypes are common in rapidly dividing immature tissues such as root tip. In the more differentiated root and pollen there is more tua2, tua3, and tua4. In pollen tua2 and tua3 are common, but there is very little tual (Joyce et al., 1992). Maize has eight P isotypes. All the P isotypes are present in most tissues, but their relative abundances are different. Tub6 is relatively high in most tissues. TubZ is high in tassel primordia, but is very low in anthers and pollen. It is slightly higher in root tip but not expressed in the mature leaf, suggesting it may be higher in tissues whose cells are going through frequent mitoses. Tub2 is low in the root tip but is present in the mature leaf. Tub3 and tub4 are very high in pollen but are also present in the shoot and the root, but not in the leaves or the endosperm. Tub5 is present at low levels in the endosperm and pollen and at higher levels in the shoot and root, but not in the leaves. In general, tub6, tub7, and tub8 are higher in the shoot and less important in the pollen (Hussey et al., 1990; Rogers et al., 1993; Villemur et al., 1994). Three of the detectable four or five a-tubulin isotypes of tobacco appear to increase during pollen tube growth (Sorri et al., 1996). Similarly, in the plum, Prunus amygdalus, there is an a isotype very similar to maize a l . It is expressed largely in the seed and the root and very little in the leaf (Stocker et a1.,1993). Along the same lines, in cultured cells from Zinnia elegans one /3 isotype is expressed constitutively at low levels, whereas expression of the other two is induced by phytohormones such as auxin and cytokinin. Finally, some differences in expression among P-tubulin isotypes have been seen in rice and lupin in response, respectively, to anoxia and continuous light (Giani and Breviario, 1996; Vassilevskaia et al., 1996).

MULTIPLE FORMS OF TUBULIN

217

4. Animals

a Nematodes Multiple isotypes of a- and P-tubulin have been observed in the nematodes Caenorhabditis, Brugia, Haemonchus, and Trichostrongyfus (Driscoll et al., 1989; Roos et af., 1990; Guenette et af., 1992; Grant and Mascord, 1996). In Brugia there are two 0-tubulin genes. One gene is expressed largely in adult male worms but not in microfiliariae; the other is expressed in microfilariae in roughly equal amounts in adult males and females (GuCnette et al., 1992). In Caenorhabditis elegans the a1 isotype is expressed largely in one set of neurons; a 2 occurs in all intestinal cells, but only in certain neuronal cells (Fukushige et uf., 1993,1995). Caenorhabditis also contains unusual MTs in its touch-receptor neurons. These MTs have 15 protofilaments, in contrast to the other MTs of C. efeguns, which contain 11 protofilaments. These “giant” MTs contain a particular isotype of P (Savage et af., 1989). b. Molluscs Evidence for isotype distribution in molluscs is somewhat indirect at this point. The cell body of the squid giant neuron was found to contain two P isotypes, as defined by their peptide maps. Only one of these two isotypes was present in the peripheral axoplasm (Arai and Matsumoto, 1988). Because the amino acid sequences of these isotypes are not known it is possible that the differences between the two isotypes could arise by posttranslational modification(s). In the marine mollusc Patella, one a isotype has been sequenced that is expressed only in the trochoblasts during embryogenesis (Damen et af.,1994). Undoubtedly, there must be another a gene expressed elsewhere in the organism. In the absence of sequence information, however, it is conceivable that this second a gene encodes an isotype of identical sequence. In the octopus, although a is expressed in many tissues, the particular mRNA probe used by Tomarev et al. (1993) detected only in the lens and the mantle, implying that there must be another P isotype but not demonstrating that the other P differs in sequence. c. Arthropods Much, perhaps most, of our understanding of the functional significance of tubulin isotypes has arisen from work done in the fruit fly Drosophila melanogaster, which has four a-tubulin genes and three P-tubulin genes (Theurkauf et al., 1986; Matthews et af., 1993). Two of the a isotypes, a1 and a3, although encoded by different genes, are identical in sequence. The pattern of expression of these two a’s is fairly similar in that they are highest in early embryonic development, gradually decreasing as metamorphosis begins, then rising again at the midpupal stage. a1 is expressed at higher levels than is a3. In contrast, a 2 is not expressed at all at the very beginning of embryonic development but then rises dramatically

218

RICHARD F. LUDUENA

as the levels of the first two isoforms start to fall. After the second larval instar, a 2 drops, then rises again in the midpupal stage concomitant with the rise in a1 and a 3 (Matthews et al., 1989; Theurkauf, 1992). a 2 is also expressed in the testes and sensory organs (Bo and Wensink, 1989). a4 is only 67% identical to the other three, making it one of the more divergent tubulin isotypes; a4 occurs in the oocyte and early embryo and then disappears (Matthews et al., 1993). Of the p isotypes, p3 is expressed mainly in the embryonic and pupal stages, particularly during development and differentiation of the visceral mesoderm, especially muscle. In the adult, expression of p3 is restricted to the ovaries and testes (Leiss et al., 1988; Kimble et al., 1989; Buttgereit et al., 1996; Dettman et al., 1996). As soon as contact to the epidermal attachment sites is made, pl expression is induced (Buttgereit et al., 1996). The other p isotype, p2, is testis specific and is involved in forming axonemal MTs as well as those of the meiotic spindle; it also plays a role in nuclear shaping (Kemphues et al., 1982; Fackenthal et al., 1995; Raff et al., 1997). An identical testis-specific p isotype has been observed in Drosophila hydei and a similar one in the moth Heliothis virescens (Michiels et al., 1987; Davis and Miller, 1988).

d Echinoderms In the sea urchin, Paracentrotus lividus, there are at least 4 a- and 3 P-tubulins (Gianguzza et al., 1989, 1990; Casano et al., 1996). The amino acid sequences of the a’s are at least 5.3% divergent (Gianguzza et al., 1989). a2 appears in the blastula and is apparently restricted to the oral ectoderm; its distribution is thought to correspond with that of certain neurons (Gianguzza et al., 1995). pl and p2 are expressed largely in the ciliated band and gut. p3 is expressed in the apical and oral ganglia and in the ciliated band. a1 is expressed largely in the ciliated band and foregut, whereas a2 is expressed exactly as is p3, suggesting that they may pair preferentially with each other. a10 is expressed in the ciliated band and gut. All 3 p isotypes contain the axonemal consensus sequence to be discussed below (Harlow and Nemer, 1987b; Casano et al., 1996). In the sea urchin, Stronglyocentrotus purpuratus, there are at least 4 expressed P-tubulin genes (out of a total of 9-12 p genes), although the divergence of their amino acid sequences is not yet clear. The expression of these genes is both tissue and stage specific. 61, p2, and p3 are expressed throughout development. 64 only begins to be expressed in the gastrula stage. In the pluteus (the larval stage), p2 and p3 are expressed more in the ectoderm than in the endomesoderm. In contrast, pl is very specific for ectoderm, whereas p4 is very highly expressed in the endomesoderm (Harlow and Nemer, 1987a,b). e. Vertebrates In vertebrates, there are multiple isotypes of both a and

p. In mammals, there appear to be six a’s and seven p’s. The a isotypes

MULTIPLE FORMS OF TUBULIN

219

are distributed as follows (Lewis et a1.,1985b;Lewis and Cowan, 1988,1990): a1 is found mostly in brain, some in lung, less in testis, and still less in heart, kidney, muscle, spleen, stomach, and thymus. In rat brain, a1 expression appears to be associated with neurite outgrowth and is most prominent in the nervous system of the embryo. a2 has a somewhat similar pattern, with highest expression in the brain and the thymus, some in the lung and spleen, less in testis, stomach, heart, kidney, and liver, and very little in muscle (F. D. Miller et al., 1987; Przyborski and Cambray-Deakin, 1996). Another isotype, a3/7, is expressed only in the testis and at very high levels. a4 is more evenly distributed with low levels in the heart and muscle, lower levels in brain, kidney, liver, spleen, stomach, and thymus, and still lower levels in lung and testis. a6 is a minor isotype, expressed at low levels and stomach and at very low levels in heart, kidney, lung, spleen, testis, and thymus. There is also a highly divergent form of a (aTTl), approximately 70% identical to the other a’s, that is testis-specific and found only in the manchette and the meiotic spindle (Hecht et al., 1988). Interestingly, a3/ 7, and not aml, is the major a in the testis. The P isotypes are more complex. PI is expressed at high levels in the brain and the thymus, at lower levels in the lung and spleen, and still lower levels in heart, kidney, liver, muscle, stomach, and testis. fill is found mostly in the brain. Lower levels of PI1are seen in the lung, still lower levels in the stomach, thymus, kidney, and liver, and even lower levels in other tissues. PrIlis expressed at high levels in the brain and Sertoli cells, at lower levels in the lung, at still lower levels in kidney, liver, stomach, and thymus, and at even lower levels in heart, muscle, spleen, and testis. PIvais expressed only in the brain. PrVbis expressed at high levels in the testis, lower levels in the thymus, and very low levels in the other tissues. Pvr appears to be restricted to hematopoietic tissues. It is found in platelets, megakaryocytes, and erythroblasts and is expressed in the spleen at low levels with trace amounts in liver and lung (Lewis et al., 1987: Lewis and Cowan, 1990). Although Pv has been observed to be expressed abundantly in birds, its distribution in mammals is still unclear. It is expressed by cultured Chinese hamster ovary cells, but its normal distribution is unknown (Ahmad et al., 1991). Looking more closely at certain tissues, although PrIand Plva are expressed in both neurons and glia, PrIris expressed only in neurons (Burgoyne et al., 1988; Caccamo et aL, 1989). PIIIalso is expressed in a variety of tumors, including tumors that have developed from tissues in which the nontransformed cells do not express Prll(Matsuzaki et al., 1987; Asai and Remolona, 1989; Scott et al., 1990; Katsetos et al., 1991; Maraziotis et al., 1992; Furuhata et al., 1993). Also, certain cells that do not normally express plIIwill begin to do so after being in culture for extended periods of time (Vinores et al., 1995).

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In evaluating the possible significance of different levels of the isotypes, it is important to consider that differential synthesis may not be the only factor but that different isotypes may differ in their stability in the cell, as implied by the preliminary work of Schwarz and Ludueiia (1996), who find that a& decays more slowly in vitro than does apII.Decay is a process whereby tubulin loses its functional properties while concomitantly exposing hydrophobic areas and sulfhydryl groups (Wilson, 1970; Prasad and Ludueiia, 1986; Ludueiia et al., 1996). A difference in propensity to decay in vitro could conceivably translate in vivo into a different susceptibility to proteolytic digestion. In addition, different isotypes could be transported at different rates from one part of a cell to another (Hoffman and Ludueiia, 1996). Within the mammalian brain and nervous system there are some clear developmental differences in the expression of the different isotypes. The pattern of expression of different p isotypes changes during development of the peripheral and central nervous systems. In both systems, PI and prr are higher at early stages than in the adult. In contrast, plv increases during development of the central nervous system but decreases during development of the peripheral nervous system, pIII,meanwhile, decreases in the central nervous system and increases in the peripheral nervous system participates (Kost and Oblinger, 1993). In the spinal cord of rat embryos, pIII in the mitotic spindle (Memberg and Hall, 1995). In rat brain, both the development and the distribution of various a and p isotypes respond in different ways to thyroid hormone (Aniello et al., 1991). The process of neuronal regeneration after injury results in differences in expression among the isotypes. In neurons of the peripheral nervous system axotomy results in an increase of a l , &I, and &II (while PI and plv remain the same). In the central nervous system axotomy causes a decrease in a1 and PIrI(Hoffman and Cleveland, 1988: Jiang and Oblinger, 1992; Moskowitz and Oblinger, 1995). The PrI1isotype may play a special role the development of neurons and neurites (Lee et al., 1990). pIIlis not incorporated into MTs of cultured brain neurons until differentiation when the neurons start generating axons and dendrites. This incorporation is correlated with the expression of high-molecular-weight MAPS (Ferreira and Caceres, 1992). Transport of plrIappears to be distinct from that of the other p isotypes; it is transported in the slow component b of axonal transport, slightly ahead of pII (Hoffman et al., 1992; Moskowitz and Oblinger, 1995). Similar observations were made when embryonal P19 carcinoma cells were allowed to differentiate into neurons (Falconer et al., 1992, 1994). Expression of pIIIincreased and was correlated with that of MAP lB, MAP 2C, and tau; expression of PI and pIvdropped as differentiation progressed. In these cells pIIaccumulated into the stable MTs and pill into the unstable

MULTIPLE FORMS OF TUBULIN

22 1

ones, again suggesting some discrimination between and PIII.When the P19 cells differentiated into muscle cells, PII synthesis increased but the stable MTs tended to incorporate PIv. At the subcellular level little is known about localized differences in expression of the tubulin isotypes. It is has been reported that retinal rod cells and tracheal cells, which express both PI1and PlV, appear to use only PIVin their axonemal MTs (Renthal et al., 1993). In addition, as described previously, differentiating neurons and muscle cells tend to incorporate certain P isotypes preferentially into the more stable MTs (Falconer et al., 1992, 1994). In rat PC-12 cells, which express all the ,tlisotypes except for Pv1, more PI, PI],and PIv and less PIIIand PV are used in MT formation (Joshi and Cleveland, 1989). All these observations suggest that cells are able to discriminate among the isotypes, though the reasons for this discrimination are still unclear. Birds have a similar isotype distribution as do mammals. Five a and seven P isotypes have been found (Monteiro and Cleveland, 1988; Pratt and Cleveland, 1988). There is a relatively divergent a found in chicken testis; bearing 83% identity to the other a’s, it has no particular relationship to the highly divergent mammalian testis-specific CY (Pratt et al., 1987). As with mammals PI is widespread and pllIis found in neurons. In the case of Pv, its distribution is better known in birds than in mammals, being found in a variety of tissues (liver, intestine, testis, spleen, thymus, and bursa) but not in the brain or in neurons (Sullivan et al., 1986b). Avian PVI is found in erythrocytes; its sequence does not have a particularly close resemblance to that of mammalian PvI (Murphy et al., 1987). In birds, unlike mammals, there is only one form of pIv,with widespread distribution (Havercroft and Cleveland, 1984). Again unlike mammals, birds have two forms of PI], differing at only two positions in their sequence but with dramatically different distributions. One form of PIIis found in many tissues but not in the adult brain; the other PIIis found mostly in the brain (Havercroft and Cleveland, 1984; Sullivan et al., 1985). Less is known about isotypes in amphibians. PI1has been observed in the frog Xenopus and is associated with axonal development (Good et al., 1989; Moody et al., 1996). There is also a P similar to mammalian PIVbthat occurs in Xenopus oocytes (Bieker and Yazdani-Buicky, 1992). Amphibian a-tubulins are currently more difficult to classify. In Xenopus there are at least two a-tubulin genes. One of them, XaT14, is 98% homologous to mouse a1 and is expressed mostly in the ovary and to a lesser extent in the egg, testis, spleen, and kidney. It is expressed at still lower levels in the liver but is not detected in the muscle (Middleton and Morgan, 1989). The other, XaT207, is apparently expressed only in the ovary. XaT207 is 88% identical to mouse a1 and 88% identical to XaT14. It is interesting that XaT207 is ovary specific because Drosophila has an a isotype, a4, that is

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RICHARD F. LUDUENA

also ovary specific. The a 2 isotype in chicken and the a4 isotype in Drosophila vary in the same regions as does XaT207, although they are not otherwise closely related (Wu and Morgan, 1994). Partial knowledge is available about fish tubulin isotypes. The vertebrate PrIIisotype, when reduced and carboxymethylated, has an unusual electrophoretic mobility (Little, 1979). This allows its tentative identification in various tissues and organisms. It is seen only in vertebrate brains and is not present in sea urchin sperm or eggs nor in squid brains (Ludueiia et al., 1982; Little et al., 1981). It has been observed in the brains of various bony and cartilaginous fishes, including the dogfish shark, the channel catfish, and three species of Antarctic fish (Gobionotothen gibberifrons, Notothenia coriiceps, and Chaenocephalus aceratus) (Ludueiia et al., 1982; Detrich et al., 1987); PrIlis not expressed in the eggs of Antarctic fish (Detrich et al., 1992). The Antarctic fish N . coriiceps has a 0-tubulin whose nucleotide sequence is most similar to mammalian and avian PII.In terms of amino acid sequence it is similar to both PII and Prv and much less similar to PI and Prrr.It thus appears that PII,at least, is present in fish (Detrich and Parker, 1993). There is a neuron-specific a-tubulin in the electric lobe of the electric eel Torpedo marmorata. This is 99.3% identical to rat al, 99.3% to mouse al, 99.6% to mouse a2, and 95.6% to an a-tubulin from the Siberian salmon. Thus, the neural-specific a-tubulin from mammals appears to occur in fish as well (Canals et al., 1995). The trout Salmo gairdneri has a testis-specific a-tubulin that is homologous to mammalian a3/7, which is also testis specific (Garber et al., 1991).

B. Functional Significance of Tubulin lsotypes Tubulin isotypes are widespread among eukaryotes. It is naturally tempting to imagine that they have functional significance, namely, that they differ in certain properties and that these properties can modulate their behavior so that they perform specific functions. There are three possible models to consider about the functional significance of isotypes. One is that they have no functional significance whatsoever. By this model, one would postulate that the only reason that different isotypes are expressed in different tissues is that a particular tubulin gene is under the control of a system that is activated when that tissue develops. In other words, if a certain isotype is found, for example, only in the root of a plant and not elsewhere, then one could imagine that there is a particular tubulin gene that is activated at the same time that the other genes are activated that causes the root to develop. Over time, some mutations have arisen that give the root isotype a different sequence, but these differences are incidental. A second model is that isotypes may have no specific function per se, but their presence may

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increase an organism’s repertoire of responses to environmental challenges. The third possibility is that different isotypes can actually perform different functions. It is also conceivable that all these possibilities are true, namely, that each model is applicable to certain families of isotypes. Let us examine each model in turn. 1. Model 1:Isotypes Have no Functional Significance We have seen in Tables 1-111 several cases of isotypes that differ at only one or two positions; generally these are conservative differences. This is the case not only with protists such as Chlamydomonas, Polytomella, Volvox, and Paramecium, but also in some higher organisms, such as in the two forms of PIIin chickens (Silflow et al., 1985; Harper and Mages, 1988; Conner et al., 1989). It is hard to imagine that such minor differences would alter the functional properties of the isotypes. Although for example, the two forms of chicken pIIare expressed in different tissues and may therefore be involved in different processes, it is unlikely that the sequence differences are what determine participation of these isotypes in those processes. Several observations have supported this model. For example, all the mammalian P isotypes, including the very divergent Pv1, have been seen to form the interphase network and the mitotic spindle. Therefore, the capability of participating in these organelles is likely to be inherent in all isotypes (Joshi ef al., 1987; Lopata and Cleveland, 1987; Lewis et al., 1987; Lewis and Cowan, 1988). Also, when the (Y isotypes a3/7, ( ~ 4 and , a6 were transfected into HeLa and 3T3 cells, they were all incorporated into interphase and spindle MTs (Gu et al., 1988). In an analogous experiment, a chicken-yeast P-tubulin chimera was transfected into 3T3 cells and was incorporated into interphase, spindle, and midbody MTs (Bond et al., 1986). This chimera had the yeast C terminus, which is very different from any avian P C terminus and therefore lacks the major isotype-defining region of the p isotypes. The authors concluded that the isotypes are functionally interchangeable. A limitation on interpreting experiments such as these is that cultured cells may have less stringent requirements in assigning functions to isotypes than cells in the whole organism. Similarly, there could be MT-containing organelles or a population of MTs that require a particular isotype; this could conceivably be true even for MTs that, to the observer, do not seem very unique. If a given organism has two isotypes and they can be shown to be interchangeable, this would argue that they are not adapted to perform specific functions. This approach has been taken with various fungi. For example, the P3 in Aspergillus appears to be involved in conidiation, but it can be replaced by pl and p2 (Weatherbee ef al., 1985). Schatz et al. (1986b) examined the two a-tubulins of the yeast Saccharomyces cerevisiae.

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RICHARD F. LUDUENA

Removal of one gene caused hypersensitivity to benomyl; removal of the other inhibited growth. However, the authors suggested that the problem was simply caused by a decrease in the total amount of tubulin expressed because overexpression of one gene could overcome defects in the other gene. Apparently the reason for having two genes is to maintain the requisite level of expression. A very similar experiment was performed by May (1989) using the fungus Aspergillus niduluns, which has two P-tubulin genes, called benA and tubC. In essence, May constructed a chimeric P-tubulin gene with the benA promoter connected to the tubC gene. He replaced the benA gene with the tubC gene. The resulting organism had only the tubC gene and was perfectly viable. The only problem was that the cells lacking benA were slightly more sensitive to benomyl. Again, it appeared that what is most important about the isotypes is their number rather than their specific sequences. One way to approach this question is to knock out one isotype and replace it with another. This is what Kirk and Morris (1993) did with A . niduluns, which has two genes for a-tubulin. TubA is involved in mitosis and nuclear migration during the vegetative growth phase; tubB is involved in sexual development before the first meiotic division. Kirk and Morris knocked out one gene and replaced it by the other at the appropriate stage. They found that the Aspergillus could grow normally even if their isotypes were exchanged. However, some subtle differences were noted. In the strain in which tubB was knocked out and replaced by one copy of tubA ascospores formed but they were not completely normal in that some were not viable. However, when three copies of tubA were used, then tubB could be safely replaced without any effect. In the converse experiment, they tested to see whether tubB could replace a mutated tubA in which the organism became supersensitive to benomyl during the vegetative growth phase. They found that tubB could completely abolish supersensitivity to benomyl. Although it is possible that tubA is performing some more subtle function that cannot be carried out by tubB, it appears that tubB and tubA are functionally almost interchangeable. The functional replacement is not perfect, however, as shown by the fact that more copies of tubA are needed to replace tubB. Nevertheless, this experiment does suggest that there is some interchangeability of isotypes. 2. Model 2: Isotypes Are Adaptive but Do Not Perform Specific Functions By this model, having a pool of different isotypes could help an organism cope with environmental challenges, but the isotypes are not preadapted to perform specific functions. One example of this model may be the higher plant Arubidopsis, which has six a-and nine P-tubulin genes. Transcription

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of some of the different 0-tubulin genes is temperature dependent. At low temperatures, transcription of TUB2, TUB3, TUB6, and TUB8 decreases and that of TUB9 increases. TUB9 appears to be more stable in the cold. It therefore appears that Arabidopsis uses its tubulin isotypes to adapt to its environment (Chu et al., 1993). Similarly, Joshi et al. (1987) observed that the presence of PvI confers some cold-stability on MTs. The same model clearly applies to nematodes. In the nematode Haernonchus contortus, there are several isotypes of P-tubulin, whereas in the benzimidazole-resistant strain there is only one (Roos et d., 1990; Kwa et d., 1993). Benzimidazole has a similar effect on the nematodes Caenorhabditis and Trichostrongylus (Driscoll et al., 1989; Grant and Mascord, 1996). This implies that the presence of isotypes helps to mediate resistance to environmental factors. The work of May (1989) and Kirk and Morris (1993) in the fungus Asperigillus is also consistent with this model. Analogous arguments could be made for cultured mammalian cells. In human prostate carcinoma cell lines there is an increase in the expression of PIIIand PIvawhen the cells become resistant to estramustine. PIIis only slightly increased with estramustine resistance (Ranganathan et al., 1996). Taxol also alters expression of p isotypes in other cell lines (Haber et al., 1995; Dumontet et al., 1996). Thus, the differential expression of tubulin isotypes may help tumor cells develop resistance to antitubulin drugs exactly as constitutive differences in isotypes help nematodes and fungi adapt to these drugs. 3. Model 3: Isotypes Perform Specific Functions

One argument for the functional significance of tubulin isotypes is the strong conservatism of isotype differences in evolutionary time. This is noticeable in plants, in which certain isotype differences have been conserved between monocots and dicots, and even more striking in the vertebrates, in which very specific differences are highly conserved. For example, there are six differences in amino acid sequence between chicken and mouse PrIand two differences between chicken and mouse PIII;in contrast, pIIand pIIIdiffer in chickens at 33 positions and in mice at 34 positions. If one were to propose that isotypes have no particular function and that the differences between them are incidental, the natural corollary is that the differences have arisen at positions that can tolerate changes. One would therefore expect that the positions that vary between isotypes would vary greatly in evolution as well. The fact that this is not always the case is a strong argument for specific structure-function correlations among tubulin isotypes. Another argument for the functional significance of isotypes is that often two isotypes occur in different parts of the same cell. Joshi and Cleveland

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(1989) found that developing neurites of rat PC-12 cells express five P isotypes (PI-pv) but preferentially incorporate PI, &I, and PIVinto their MTs. In fact, Asai and Remolona (1989) report that PIIIappears in a granular pattern in these cells and does not actually appear to be incorporated into MTs. Along the same lines is the previously mentioned work of Renthal et al. (1993) showing that bovine retinal rod and tracheal cells, which express both PIIand PIv, use only PIv in their axonemal MTs. If a cell is in essence given a choice of two isotypes and yet uses only one for a given function, or incorporates one preferentially into MTs, then it is only reasonable that there is some structural feature of that isotype that adapts it to that purpose. However, one could still argue that the synthesis of a structure as complex as an axoneme may require expression of a large set of genes, one of which happens to be the PIvgene, and that the isotypespecific differences are incidental. One could even apply this argument to the assembly of a particular group of MTs in a cell, a population that to our eyes may not seem exceptional but that may require a unique set of genes to be expressed. Gene knockout or replacement experiments have been used to approach the question of isotype functional specificity. The nematode C. elegans contains unusual MTs in its touch-receptor neurons. These MTs have 15 protofilaments instead of the usual 11 (for C. efegans).These MTs contain a particular isotype of P. Mutations in this tubulin cause disappearance of these MTs and loss of touch sensitivity (Savage et al., 1989; Hamelin et al., 1992). A series of experiments in Drosophilu have indicated that isotypes may be uniquely adapted to perform specific functions. In one experiment, Fackenthal ef af. (1993) removed the last 15 amino acids from the testisspecific p2 isotype. They found that, although this isotype could assemble into doublet MTs in vivo, it was unable to form functional axonemes. The MTs of the axonemes were completely disorganized. This suggests that the isotype-specific C-terminal region plays a role in mediating interaction of tubulin with other axonemal components. Conceivably, this region plays a role in interactions that may take place in the basal body. Analogous results have been observed with other mutations in the p2 isotype (Fuller et af., 1987; Rudolph et a1.,1987; Fackenthal et al., 1995). In another experiment, Hoyle and Raff (1990) replaced the testis-specific P2 isotype with the P3 isotype. Normally P2 forms meiotic spindles and axonemes and some cytoplasmic MTs. When they expressed p3 in cells lacking P2, they found that in these cells some cytoplasmic MTs get made but axonemes do not form, meiosis does not happen, and nuclear shaping is abnormal. When they coexpressed P3 and P2 in the same cells they found that if P3 was less than 20% of the total P-tubulin in the testis, there were

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no apparent abnormalities but if p 3 exceeded 20% then the axonemal MTs were morphologically abnormal. In a further experiment, Hoyle et uf. (1995) created a chimera in which the N-terminal portion of 02 (positions 1-344) is replaced by that of p3. Although, unlike normal p3, or p2 lacking the C-terminal region, the chimeric protein can partly support doublet tubule formation and axoneme elongation, it is unable to support meiosis or nuclear shaping. This suggests that there are some functionally important differences between p2 and p3 in the region before position 344. In other words, the C terminus may not be the only region of the protein that contains critical differences among the isotypes. Interestingly, a construct of p2 lacking the C terminus supports both nuclear shaping and meiosis, although not as well as wild type (Fackenthal et uf., 1993). In short, the specific functions of p2 require regions both in the C terminus and elsewhere. An analogous group of experiments showed that deletion of the p3 isotype, normally expressed only in the embryonic and pupal stages, was deleterious for viability and fertility. Furthermore, p3 could not be replaced by p l , the constitutive isotype (Kimble et af., 1989, 1990). It seems that although the testis-specific isotype p 2 can participate in a variety of functions, including meiosis and nuclear shaping in which it could be replaced by another isotype, there are some functions that only 62 can perform (Kemphues et af., 1982). In another experiment, Fackenthal et af. (1995) prepared a mutant of p2 in which an aspartate is substituted for a glycine at position 56. This is a “hot spot” for isotype variation; Drosophilu p3 has a six-residue insertion between positions 56 and 57. The presence of this mutation does not prevent MT assembly, but it does alter the morphology of the axonemal MTs. The phenotype change of this mutant is subtle. In wild-type Drosophifu, in addition to the usual nine doublet MTs, there is also a set of nine singlet MTs in the axoneme. The centers of these extra MTs appear to contain luminal filaments of unknown nature that are absent from the outer doublet and central pair of MTs. In the mutant, more of the MTs contain luminal filaments. The authors speculate that the mutation slows down assembly of the doublet MTs relative to assembly of the singlet MTs so that the luminal filament assembly mechanism “thinks” the doublets are singlets and assembles the filaments in those MTs. In sum, the experiments on Drosophifa 02 indicate that a variety of positions beside the C-terminal region may determine the functional properties of specific isotypes. Working with the a isotypes of Drosophifu, Komma and Endow (1997) showed that the aTub67C isotype is specifically required for mitosis and binds to the kinesin-like motor protein Ncd. Defects in another a isotype, aTub84B, did not affect Ncd, suggesting that Ncd has a specificity for a particular a isotype. This is consistent with the earlier results of R. H. Miller et uf. (1987) who found that, in lobster axons, certain MTs, but not

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others, were involved in vesicle transport; it is tempting to hypothesize that motors may select the MTs to which they bind in vivo in part according to the isotype composition of the MTs.

C. Structure-Function Correlations in Tubulin lsotypes If tubulin isotypes have some functional specificity, then it should be possible to identify certain regions in the molecule that give rise to this specificity. Raff et al. (1997) have proposed that the sequence EGEFXXX, where X is an acidic amino acid, located close to the C terminus in a variety of tubulins (positions 433-439 in vertebrate PIm) may allow a tubulin isotype containing that sequence to be incorporated into an axonemal MT. In Drosophila, only the testis-specific P2 isotype, and in vertebrates, only Prv, both known to be expressed in flagella, contain this sequence. The PIv isotype also forms the axonemal MTs of retinal rod cells and the ciliated tracheal epithelial cells (Renthal et al., 1993). Because these cells also express the closely related PIIisotype, it is tempting to postulate that this signal sequence determines the participation of Prvin the axonemal MTs. This is corroborated by the observation that ciliated protists, which have only a single or almost identical P-tubulins, also have the EGEFXXX sequence or something very similar (Table IV). Interestingly, fungi and certain groups of protists that do not have cilia or flagella totally lack this sequence or anything like it. The correlation is not necessarily perfect, as is indicated in Table IV. Organisms such as nematodes, which presumably have cilia or flagella, do not appear to have the signal sequence in their known P-tubulins. It is conceivable, however, that other regions could substitute for this signal sequence in these organisms. Exactly what this signal sequence may specify is an interesting question. When Fackenthal et al. (1993) removed the last 15 amino acids from the Drosophila P2 isotype, they perforce removed this signal sequence. The resulting tubulin was able to assemble not only into MTs but also into the doublet MTs of the axoneme; these axonemes, however, were not functional. This finding implies that the signal sequence may not be necessary for doublet MT formation per se but may be involved in the interaction of the axonemal MTs with some other component critical for function. This in turn raises a question about the observation of Renthal el al. (1993) that cells that express both &I and PIVuse only P1v in their axonemal MTs. Clearly, if the signal sequence (present in PIVand not in Pll)is not necessary for incorporation into axonemal MTs, then there must be other portions of the sequence where PIIand PIVdiffer that play a role in this discrimination. There are indeed other places in the sequence where PIVdiffers from 011.Burns and Surridge (1990) have suggested a correlation between the

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phenylalanine at position 436 and the sequence at positions 217-218 (Table V). Although the correlation applies to the presumably nonaxonemal PI, PI1,and Pv, as well as to PIv,it does suggest that there may be some contact between the signal sequence, which contains Phe436,and other parts of the molecule. Conceivably, these other parts could also play a role in determining axonemal specificity.

D. Behavior of Purified Tubulin lsotypes in Vitro If tubulin isotypes have different functions in vivo, one should be able to design experiments in which the purified isotypes can be shown to behave differently in vitro. Such has proven to be the case. In one approach, purified vertebrate brain tubulin that contains the PI,PI,,PII~, and PIv isotypes has been compared to either mammalian platelet tubulin or avian erythrocyte tubulin, both of which contain the PVIisotype. Erythrocyte tubulin and brain tubulin assemble with different kinetics in vitro. Erythrocyte tubulin also forms a ring oligomer at 5°C that is not formed by brain tubulin (Murphy and Wallis, 1983). Microtubules formed from erythrocyte tubulin are much less dynamic in vitro than are those formed from brain tubulin (Trinczek et af.,1993). Inhibition of MT assembly by the sulfhydryl-directed reagent N,N'-ethylenebis(iodoacetamide) (EBI) was much less marked for erythrocyte than for brain tubulin (Ludueiia et al., 1985), but this may be explained by the fact that erythrocyte PvI lacks cys239,which has an assembly-critical sulfhydryl that reacts very well with EBI (Little and Ludueiia, 1985; Bai et aL, 1989). Also, chicken erythrocyte tubulin has a much greater propensity to assemble into spirals with vinblastine than does brain tubulin and it aggregates in the presence of nocodazole, a phenomenon not noted for brain tubulin (Ludueiia etal., 1985,1986). In addition, Serrano et af. (1989) find that chicken erythrocyte tubulin binds 0.9 calciums per dimer as opposed to 1.4 per dimer for brain tubulin. Mammalian platelet tubulin contains the PvI isotype and has some unusual properties in vitro; it appears to contain a disulfide bridge not noted in brain tubulin. Because pVIhas two more cysteines than d o the other /3 isotypes, presumably one of these, at least, is involved in the disulfide (Ikeda and Steiner, 1978). Also, platelet tubulin binds irreversibly to the GTP affinity analog, p-fluorosulfonyl benzoylguanosine (FSBG) (Steiner, 1984). In contrast, brain tubulin binds FSBG reversibly (Prasad and Ludueiia, 1987). FSBG reacts with a sulfhydryl group so the fact that it binds reversibly to brain tubulin implies the presence of a second sulfhydryl group nearby that could displace the FSBG. Presumably this work on FSBG involves the p subunit because the exchangeable GTP binding site is on the subunit close to Cys12 (Shivanna er al., 1993). Therefore, these results

TABLE IV

Axonemal Signal Sequences" Isotype

Organism Animals Chordate Human

PI PI11

PlV.

Plvb

Mouse

B1

Bn Plll PlVa. Iv

0 0

PVl

Hamster Chicken

PV PI PI1 PI11

PIV PV PVl

Xenopus

Plvb 811

Notothenin (fish) Echinoderm Stronglyocentrotus Paracentrotus Lytechinus Mollusc octopus

PI

Prvb

Sequence

Tubulin forms axonemal microtubulesb

Reference

EEDFGEE GEMYEDD QGEFEEE EGEFEEE EEDFGEE QGEFEEE EGEMYED EGEFEEE EDAEEAE DGEEAFE EEDFGEE QGEFEEE EGEMYED EGEFEEE EEAFEDD VEEYEEA EGEFEEG QGEFEEE EGEFEEE

No No No Yes No No No Yes No ND No No No Yes ND No ND No ND

Lewis and Cowan, 1990 Sullivan and Cleveland, 1986 Lewis et al., 1985a Sullivan and Cleveland, 1986 Wang et al., 1986; Lewis and Cowan, 1990

EGEFDEE EGEFDEE EGEFDEE

Yes ND Yes

Harlow et al., 1988 Casano etal., 1996; Di Bemardo et al., 1989 Alexandraki and Ruderman, 1983

SDEYDNE

ND

Tomarev et al., 1993

Ahmad ef al., 1991 Monteiro and Cleveland, 1988 Sullivan et al., 1985 Sullivan er al., 1986a Sullivan et al., 1986a Sullivan et al., 1986b Murphy et aL, 1987 Bieker and Yazdani-Buicky, 1992 Good et al., 1989 Detrich and Parker, 1993

Arthropod Drosophila

P1

P2 P3 Homarus (lobster) Nematode Haemonchus Caenorhabditis

Pgru-1,/38-9 @12-16,12-164 pben-1 @tub-1 pmec-7

Brugia Teladorsagia Onchocerca

Plants Angiosperms Glycine (soybean)

PI p2

Arabidopis (mouse-ear cress)

TUB1 TUB2, TUB3 TUB4 TUB5 TUB6 TUB7 TUB8 TUB9

Zinnia

Pl P2 03

Solanurn (potato) Lupinus (lupin) Daucus (carrot)

PSTl,PST2 PLP2

DAEFEEE EGEFDED EFDPEVN EAEFEEE

No Yes No ND

Michiels et al., 1987 Rudolph et al., 1987

MGDLDAE EGEMEGA DGELDGT DVDGYAE DAAEAFD EGDLQEG MGDLDAE ELNETIE

ND ND ND ND ND ND ND ND

Kwa et al., 1993

DDHEDED EDEYEEE EDEYDEE EGDYEDE EEYEEEE EGEYDVE EGEYEED EGEYEEE EEGYEYE EEYEEDE EGEYEED EYDEEDG DDGEYEE EEEYDDE DGYEYED EEYYEDE

ND ND ND ND ND ND ND ND ND

Guiltinan et al., 1987

ND ND ND ND ND ND

Yoshimura et al., 1996

Demers et al., 1996

Driscoll et al., 1989

Helm et al., 1989; GuCnette et al., 1991 Elard ef al., 1996 Marguitti-Pinto et al., 1995

Snustad et al., 1992

Taylor et al., 1994 Vassilevskaia et al., 1996 Okamura and Azumano. 1988 (continued)

TABLE IV (continued)

Organism

Isotype

Tubulin forms axonemal miaotubulesb

Reference Rogers et al., 1993; Villemur et al., 1994

EGEYEDE EADYEEE EEEQDGE AEEYEEE EAEYEDE EEEYEDE DEEYEEE EAEYEDE EGEYEDE EYEDEEE EEEDYGD

ND ND ND ND ND ND ND ND ND ND

P lB 2 S 3 Pl

EGEFGEE EGEFEGE EGEFEGE

ND Yes Yes

Conner et al., 1989 Harper and Mages, 1988 Youngblom et al., 1984

P5, P6

EGEFDED

ND

MacKay and Gallant, 1991

VEGYEEE

No

Liaud et al.. 1995

EGEFEEE EGEMDEE EGEFDDE EGEYVED E EE MDE E EGEFEEE

Yes ND ND ND Yes ND

Gaertig et al., 1993 Liang et al., 1994 Miceli et al., 1994 Harper and Jahn, 1989 Conzelmann and Helftenbein, 1987 Dupuis, 1992

Zea (corn)

tub1 tub2 tub3 tub4 tub5 tub6 tub7 tub8

Oryra (rice)

Pl pz R2242

Protists Chlorophyta Polytornella Volvox Chlamydomonas Phaeophyta Ectocarpus Rhodophyta Chondrus Ciliophora Tetrahymena Euplotes octocarinatus Euplotes focardii Euplotes crassus Stylonichia Paramecium Zoomastigina

Sequence

B1,pz

PL pz

Pl

Kang et al., 1994; Koga-Ban et al., 1995

ND

Naegleria Trichomonas Trypanosoma Oomycota Achlya Apicomplexa Plasmodium Toxoplasma Leishmania Eimeria Babesia Myxomycota Physarum

IU

w

0

Euglenophyta Euglena Diplomonada Giardia Acraisomycota Dicty ostelium Rhizopoda Entamoeba Reticulomyxa

Pl p2'

Microsporidia Encephalitozoon Fungi Hyphomycetes Geotrichum Trichoderma

EGEFDEN EGEEDEE EGEFDEE

Yes ND ND

Lai et aL, 1994 Katiyar and Edlind, 1994 Kimmel et al.. 1985

E G E FD E D

Yes

Cameron et al., 1990

EGEFEEE EGEFDEE EGEYDEE EGEFDEE DADDMVN

Yes Yes ND Yes ND

Delves et al., 1989 Nagel and Boothroyd, 1988 Coulson et al., 1996 Zhu and Keithly, 1996 Casu, 1993

E E GG E E E EAMEDDA

Yes No

Paul et al., 1992 Burland et al.. 1988

EGEFDEE

Yes

Schantz and Schantz, 1989

EGEEFEEE

ND

Kirk-Mason er al., 1988

GGEYQEE

No

Triviiios-Lagos et al.,, 1993

DDEQPDQ EDTEEAE ENAENAE

No No No

Edlind et al., 1996 Linder et al., 1991

AEEFLVN

No

Edlind et al., 1996

DMEYEDE EELMDHE DGEEYEE EEEEYED

No No No No

Gold et al., 1991 Goldman et al., 1993 (continued)

TABLE IV (continued)

Organism

Isotype

Acrernoniurn Deuteromycota Colletotrichurn Histoplasma Candida Aspergillus

N

Fusariurn Septoria Ascomycota Saccharornyces Schizosaccharornyces Neurospora Epichloe Erysiphe Pneurnocystis Basidiomycota Schizophyllurn

PbenA @ubC

Sequence

Tubulin forms axonemal microtubulesb

Reference

EEEYEEE

No

Now& and Kiick, 1994

AEEAYEE EEEYEEE GEDEYFD EELEYAD EEEYAEE GAYDAEE EEEYEEE EEEYDEE

No

Buhr and Dickman, 1994

No No No

Hams et al., 1989 Smith er aI., 1988 May et al., 1987

No No

Yan and Dickman, 1996 Cooley and Caten, 1993

EEVDENG GDEDYEI EEEYEEE EEEYEEE EEEYPEE EVELDDE

No No No No No No

Neff et al., 1983 Hiraoka et al., 1984 Orbach et al., 1986 Byrd et al., 1990 Shenvood and Somerville, 1990 Dyer et al., (1992)

EGEYEEE

No

Russo et al., (1992)

’The table gives the signal sequences as defined by Raff et al., (1997) for a variety of P-tubulin isotypes. In mammalian PrVb.this corresponds to positions 433-439. In other organisms, the numbers will be different, but they represent the best match of corresponding positions. In many cases, although not all, isotypes that occur in axonemes have the signal sequence EGEFEEE, where X is an acidic amino acid. “Yes” means that particular tubulin has been seen to form axonemes, that it is particularly strongly expressed in ciliated cells, or, in the case of ciliated or flagellated protists, that it is the only 0-tubulin expressed, therefore, it must participate in axonemal microtubules. “No” means that the isotype is known not to be expressed in axonemes, or that it has only been found to be expressed in cells that lack axonemes, and there is another isotype that is expressed in these cells, or that it is an organism, such as a fungus, known not to have axonemes. ND (not determined) means that there is no real evidence either way. With only 55% identity to other P-tubulins, P2 of Reticulornyxa jilosa is the most divergent P-tubulin yet discovered.

235

MULTIPLE FORMS OF TUBULIN TABLE V

Some “Hot Spots” of Differences among Vertebrate P-Tubulin Isotypes”

Isotype class PI

Plva PlVb

PI1 Plll

Position in sequence 35

55-57

124

T T T S S

TGG TGG TGG AGN SSH

A A A S C C C S C

PV

?

???

PVI

G S N

SSQ AYG AYS

217-218

T T T T

T T T T AT T T T T T T TN

436

C terminus EEEEDFGEEAEEEAh EEGEFEEE AEEEVA” EEE~EFEEEAEEEVA~ DEQGEF~EEEGEDEA~ EEEGI~MYEDDEEESESQGPK~

VNDGEEAFEDEDEEEINE~ ANDGEEAFEDDEEEINEg SEEDAEEAEVEAEDKDHg ADVEEY EEAEASPEKET~

a The table shows various “hot spots” including the C-terminal end. Position 436 is shown separately to permit quick comparison with positions in other hot spots, but it is also located in the C-terminal region (Sullivan and Cleveland, 1986; Burns and Surridge, 1990). Sequences shown are from the mouse, except for the two Pv sequences, which are from the Chinese hamster (top) and the chicken (bottom) (Ahmad eta/., 1991). Of the two PvI sequences, the top one is from the mouse and the bottom one from the chicken. Underlined residues are posttranslationally modified (A. Riidiger et al., 1995). GIuU1 is polyglutamylated (Mary et al., 1994). G I u ~ ~is’polyglutamylated (Mary et al., 1994). Mammalian Plvb is known to be polyglycylated and polyglutamylated, but the modified residues have not been identified. By analogy with other organisms, G ~ is uassumed ~ ~to be ~ the polyglycylated residue (M. Riidiger et al., 1995). ‘G I u is~ polyglutamylated ~ ~ (Riidiger et al., 1992). fTyr437is phosphorylated, polyglutamylated, SerU4 phosphorylated (Alexander et al., 1991). The sequence of PIIrhas an inserted methionine at position 436, which means that Tyf‘37 is actually homologous to Phe436in the other isotypes. g Not known to be posttranslationally modified. * Sera’ is phosphorylated in turkey erythrocytes (Riidiger and Weber, 1993).

imply that Pvr differs from the other isotypes in the distribution of its sulfhydryl groups. Because the affinity group of FSBG replaces the phosphate moiety of GTP, these sulfhydryl groups may be involved in the GTPase activity of tubulin. Mejillano et al. (1996) have indeed suggested that the hydrolysis of GTP by tubulin is catalyzed by sulfhydryl groups. It is difficult to draw too many conclusions about isotype-specific functions from the previous studies for several reasons: (i) In these studies, aPVlwas compared to what is essentially a mixture of tubulin dimers; (ii) the functions assessed (such as assembly inhibition by EBI) may not be of physiological significance, and (iii) because the highly divergent Pvl apparently performs fewer functions than other tubulins, it can change rapidly in the course of evolution and one can readily imagine that it could

236

RICHARD F. LUDUENA

acquire very different functional properties that could be manifested in vitro, properties that have no relevance to the situation in viva A more meaningful approach would be to purify and compare tubulin isotypes that occur in the same cell. It is reasonable to postulate that isotypes from the same cell are likely to be exposed to the same environment in vivo. Thus, if the properties of the isotypes differ in v i m , one might argue that they may also differ in vivo, even if the precise parameters of the difference are not the same in vivo. For example, isotypes that differ in their ability to bind to kinesin in vitro might reasonably differ in that respect in vivo as well even though the Kd values of the binding might be not the same in vivo as they are in vitro. Bovine cerebral P-tubulin consists of PI (3%),PII(58%),PIII(25%),and& (14%) (Banerjee et al., 1988).To separate these isotypes, a series of monoclonal antibodies specific for the fill, PIII,and PIvisotypes have been developed that appear to be specific by immunoblot assays (Banerjee et al., 1988,1990,1992; Lee et al., 1990). These antibodies have been used to purify from bovine or porcine brain the a/31~~, and aPIVdimers by immunoaffinity chromatography (Banerjee et al., 1992; Lobert et al., 1995). No antibody specific for PI has been used in these purifications; therefore, it is possible that small amounts of apt have been present in some of the purified dimers. The purified dimers all assemble more rapidly than does the unfractionated material (Banerjee et al., 1992; Lu and Ludueiia, 1994). Even partially purified tubulin assembles more rapidly than does unfractionated tubulin (Banerjee et al., 1990; Lu and Ludueiia, 1993a). There are two possible explanations for this: (i) Isotypically homogeneous tubulin dimers may fit together more readily, and hence assemble more rapidly, than would dimers in a heterogeneous population; and (ii) dimers that do not fit together readily may instead decay faster and hence more quickly lose their ability to assemble into MTs. Even among the purified dimers, however, some assembly differences are noted. In the presence of the MT-associated proteins tau or MAP2 both aPIland aPIIIassemble faster, and to a larger extent, than does aPIv(Banerjee et al., 1992);this difference is not observed when the isotypes assemble in the absence of MAPs (Lu and Ludueiia, 1994). Because tau and MAP2 both occur largely in neural tissue, as do PIIand Pill, it is reasonable that an association between the tubulin dimers and these two MAPs would have evolved. In contrast, PIv is widespread and occurs in many tissues that do not appear to express either tau or MAP2, so it may not have such a strong interaction with tau and MAP2. Perhaps one of the more dramatic differences between the purified isotypes is in their dynamic behavior (Panda et al., 1994). Microtubules assembled in vitro from aPIIIare more dynamic than those assembled from apII or aPIVor from unfractionated tubulin. The assembly and disassembly rates of aPIIIMTs are, respectively, 51-57 and 75-92% greater than those of the

MULTIPLE FORMS

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237

other MTs, and aPIIIMTs spend 24% of their time in attenuation (neither growing nor shortening detectably). This is much less than the 42-47% spent by the other types of MT. In short, the dynamicity of aPIIIMTs is 67 ? 7 sec-', whereas the dynamicities of aPIIMTs, aPIv MT, and MTs made of unfractionated tubulin are, respectively, 28 2 3,30 ? 2, and 30 2 2 sec-'. The effects of combinations are complex. Microtubules composed of 20% aP11 and 80% a& exhibit the dynamic properties of pure a0111; however, MTs made of 50% a& and 50% aPIllare very much like MTs made of pure a&. It thus appears that the effects of aPIIare more significant than those of a&II.It is difficult to extrapolate directly from these results to the situation in vivo. In the cell, conditions could be such as to greatly alter the dynamics of all the tubulin isotypes. These results do, however, argue that in the cell the different isotypes could indeed have different dynamics and raise the possibility that a cell could control the dynamic behavior of its MTs by adjusting the relative proportions of different isotypes. For highly orchestrated processes such as mitosis, in which MTs play a major role, it is likely that careful regulation of MT dynamic behavior is critical for a successful outcome. The work of Mejillano et al. (1996), mentioned previously, suggesting that sulfhydryl groups may be involved in tubulin's GTPase activity, may perhaps explain the different dynamic behaviors of the isotypes. It has been postulated that the GTP binding site in P-tubulin includes the region 125-133 and that that segment is a loop that is exposed in the dimer but not in the MT. This implies that this area is involved in tubulin-tubulin interactions in MT assembly (de Pereda and Andreu, 1996; de Pereda et al., 1996). It is interesting that the cysteines at positions 127 and 129 are highly conserved, particularly among the animals. However, PIIl,Pv,and PvI have cysteines at position 124; no other known tubulin has a cysteine Pv, and PvI,there is a three-cysteine at position 124. This means that in PIIlr cluster at this position. The only other cysteine in the GTP-binding region is Cysl*, which is known to be very close to the guanine moiety and not to the phosphate moiety (Shivanna et a1.,1993). It is interesting that the colchicine-induced GTPase activity of aPIIIis significantly higher than those of aPIrand aPIV(Lu and Ludueiia, 1993b; Banerjee, 1997). Because a& has the lowest affinity for colchicine (Banerjee and Ludueiia, 1992), this difference in GTPase activity may reflect a difference intrinsic to the GTPase site rather than to the colchicine binding site. It is tempting to speculate that the region including positions 124-129, which is likely to be involved in GTP hydrolysis and which differs among the isotypes, may play a role in determining the dynamic properties of the isotypes. If a cell is able to choose between two closely related isotypes, such as PII and PIv, for incorporation into axonemal MTs, there must be some difference among the isotypes that could be perceived by the factors in the

238

RICHARD F. LUDUENA

cell that are involved in this selection. An experiment by Sharma and Ludueiia (1994) may illuminate this point. The sulfhydryl-specific bifunctional cross-linker EBI forms two cross-links in P-tubulin; these cross-links are readily observed because they alter the electrophoretic mobility of P-tubulin in distinct fashions (Roach and Ludueiia, 1984). One of these cross-links, designated P*, is between Cys239and Cys354;the other, designated PS,is between Cys12 and either CysZo1or Cys211(Little and Ludueiia, 1985, 1987). In the experiments of Sharma and Ludueiia (1994), each of the isotypically purified tubulin dimers (a&, aPIll,and aPW)was treated with a series of analogs of EBI of increasing chain length. As expected, both aPIland aPrvform the cross-link between Cys239and Cys354.However, aPIIIdoes not form the P* cross-link, presumably because it has a serine at position 239 instead of a cysteine. Although the sequences of each isotype of the bovine tubulin used in this experiment have not been individually determined, one would imagine by extrapolation from other mammals and birds that bovine P-tubulin has all three cysteines (Cys12,Cys201,and Cys211) involved in the Ps cross-link. However, crPIIIdid not form this cross-link at all, regardless of the chain length of the cross-linker. The following possibilities could account for this result: (i) a disulfide bond in aPII1involving one of these cysteines, (ii) a conformation in aPIIIin which at least one of the cysteines involved is inaccessible to the cross-linker, (iii) a conformation in aPrrIin which the distance between the sulfhydryls is too great to be spanned by even the longest of the EBI analogs used in this experiment; or (iv) a bulky region between the sulfhydryls that prevents EBI from connecting them. Each of these possibilities implies a major structural difference between aPIIIand the other two dimers. Another, perhaps more interesting, result was that aPIIand a& also differed in their reactions with the cross-linker. With aOrI,a high yield of the Ps crosslink was obtained with EBI and with analogs containing two to five more methylenes than EBI; with the analogs containing one more methylene or eight more methylenes, however, very little cross-link formed. With a&, on the other hand, the highest yield of PS cross-link was obtained with EBI, but all the other analogs were able to generate the cross-link in roughly equal amounts, including the ones that could not generate it in a&. These results imply that the cysteines may be somewhat less accessible in a& than in aPIv and suggest that there may be a subtle conformational difference between a& and a& consistent with the observation that certain cells that express both aPIIand aPIv discriminate between them and specifically choose the latter for their axonemal MTs (Renthal et d.,1993). The purified isotypes differ markedly in their interactions with antitubulin drugs. Estramustine binds less well to the PIIIisotype than to the others (Laing et al., 1997). Colchicine binds to aPIIIwith a dissociation constant ( K d ) of 0.12 pM, only half as much as its affinity for upII(0.24 p M ) and

MULTIPLE FORMS OF TUBULIN

239

as much as its affinity for aPlV(3.31 p M ) (Banerjee and Ludueiia, 1992). The apparent on-rate constants for the binding to ~ P I I( Y,~ I I I , and ~ P I V are, respectively, 132 ? 5 , 30 ? 2, and 236 t 7 M - ’ sec-’ (Banerjee and Ludueiia, 1992). The significantly slower binding seen with a& explains why the colchicine binding kinetics are biphasic in brain tubulin (which contains all three dimers) but monophasic in kidney tubulin (which lacks pIII)(Banerjee and Ludueiia, 1987); in fact, recombination of aPIIlwith tubulin lacking ~ 1 1 restores 1 biphasic kinetics (Banerjee and Ludueiia, 1991). Using stopped-flow fluorimetry, Banerjee et al. (1994)dissected the drugtubulin interaction. Employing a more rapidly binding drug analog desacetamidocolchicine (DAAC), Banerjee et al. (1994) measured the binding parameters shown in the following equation: K’ k2 T + DAAC H T-AAC (T-DAAC) k-2

They found that the isotypes showed very little difference in their values for K l and k,; the largest difference was in the rate constant, k2,a measure of the rate of the conformational change ensuing in the tubulin molecule when the drug binds. For aPI1,aPrrr,and apIv,the values for k2 were, respectively, 0.67 t 0.05,0.053t 0.006,and 0.59 ? 0.07sec-’. Another colchicine analog, 2-methoxy-5-(2’,3’,4’-trimethoxyphenyl) tropone (MTPT), gave an analogous pattern in which the k2 values for the a& apIII,and cuplv dimers were, respectively, 4.22 ? 0.4,2.07 ? 0.17,and 5.28 t 1 sec-’ (Banerjee et al., 1997). It thus appears that apIIImay be more rigid than the other isotypes in the region of the colchicine binding site. It is striking that in the case of DAAC, the k2 values for apll and a& were 11.2-12.6 times greater than that of aPlll.In contrast, for MTPT, the k2 values for a& and aPrvwere only 2.0-2.6 times greater than that for aplll.DAAC lacks the side chain on the B ring of colchicine, whereas MTPT lacks the entire B ring; these results suggest that the rigid portion of a& involves the site where colchicine’s B ring binds (Banerjee et al., 1997). The interaction of tubulin isotypes with taxol was measured indirectly by examining the effect of taxol on their dynamic behavior (Derry et al., 1997). The authors calculated the “suppressivity,” which is related to the effect of taxol on the individual dynamic parameters as a function of the stoichiometry of bound taxol. Regarding the effect on the shortening rate, taxol’s chief effect on MT dynamics, apln and aPlVare the least sensitive. Suppressivity values for apI1,a&l, and apIvare 3626,765,and 784,respectively. When Lobert et al. (1995)examined vinblastine-induced aggregation of unfractionated tubulin and of aPlrlthey found no difference in the dependence of sedimentation coefficient as a function of vinblastine concentra-

240

RICHARD F. LUDUENA

tion. In other words, the effect of vinblastine on aggregation into non-MT forms appeared to be the same for both aPIIIand unfractionated tubulin. In apparent contrast, preliminary results suggested that when &I, ~ P I I I , and upIv were compared in the presence of vinblastine, MT assembly by aPIrwas the most sensitive to vinblastine inhibition and crPIIIwas the least likely to undergo vinblastine-induced aggregation into non-MT polymers (Khan and Ludueiia, 1995). The tubulin isotypes differ in their folding pattern as shown in an experiment in which Fontalba et af. (1995) expressed six mouse P-tubulin isotypes in vitro in a rabbit reticulocyte cell-free system. They found that the isotypes differed in their ability to be released from chaperonins and to form dimers with a. PIvaand Pvr were not released from the 300-kDa complex. PIrrwas released most readily. PI,PII,and PIVbwere partly released. PIVbshowed the greatest propensity to form a dimer. PIIlformed a dimer less well; mostly, it remained a monomer. Could these results be related to PIII’s greater conformational rigidity? To identify specific regions involved in determining the different behaviors of the isotypes in this system the authors constructed hybrids of P I V b and PvI and found that these hybrids (with P I V b constituting the C-terminal 156 residues) were intermediate in dimer formation between PrVb and &I. When the last 12 amino acids were removed from &b, incorporation into dimers was slightly inhibited. These findings suggest that the last 12 residues in the C-terminal region of p-tubulin may be involved in release from chaperonins but also imply that the other parts of the last third of the molecule may play a role as well, as indicated by Dobrzynski et af. (1996). A possible conclusion from some of the work described here is that PIII may have a less flexible conformation than some of the other isotypes. If one assumes that decay involves a conformational change, then the hypothesis that PIIIis less flexible is consistent with preliminary work indicating that aPIIIdecays more slowly than aPII (Schwarz and Ludueiia, 1996). If tubulin undergoes a conformational change during the assembly and disassembly process, then it is conceivable that a more rigid form of tubulin such as aPIIIwould have different dynamics, as was reported by Panda et af. (1994). Burns and Surridge (1990) have found hot spots of variation among the vertebrate /3 isotypes including the C-terminal peptide and residues 35, 55-57, and 124. These variations appear to be coordinated. Additionally, there is a good correlation between the presence of either phenylalanine or tyrosine at position 436 and the sequence at positions 217-218. These correlations are shown in Table V. Based on the differences shown in Table V, Burns and Surridge (1990) propose that tubulin isotypes in vertebrates can be grouped into three types: type 1, including PI,PIva, and PI^; type 2, consisting of PI];and type

MULTIPLE FORMS OF TUBULIN

241

3, comprising PIII,Pv, and pvI.They argue that the C terminus and positions 35, 55-57, and 124 are likely to be located on the exterior of the tubulin molecule. They also point out that, except for Phe436,every position shown in Table V is very close to a highly conserved basic residue (His37,L y P , L Y S ' ~and ~ , Arg215).It is possible that there could be some electrostatic interactions between the acidic C terminus and these basic residues. Because the C terminus differs greatly among the different isotypes it is possible that these interactions could be affected by the nearby variable residues; therefore, the interactions and conceivably the flexibility or stability of the molecule could vary among the isotypes. The isotypes also differ in the nature of their posttranslational modifications. In those organisms that have a specific isotype in their flagella, that isotype is also apparently the only one to undergo polyglycylation (M. Riidiger et al., 1995). The mammalian PIrrand avian Pv1 isotypes are phosphorylated at SerW and Ser441,respectively, whereas mammalian PIII is also phosphorylated at Tyr437(Diaz-Nido et al., 1990; Alexander et al., 1991; Riidiger and Weber, 1993). These particular modifications cannot occur in the other /3 isotypes because they lack those residues (Table V). As a matter of fact, the other isotypes have no serine or tyrosine residues in the C-terminal region. Finally, the vertebrate isotypes differ in their polyglutamylation. Among the a's, a1 and a 2 are polyglutamylated at G ~ uthe ~ polyglutamylation ~ ~ ; of the others is not clear. Of the p's, PI,PII, Pin, and pIv are polyglutamylated, whereas PvI is not (EddC et al., 1990; Alexander et al., 1991; Rudiger et al., 1992; Redeker et al., 1992; Mary et al., 1994). Whether pv experiences this modification is unknown. Even among the polyglutamylated isotypes there are intriguing differences: PIis modified at G1uM1,whose location in the sequence of the protein is not equivalent to those of the polyglutamylated residues in PI1,Prn,and pIv.This fact suggests that different enzymes may polyglutamylate different isotypes and even raises the possibility that the function or regulation of polyglutamylation differs among the isotypes (Redeker etal., 1992;Riidiger et al., 1992).Posttranslational modifications will be discussed in more detail later.

E. y-Tubulin In addition to a and p, there is a third form of tubulin, y, originally described in A . nidulans (Oakley and Oakley, 1989). The sequence of y-tubulin is 28-35% identical to those of a and p; y has been found in animals, plants, fungi, and protists (Fuchs et al., 1993; Liang and Heckmann, 1993; Luo and Perlin, 1993; Liu et al., 1994; Oakley, 1994; Lopez et al., 1995; Scott et al., 1997). y-Tubulin is located in the pericentriolar material and in the core of the centriole (Fuller et af., 1995); it is also part of the basal body in

242

RICHARD F. LUDUENA

ciliates (Liang et al., 1996). y-Tubulin is involved in nucleating MTs and binds to their minus ends (Li and Joshi, 1995; Moritz et al., 1995; Zheng etal., 1995; Sobel and Snyder, 1995; Marschall et al., 1996;Spang et al., 1996). Interestingly, there is now evidence that y , like a and p, exists in isotypic forms. In Arabidopsis thaliana there are two isotypes of y-tubulin that are 98.1% identical (Liu et al., 1994). It is not known if these isotypes differ in their cellular or subcellular distribution. Two of the nine differences among the two isotypes are potentially significant: a Gly-Asn difference at position 211 and a Glu-Gly difference at position 455. In Physarum polycephalum there is a single y-tubulin gene but it encodes two different y-tubulins of molecular weights 52,000 and 50,000 (Lajoie-Mazenc et al., 1996). The larger of these two forms binds very tightly to the MT-organizing centers, whereas the latter binds more weakly. It is not clear whether the Physarum isotypes arise from different mRNA splicing or from different posttranslational modifications. Dibbayawan et al. (1995) treated HeLa cells with a polyclonal antibody against y-tubulin and found that the antibody stained the centriole pair in HeLa cells as well as the basal body in Chlamydomonas. Other anti-y-tubulin antibodies stained the whole pericentriolar region. Could this reflect multiple y-tubulin genes? Perhaps, but it could also reflect different posttranslational modifications or perhaps uneven masking of epitopes for different antibodies. At any rate, although it is clear that there are at least two isotypes of y-tubulin in certain organisms, the possible functional significance of these isotypes is still completely unclear.

F. Evolution of lsotypes The evolution of tubulin isotypes is intimately connected with the evolution of tubulin itself. The origins of tubulin are still a mystery. a and p share 36-42% identity with each other and 28-35% identity with y, suggesting that y diverged first and then a and separated from each other (Little and Seehaus, 1988; Oakley, 1994). Because all eukaryotes have both a and p and appear to have y, these separations must predate the eukaryotic divergence. Recently, a bacterial GTPase, FtsZ, has been described that shares some similarities with a- p-, and y-tubulin; the similarities involve the GTP binding sites and other regions in the N-terminal portion (Mukherjee and Lutkenhaus, 1994; Erickson, 1995). FtsZ is involved in cell division in Escherichia coli and can form rings, as indeed does y-tubulin. It also forms sheets of protofilaments (Erickson et al., 1996). It is tempting to speculate that tubulin may have evolved from a bacterial, possibly archaebacterial, FtsZ and somewhere along the way changed the morphology of its polymer from a ring to a tubule (Margolin et al., 1996).

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Isotypes of a- and 0-tubulin may have appeared very early in eukaryotic evolution because they occur in all four kingdoms of eukaryotes. An intriguing set of observations is that the putative axonemal signal sequence in P-tubulin is highly conserved (Raff et af., 1997) and that in many organisms with isotypes, the isotypes that occur in flagella are more closely related to tubulins in other organisms (Gaertig et af., 1993). This relatedness is not always seen by comparing the entire sequence but rather by focusing on the hot spots of variation within a set of isotypes (Burns and Surridge, 1990). For example, of the three a isotypes in Physarum the one that occurs in the flagellated form is most similar to a’s from mammals, plants, fungi, and other protists (Cunningham et al., 1993). It is tempting to conclude that the original function of MTs was to form axonemes and that other functions, such as their participation in mitosis, evolved later. This is also consistent with the hypothesis of Margulis (Sagan, 1967) that undulipodia (cilia and flagella) originated as a symbiont early in the history of the eukaryotes, with the MTs being involved originally in axonemes and then later being used for mitosis. One could also postulate that participation in a highly complex and organized structure such as an axoneme constrains the divergence of tubulin. In general, isotype families are organized at the level of the phylum rather than the kingdom (Fig. 1). As mentioned previously, Burns and Surridge (1990) grouped the vertebrate 0-tubulin isotypes into three classes. Amphibians express pIv(class 1) and Prr(class 2) (Good et al., 1989; Moody er al., 1996), and fish express PrII(class 3) and a P that could be related to either PIIor Prv(Detrich et al. 1987; Detrich and Parker, 1993). Thus, the divergence between class 3 and the other two classes dates to at least the origin of the vertebrates more than 500 million years ago. The divergence between classes 1and 2 must date back at least to the time of the amphibians. Divergence within the classes occurred later. Pv and probably PvI would have originated prior to the divergence of birds and mammals about 280 million years ago. The separation into two isotypes of PIv in mammals and of pIIin birds is probably much later (Fig. 2). An analogous evolution occurred among the a isotypes. There is a neuron-specific a in the electric eel that is most closely related to mammalian a1 and a2 and the trout has a testis-specific a that resembles mammalian a3/7 (Canals et al., 1995; Garber et al., 1991). Therefore, some divergence among the a isotypes also happened about the same time that the isotypes were beginning to diverge. Further divergence among the a isotypes may have occurred later in evolution. Plants have a similar story. The five a-tubulin isotype genes of maize have been grouped into three subfamilies according to the amino acid sequences of their encoded proteins. Subfamily I1 (Tua5 and Tua6) is similar to Arabidopsis TUAI and almost as close to Arabidopsis TUA.5 and TUA3.

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Ancesh a

Anceitral f3 (Axonemal?)

Ancestral Protein FIG. 1 Evolution of tubulin and its isotypes. The figure postulates that there was, in prokaryotes, a primitive protein, possibly serving some kind of cytoskeletal role, which was ancestral to bacterial FtsZ and to the original tubulin. At some point this ancestral tubulin gave rise t o y and then a! and p diverged. The figure also shows the speculation that the original tubulin may have had an axonemal role. Although isotypes must have appeared soon after the origins of a! and p, the major isotype families do not seem to extend across phyla, hence the figure shows the divergence into isotypes after the divergence of the eukaryotic kingdoms and phyla. The figure shows a,@, and y diverging after the rise of the eukaryotes. That is a conservative hypothesis: it is conceivable that this happened at the prokaryotic stage.

Maize subfamilies I (Tual and Tua2) and I11 ( T u d )are close to Arabidopsis TUA2, TUA4, and TUA6. Thus, subfamilies I and I1 diverged prior to the divergence of the monocots and dicots; subfamily I11 may have arisen after this divergence (Villemur et al., 1992). Analogous groupings occur with the p isotypes. One group comprises maize tub4 and tub7, Arabidopsis TUBl, TUB4, TUBS, and TUB9, and soybean p l ; the other contains maize tubl, tub2, tub6, and tub& Arabidopsis TUB2, TUB3, TUB6, TUB7, and TUB&, and soybean p2 (Villemur et al., 1994; Taylor et al., 1994). Thus, some of the p isotypes diverged prior to the separation of monocots and dicots. Other than the slight conservatism of the flagellated isotypes, there appears to be little relation between isotypes in one phylum and those in another. Thus, the isotypes of Drosophila, although they show some relation to those of other insects, have no particular relationship to those of vertebrates. It is tempting to imagine that isotypes started their major evolution during the Cambrian radiation. As has already been mentioned, within the larger families of isotypes there is often one that is highly divergent, both from the other members

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\f

_ _ _ _ _ _ _ - - - _ _ - - _ ------------ -

--__-_-_______________

P (PlV?)

PRIMITIVE CHORDATES

FIG. 2 Evolution of the vertebrate P-tubulin isotypes. The figure postulates that plv,serving as an axonemal component, was the original vertebrate /3 isotype. It is clear that fish have both P ~ and v PIII.By the amphibian stage, fill had appeared, thus, all three classes of vertebrate isotypes are present in amphibians. The divergence of class 1 into PI and Plv and of class 3 into Pill, &. and Pvl must have occurred in reptiles, since these isotypes are present in both birds and mammals, which evolved from reptilian ancestors independent of each other. These separations need not have occurred at the reptile stage, as the figure suggests, but may be even earlier. The figure shows the further divergence of Plv in mammals into Plva and PrVband of Pll in birds into Plla and Plllh. These divergences could have occurred more recently, not necessarily at the reptile stage. Plvaand &, occur in both rodents and primates, so their origins are likely to date from the Mesozoic, when these lines diverged; the two forms of PII have only so far been seen in chickens and hence could be a very recent development.

of the families and from tubulin in other organisms. The mammalian and avian pv,, found in blood cells, are good examples. Bearing little resemblance to each other, they are also quite divergent from other tubulins. One could imagine that an isotype that occurs only in a few tissues and perhaps performs fewer functions than other MTs would be able to evolve more rapidly (Gaertig et al., 1993). It is interesting that some of the more divergent isotypes occur in the gonads, such as a4 in Drosophila oocytes, aTTl in mouse testis and chicken testis a, and Tual in Arabidopsis pollen (Pratt et al., 1987; Hecht et al., 1988; Carpenter et al., 1992; Matthews et al., 1993). None of these are particularly related to each other so there cannot be any common function that puts strong constraints on their evolution. One specific role these all appear to have is that they form the meiotic spindle. Conceivably, if this is their only role, then the structural requirements for the meiotic spindle may be much less and hence they could diverge rapidly.

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111. Postrensletional Modifications of Tubulin The tubulin molecule is subject to a large number of varied posttranslational modifications, some of which also occur in other proteins, whereas others have so far only been found in tubulin (Sullivan, 1988). These will be described briefly below.

A. Phosphorylation Phosphorylation of P-tubulin in rat brain was first observed by Eipper (1972). She localized the phosphorylation site to the C-terminal region and was careful to distinguish between phosphorylation in vivo and in vitro, the latter being of low specific activity and occurring at various sites on tubulin (Eipper, 1974a,b). The phosphorylated isotype was later identified as Prrl(Ludueiia et af., 1988; Diaz-Nido et af., 1990). The phosphorylated residue is Ser444(Diaz-Nido et af., 1990; Alexander et af., 1991) (Table V). It is not clear exactly which enzymes carry out this phosphorylation in vivo, but casein kinase I1 appears to mimic the phosphorylation in vitro (Serrano et af., 1987). A casein kinase-like kinase that phosphorylates P-tubulin has been described in PC12 cells and is apparently associated with tubulin (Crute and Van Buskirk, 1992). Another kinase that phosphorylates both P-tubulin and tau has been observed in bovine brain (Takahashi et af., 1995). Phosphatases 2A and 2B are both associated with MTs, suggesting that there may be a cycle of phosphorylation (Dudek and Johnson, 1995). The function of phosphorylation of PII1is not yet clear. It apparently accompanies differentiation of neurons (Gard and Kirschner, 1985; Aletta, 1996). Removal of the phosphate inhibits MAP-induced MT assembly but does not affect assembly of pure tubulin in glycerol, suggesting that the phosphate may mediate the interaction of PIIlwith MAPS (Khan and Ludueiia, 1996). This is a reasonable hypothesis, because the C terminus of PIII,which has a lysine residue at the very end, is somewhat less acidic than the C termini of several other P isotypes. It is possible that adding a phosphate restores some of the negative charge in *a regulated fashion. Khan and Ludueiia (1996) analyzed PIIIby direct phosphate determination and measured 1.52 mol of phosphate per mole, raising the possibility that another residue in addition to Ser444may be phosphorylated in PIII.Tyr437in PrIIis also phosphorylated (Alexander et af., 1991). Human platelet tubulin, presumably consisting largely of PvI,is not phosphorylated even after thrombin stimulation of the platelets (Janiak et af., 1995). In contrast, approximately 10% of turkey erythrocyte PvI is phosphorylated at Ser441(Riidiger and Weber, 1993) (Table V). Tubulin has

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been reported to be phosphorylated by a casein kinase-like kinase in the tobacco hornworm Manduca sexta at an as yet unidentified position (Song et al., 1994). Carrot tubulin is also reported to be phosphorylated (Koontz and Choi, 1993). In activated human B lymphocytes, a-tubulin appears to be phosphorylated on a tyrosine residue near the C terminus by the tyrosine kinase Syk (Peters et al., 1996). It is not clear whether the phosphorylated residue is the C-terminal tyrosine or Tyr432.Tyrosine phosphorylation of both a- and P-tubulin in fetal rat nerve growth cones has also been reported (Matten et al., 1990; Atashi et al., 1992). Tyrosine phosphorylation may be a response to nerve growth factor because it appears to accompany differentiation of neurons (Cox and Maness, 1993). One study noted phosphosphorylation of P-tubulin in rat uterine smooth muscle. Interestingly, during labor, a became phosphorylated instead of P; the labeled isotypes or residues were not determined (Joseph et al., 1982). In certain axonemes, tubulin appears to be phosphorylated. In sea urchin sperm outer doublets, a and p contain, respectively, 1.2-1.4 and 1.0 phosphorylated serines; there is no difference in this respect between the two subfibers of the doublet (Stephens, 1975). In Chlamydomonas axonemes, only a is phosphorylated, but the phosphate is on both serine and threonine (Piperno and Luck, 1976).

B. Acetylation A wide variety of a-tubulins have an acetyl group added to the &-amino group of Lys40.This modification has the effect of neutralizing the positive charge of the &-aminogroup and has been observed in a’s from vertebrates, insects, echinoderms, nematodes, plants, and the protists Physarum, Trypanosoma, Chlamydomonas, Trichomonas, and others (L’Hernault and Rosenbaum, 1985; Gallo and Precigout, 1988; Wolf et al., 1988; Siddiqui et al., 1989; Wilson and Forer, 1989;LeDizet and Piperno, 1991; Souto-Padron et al., 1993;Delgado-Viscogliosi et al., 1996;Wolf, 1996a,b). Tubulin containing the modified lysine forms MTs that are more stable in the presence of antitubulin drugs (Geyp et al., 1996). Acetylation is particularly notable in axonemal MTs, which are generally quite stable structures. Acetylation has been observed in the flagella of Chlarnydomonas and Physarum and in Drosophila and human sperm, and in cilia from Tetrahymena, sea urchin blastulae, and retinal rod cells (Piperno and Fuller, 1985; LeDizet and Piperno, 1986; Sasse et al., 1987; Diggins and Dove, 1987; Sale et al., 1988). An a-tubulin acetyltransferase and a tubulin deacetylase have been characterized (Greer e f al., 1985; Maruta etal., 1986). Many other MT populations also contain acetylated tubulin (LeDizet and Piperno, 1986; Diggins and

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Dove, 1987; Piperno et af., 1987; Siddiqui et al., 1989). In Trypanosoma cruzi, the flagellar and subpellicular MTs are acetylated (Souto-Padron et af., 1993). Other stable MTs, such as those of inner and outer pillar cells and Deiters cells of the mammalian cochlea, contain acetylated tubulin (Slepecky et af., 1995). When microglia acquire the less active ramified morphology, their tubulin becomes more highly acetylated (Ilschner and Brandt, 1996). Acetylation appears to occur to tubulin after it is incorporated into MTs (Wilson and Forer, 1989). Other than causing an increase in stabilization of MTs, the precise function of acetylation is not clear. In one experiment, Lys40 of Chfamydomonas a was replaced by either an arginine (to conserve the positive charge) or an alanine-both are residues that cannot be acetylated. When the mutagenized a was overexpressed in Chfamydomonasit was incorporated into all the MTs but no change in the organism’s phenotype was seen, suggesting that acetylation may have a subtle function (Kozminski et af., 1993). Because some nonmutagenized a was also present, however, it is conceivable that the small level of acetylation could have been sufficient to carry out the stabilization or other function for which acetylation may be required. C. Tyrosination/Detyrosination Cycle Except for a4, all the isotypes of mammalian a-tubulin are encoded with a C-terminal tyrosine residue. The tyrosine is often removed by a tubulinspecific carboxypeptidase. Another enzyme, tubulin-tyrosine ligase, can add the tyrosine back to all the a isoforms, including a4, which lacked it in the first place (Barra et al., 1974; Gu et af., 1988). This modification has been seen in a variety of vertebrates, brine shrimp, and trypanosomes (Gallo and Precigout, 1988;Xiang and MacRae, 1995; Rutberg et af.,1996). The tubulin carboxypeptidase has been partly purified and found to act preferentially on polymerized tubulin (Argaraiia et al., 1980; Kumar and Flavin, 1981; Arce and Barra, 1983). The tubulin-tyrosine ligase has been well characterized and sequenced; it requires ATP but not a nucleic acid (Raybin and Flavin, 1977; Ersfeld et af., 1993). The enzyme is specific for native tubulin but can also incorporate phenylalanine and ~-3P-dihydroxyphenylalanine instead of tyrosine (Arce et al., 1975). Although the ligase acts on a,it has a binding site for p, raising the possibility that the nature of the p isotype could influence the action of the ligase (Wehland and Weber, 1987). Tyrosinated and nontyrosinated tubulin often form different MTs in the same cell; tyrosinated tubulin is common in the interphase network and in the spindle. The nontyrosinated tubulin occurs in some interphase MTs (Gundersen et al., 1984). In cultured chick retinal neurons, tyrosinated MTs

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were found in all parts of the neuron, whereas the nontyrosinated MTs were restricted to the longer neurites (Arregui and Barra, 1995). The detyrosinated MTs apparently constitute a more stable population in vivo; among their roles is stabilization of vimentin intermediate filaments (Kreis, 1987; Khawaja et al., 1988; Gurland and Gundersen, 1995). It is interesting that sea urchin sperm flagellar outer doublets exhibit heterogeneity of this modification; the A-tubules are only slightly detyrosinated, whereas the B-tubules are about 65% detyrosinated (Multigner et al., 1996).

D. A2-Tubulin There is a form of a-tubulin in which the last two residues ( G ~ u ~and ~O Tyr451)have been removed. This form of tubulin, called A2-tubulin, is not a substrate for tubulin-tyrosine ligase (Parturle-Lafanechbre et al., 1991). A2-Tubulin accounts for approximately 35% of brain a-tubulin and is present in neuronal growth cones and sea urchin flagella and cilia (Mary et al., 1996). Its distribution in neurons is distinct from that of detyrosinated tubulin (Paturle-Lafanechbre et al., 1994). The MTs containing A2-tubulin are thought to be very stable; the modification removes the tubulin from the tyrosination/detyrosination cycle (Paturle-Lafanechkre et al., 1991,1994).In vertebrates, this modification is particularly common in brain tubulin but is not observed, or is observed at lower levels, in a from turkey erythrocytes and rat muscle (Riidiger and Weber, 1993; Alonso et al., 1993). Redeker et al. (1996) found that in rat brain development the major posttranslational modification that occurs is the removal of the penultimate glutamate, rendering the a nontyrosinatable.

E. Polyglutamylation An unusual modification of tubulin is the addition of up to seven glutamate residues to a glutamate near the C terminus of both a-and P-tubulin (EddC et al., 1990).This major modification occurs in 40-50% of the total a-tubulin in mouse brain and in more than 85% of Pill in bovine brain (EddC et al., 1990; Alexander et al., 1991; A. Riidiger et al., 1995). In mammals the polyglutamate chains are added to the y-carboxyl group of GhM5of a1 and a2, G1uM1of PI, G ~ of pII, u G ~ ~ of Pnr, ~ u ~ and G1uMO ~ ~ of PIva (EddC et al., 1990;Alexander et al., 1991; Riidiger et al., 1992; Redeker et al., 1992; Mary et al., 1994) (Table V). In addition, a3/7 appears to be glutamylated, whereas the very divergent am1 is not (Fouquet et al., 1994). The Pvr isotype, at least in avian erythrocytes, does not appear to be polyglutamylated (A. Riidiger etal., 1995). Avian erythrocyte a1 is also not polyglutamy-

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lated (Rudiger and Weber, 1993). Polyglutamylation, particularly of a, appears to be more common in the brain in which polyglutamylation of a remains stable during differentiation, whereas that of /3 increases (Wolff et af., 1992; Audebert et af., 1994). Polyglutamylation is reversible; of the six glutamates commonly added in the brain the last three are readily removed, whereas the first three are more stable. This suggests that there may be more than one enzyme involved in deglutamylation and raises the possibility that the cycle of glutamylation and deglutamylation may be very complex (Audebert et al., 1993). It is noteworthy that the glutamylated residues in PIr,Prrl,and PIvaare adjacent to the Phe/Tyr residue at the C terminus (Table V), whose possible interaction with positions 216 and 217 has already been mentioned. This residue is also in the middle of the axonemal signal sequence previously discussed (Table IV). It is interesting that this is not the case with PI, in which the glutamylated residue is five positions removed from the phenylalanine residue; this difference raises the possibility of isotype-specific mechanisms of glutamylation, perhaps involving different enzymes and even serving different functions. Although the first glutamate to be added is joined to the polypeptide by an a,ylinkage, the subsequent glutamates are largely joined by a,alinkages (Redeker et al., 1991,1996). Polyglutamylation of tubulin has also been observed in the protist Trichomonas (Delgado-Viscogliosi et af., 1996). The function of polyglutamylation is not clear, but it obviously results in the addition of a large number of negative charges to an already highly acidic region. a-Tubulin can be tyrosinated or detyrosinated equally well regardless of the degree of polyglutamylation (Edd6 et af., 1992). There is evidence that polyglutamylation favors kinesin binding to tubulin (Larcher et al., 1996). This is interesting because the specific domain for binding to kinesin was thought to be at positions 410-436 in a and 6, which, at least in a and PI, does not include the polyglutamylated residues (Goldsmith et al., 1991). It is possible, however, that the C-terminal region may regulate the kinesin binding site. Binding of tau to both a and p from the mouse is also strongly influenced by polyglutamylation. The binding is relatively low for nonglutamylated tubulin and increases with the number of added glutamates, reaching a maximum with three glutamates; the binding then decreases when more glutamates are added (Boucher et af., 1994). The flagellar outer doublet MTs in mouse sperm are all polyglutamylated but three of them are more so than the others; these three lie on the plane of the flagellar wave. This suggests that the unequal polyglutamylation may play a role in the movement of the flagellum. In contrast, the degree of acetylation of flagellar MTs is the same for all the outer doublet MTs (Fouquet et al., 1996). The hypothesis that polyglutamylation plays a role in flagellar motility is supported by the observation that an antibody to polyglutamylated tubulin can decrease the amplitude but not the frequency

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of the flagellar wave (Gagnon et al., 1996). In neurons, it is possible that polyglutamylation of /31rImay play a role in differentiation (Falconer et al., 1994).

F. Polyglycylation A modification similar to polyglutamylation is polyglycylation, the addition of 2-40 glycines to a glutamate residue near the C terminus of either a or p. To date, this has only been observed in axonemal MTs. It has been seen in from bull sperm, in both a and p from sea urchin sperm and Paramecium cilia, and in a from Giardia lamblia (Redeker et al., 1994; M. Riidiger et al., 1995; Weber et al., 1996; Mary et al., 1996). Specific antibodies have been used to demonstrate the presence of the modification in sperm cells from humans, other mammals, birds, sea urchins, snails, and Drosophila, and in cilia from Tetrahymena and Stylonichia. Polyglycylation has not been observed in the ciliated Euglena (Bressac et al., 1995; Levilliers et al., 1995; BrC et al., 1996). The degree of modification is variable; there are 3-34 glycines added to both a and p in Paramecium, 2-23 to Giardia a, 13 to bull sperm p, and 12 and 11, respectively, to a and /3 from sea urchin sperm (M. Rudiger et al., 1995; Mary et al., 1996). As with polyglutamylation, polyglycylation is also a major modification, occurring in 60% of bull sperm fl (M. Riidiger et al., 1995). By analogy with Paramecium, the polyglycylated residue in bull sperm p would be G ~ (M.u Riidiger ~ ~ et al., ~ 1995). A single isoform can be both polyglycylated and polyglutamylated (Mary et al., 1996). The function of polyglycylation is not clear. One clue is seen in sea urchin sperm outer doublets; there is little polyglycylation in the Atubule, but both a and /3 are 40-45% polyglycylated in the B-tubule (Multigner et al., 1996). It is also interesting that the polyglycylated residue is in the axonemal signal sequence. Perhaps one function of that sequence is to provide a site for polyglycylation and to permit the unusual B-tubule to form. It is interesting that an antibody to polyglycylated tubulin inhibits sea urchin sperm motility (BrC et al., 1996).

G. Other Modifications

Tubulin is likely to be a very long-lived protein, particularly in neurons. It should not be surprising, therefore, that it becomes modified as it ages. For example, various asparagine or aspartate residues may isomerize to give isoaspartate (Najbauer et al., 1996). Also, lysinoalanine cross-links appear in neuronal tubulin (Correia et al., 1993). The physiological significance

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of these modifications is unclear but they could affect MT assembly and interactions with other proteins and hence compromise neuronal function.

H. Membrane Tubulin A review of tubulin isotypes needs to include membrane tubulin (Stephens, 1986; Niggli and Burger, 1987). This form of tubulin has not yet been sequenced so it is not clear whether this represents a form of tubulin encoded by different genes or a posttranslationally modified form of tubulin. Membrane tubulin was described by Bhattacharyya and Wolff (1975) in bovine thyroid and rat brain and by Stephens (1981) in the gill cilia of the scallop Aequipecten irradians. Stephens found that the axonemal a- and P-tubulin from this organism have different peptide maps than do a- and P-tubulin from membrane tubulin. Membrane tubulin also appears to occur in bovine retinal rod outer segments and in tobacco plants (Matesic et al., 1992; Laporte et al., 1993). Membrane tubulin could be involved in binding of MTs to membranes (Bernier-Valentin et al., 1983). In the gill cilia of the scallop A. irrudians the membrane tubulin appears to form a reticular complex that is connected to the outer doublet MTs (Stephens et al., 1987). Membrane tubulin could be involved in transport of substances within the membrane, in tethering MTs to the membrane, or in signal transduction. Stephens (1986) also speculates that tubulin could recycle out of the membrane onto the tip of a growing axonemal or other nearby MT. To summarize, there are three possible ways to account for membrane tubulin: (i) There may be an as yet undiscovered tubulin isotype that is hydrophobic and enters the membrane; (ii) a posttranslational modification, such as farnesylation, may anchor tubulin to the membrane. Such a modification could certainly be a novel one (tubulin is already known to undergo various novel modifications, so another one should not be surprising); and (iii) the tubulin molecule, which has unusual conformational properties, may undergo a conformational change that allows it to bind with high affinity to membrane phospholipids (Niggli and Burger, 1987).The isolation of membrane tubulin using detergents has recently been described (Stephens, 1995); therefore, it is likely that the structural basis of membrane tubulin will be understood in the near future.

IV. Concluding Remarks

It appears that many eukaryotes, particularly the multicellular ones, have multiple isotypes of a-and P-tubulin. The functional significance of these

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isotypes is being elucidated. It is likely that some isotopes have no functional significance whatsoever. Others may simply increase an organism’s adaptability without performing any specific function. It seems quite clear, however, that in some organisms certain isotypes are adapted to perform particular functions. This has been shown most elegantly in Drosophilu, in which the /3 isotype used in sperm flagella could not be functionally replaced by other p isotypes. Based on this and other work it seems that there is a very common, if not universal, signal in the sequence of certain p isotypes that is likely to be necessary for participation in axonemal MTs. If this was the original function of eukaryotic tubulin, then that function is still retained. It is possible that the function of tubulin isotypes is not always formation of a particular type of MT. Perhaps the isotype composition can determine the overrall chemical and physical properties of the MT including its nucleation, dynamic behavior, and susceptibility to posttranslational modification. For example, in a neuron, MTs containing aPIIrcould be phosphorylated, whereas others could not. If isotypes differ in their ability to nucleate MTs perhaps the presence of an isotype that nucleates readily could lead to formation of a large number of short MTs, whereas the absence of such an isotype would create fewer but longer MTs. One unanswered question is the nature of alp interactions. Given that higher eukaryotes have multiple a and p isotypes, do CY and p combine promiscuously or monogamously and does it matter? In other words, do particular (Y isotypes prefer to form dimers with certain p isotypes? For example, turkey erythrocytes contain a single a and a single p isotype (a1 and Pv1)but that does not necessarily imply any particular affinity between a1 and pvI.One could imagine that the developmental program for turkey erythrocytes simply includes these two isotypes for no particular reason. One could also imagine that both a1 and pvl have particular erythrocytespecific functions to perform but that they d o not actually need to be in the same dimer to perform these functions. In other words, perhaps in a turkey erythrocyte MTs containing both alpIIand a2Pv1dimers would function equally well as do the actual MTs composed of alpvIdimers. At the other extreme, if we assume that the alp interactions are stable in vivo, then we could imagine that by combining different a with different isotypes we could generate a large variety of dimers, each of them with slightly different functional properties. Isotype sorting at the subcellular level is still a mystery. Given that very different tubulins, including different isotypes, can copolymerize in vitro it is intriguing that they do not always do so in vivo as shown by the observation that vertebrate axonemal MTs are made of apIv.Also, given that there is an axonemal signal in the C-terminal region of PIv, what is it in the cell that recognizes that signal? Of course, axonemal MTs are extensions of the MTs of the basal body, an organelle indistinguishable from the centriole.

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We know that MTs made of aPI1,aPIII,and aPIvcan grow from sea urchin MTs axonemal MTs in vitro (Panda et al., 1994), so why do only appear to grow from basal bodies in vivo? It is unlikely to be the isotype composition of the basal body MTs that is the determining factor, so perhaps it is some other component of the basal body that binds specifically to aPrv. This in turn raises the question of the isotype compositions of basal bodies and centrioles, which are still unknown. Once isotype sorting occurs among different populations of MTs in the same cell, what keeps the isotypes from exchanging between these different MTs? One could argue that cilia and flagella are somewhat isolated organelles with little exchange with the rest of the cell, but there are several observations of sorting of different isotypes to different parts of the same neuron. Perhaps isotype segregation is simply a nonequilibrium phenomenon. That is, a cell forms MTs of a certain isotype for a particular temporary purpose and eventually the isotypes could exchange randomly throughout the cell. On the other hand, perhaps there are certain unknown mechanisms that “lock” the isotype into place in a particular population of MTs. The same arguments could apply to posttranslational modifications of MTs in the same cell. What is the mechanism by which detyrosinated MTs are associated with intermediate filaments while tyrosinated MTs are not? One loose end in our understanding of vertebrate isotypes is the role of Pv. Although Pv is widespread in the tissues of the chicken, its distribution in mammals is unknown. The fact that it is significantly different from the other isotypes (identity of 92 or 93% with PI, PII,PIII,and PIV)and at the same time conserved in evolution suggests that Pv may have some unique functional significance. At this point, however, aPv dimers have not been purified so we can only guess as to the function of Pv. Much work remains to be done on the mechanisms of the posttranslational modifications. The enzymes that carry out several of them, including polyglutamylation, polyglycylation, and formation of AZtubulin, have yet to be isolated and characterized. When the structures and regulatory mechanisms of these enzymes are known, we will probably be close to answering questions about the sorting of differently modified tubulins in the cell. The functions of the posttranslational modifications are still largely unknown. The fact that some of them can alter assembly properties or binding to MT-associated proteins in vitro (Boucher et al., 1994; Khan and Ludueiia, 1996), although potentially significant, may not actually be relevant in vivo, where the modifications may be responsible for much more subtle effects. In other words, interpretation of experiments in vitro is limited to the nature of the questions asked. Future research on tubulin isotypes could have some interesting and useful outcomes. There is already evidence that isotypes differ in their interactions with antitubulin drugs. Because the PlI1isotype has a highly restricted distribution but is expressed in tumors, a PIII-specific drug may

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be a potent antitumor agent. Nature has already provided us with some very useful drugs, such as taxol and colchicine, that discriminate among the isotypes. Unfortunately, these drugs discriminate the wrong way, interacting less well with a&I; however, the possibility exists of finding or designing drugs that do the opposite. Certainly, knowledge of tubulin’s threedimensional structure at high resolution will help to understand its interaction with drugs. Tubulin crystals will be required to determine this structure. To crystallize tubulin, it will be necessary to obtain a homogeneous preparation of tubulin. Isotypically purified tubulin may be very useful in this respect, particularly if it can be prepared without the posttranslational modifications that themselves introduce heterogeneity. Purified a& dimers have already been used to form tubulin sheets and to localize the taxol binding region in the three-dimensional structure of tubulin (Nogales et al., 1995). In summary, the original suggestions of Behnke and Forer (1967) and Fulton and Simpson (1976) were that different forms of tubulin could sort themselves into different organelles and mediate different functions. Much of the discussion in this review has focused on localization of isotypes into different organelles or different cells. Although little evidence is available, it is also possible that isotypes interact differently with different proteins; such differences could affect the distributions or functions of the isotypes. In addition to the much discussed motors and MT-associated proteins (for reviews, see Hyams and Lloyd, 1994; Kreis and Vale, 1993), recent work has suggested that many other proteins interact with tubulin, including chaperonins, histones, glycolytic enzymes, phosphoinositide 3-kinase, G proteins, and protein kinases. Some of the glycolytic enzymes even bind to a-tubulin in the C-terminal region where the a isotypes differ (Wang and Rasenick, 1991; Multigner et al., 1992; Volker and Knull, 1993; Volker et al., 1995; Kapeller et aL, 1995; Best et al., 1996; Tian et al., 1996; GarciaRocha et al., 1997). Exploration of isotype differences in these interactions may be a fruitful line of work for much time to come. Acknowledgments I am grateful to Dr. Asok Banerjee for his helpful comments and access to an unpublished manuscript. I thank Dr. Israr Khan, Dr. Larry Barnes, Dr. Asish Ray Chaudhuri, Rebekah Scotch, Consuelo Walss, and Patricia Schwarz for their critical reading of the manuscript. This work was supported by Grant CA26376 from the NIH and Grant AQ-0726 from the Welch Foundation.

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Translocation of Proteins across the Endoplasmic Reticulum Membrane Jeffrey L. Brodsky Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Secretory protein biogenesis begins with the insertion of a preprotein into the lumen of the endoplasmic reticulum (ER). This insertion event, known as ER protein translocation, can occur either posttranslationally, in which the preprotein is completely synthesized on cytosolic ribosomes before being translocated, or cotranslationally, in which membraneassociated ribosomes direct the nascent polypeptide chain into the ER concomitant with polypeptide elongation. In either case, preproteins are targeted to the ER membrane through specific interactions with cytosolic andlor ER membrane factors. The preprotein is then transferred to a multiprotein translocation machine in the ER membrane that includes a pore through which the preprotein passes into the ER lumen. The energy required to drive protein translocation may derive either from the coupling of translation to translocation (during cotranslational translocation) or from ER lumenal molecular chaperones that may harness the preprotein or regulate the translocation machinery (during posttranslationaltranslocation). KEY WORDS: Protein translocation, Endoplasmic reticulum, Molecular chaperones, Signal recognition particle, Sec61p, BiP, Hsp70.

1. Introduction Efficient biochemical reactions occur in vivo because the cell sequesters nutrients and enzymes at high concentrations. This sequestration is made possible because the outer membrane of the cell is selectively permeable to nutrients and impermeable to large molecules, such as proteins. However, because cells must secrete proteinaceous hormones and enzymes required for growth and intercellular communication, the barrier imposed by the cell membrane presents a problem. Thus, a mechanism exists to selectively Inrernalional Review of Cyrology, Vol. 178

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synthesize, process, and sort proteins that are either exported from the cell or that reside in cellular membranes. The organelles and protein machines required for this process constitute the secretory pathway. The biogenesis of secretory proteins begins with their insertion, or translocation, into either the lumen of the endoplasmic reticulum (ER) or into the lipid bilayer of the ER membrane. Consequently, protein translocation is the first committed step in the secretory pathway. Secreted proteins subsequently travel in vesicles that migrate sequentially from the E R to the Golgi complex and finally to the plasma membrane, at which time they may be released from or retained in the plasma membrane. The secretory pathway also transports soluble and membrane proteins that may be withheld in the E R or Golgi complex. Early models for protein translocation hypothesized either that preproteins might spontaneously insert into and transit across lipid bilayers (von Heijne and Blomberg, 1979; Wickner, 1979, 1980; Inouye and Halegoua, 1980; Engleman and Steitz, 1981) or that translocation is engineered by a pore in the E R membrane (Blobel and Dobberstein, 1975). It is now clear that translocation requires not only a specific pore but also sophisticated protein machines in the cytosol, in the E R membrane, and in the lumen of the ER. Protein translocation can be divided schematically into three steps: (i) recognition and targeting of a preprotein to the E R membrane, (ii) insertion of the preprotein into the translocation pore in the ER membrane, and (iii) energy-dependent import of the preprotein into the E R lumen. Although protein processing in the ER may occur concomitantly with translocation, proteins attain their final conformations only after translocation is complete. Thus, the biogenesis of secretory proteins is tightly coupled to translocation and may be regulated by the translocation machinery. This review will first summarize much of the current knowledge about the factors comprising the translocation machines that direct preprotein targeting, membrane insertion, and import into the ER. The genetic and biochemical techniques that uncovered the molecules required for translocation and pertinent questions that remain in this field will be detailed. Particular attention will be placed on the knowledge gained from experiments conducted with the simple eucaryote, Saccharomyces cerevisiae or “baker’s yeast.” Finally, models that currently best describe how the translocation machine functions and how energy might be used to drive proteins into the E R will be discussed. This review will not attempt to summarize the current knowledge on posttranslocational events, such as protein folding and modification, because excellent reviews on these topics are available (Gething and Sambrook, 1992; Hartl, 1996).

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II. Signal Peptides Proteins targeted to the secretory pathway almost always contain an aminoterminal extension known as a signal peptide or signal sequence (Fig. 1A). Signal peptides are required to direct preproteins to the ER translocation machine because they interact directly with components of the machinery. They also specifically interact with a cytosolic protein complex known as the signal recognition particle (SRP; Walter and Johnson, 1994) and may spontaneously insert into lipid bilayers (Shinnar and Kaiser, 1984; Briggs et af., 1985; Batenburg et af., 1988; Killian et af., 1990; Hoyt and Gierasch, 1991; McKnight et af., 1991; Martoglio et af., 1995). The preprotein forms a membrane-inserted hairpin loop, leaving the amino terminus of the signal peptide in the cytosol (Fig. 1B; Inukai and Inouye, 1983; Kuhn, 1987; Shaw et af., 1988; Mothes et al., 1994). Signal peptides contain a tripartite structure consisting of 1-5 amino acids at their extreme amino terminus displaying a net positive charge, a central hydrophobic core containing from 7 to 15 amino acids, and conclude with a more polar region composed of 3-7 amino acids (Fig. 1A; Gierasch,

A signal sequence

++

hydrophobic core

mature region

polar domain

t

cleavage site

cytosol I /

ER lumen FIG. 1 The domain structure and ER membrane-inserted topology of the signal sequence. See text for details.

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1989; von Heijne, 1990; Izard and Kendall, 1994). This final polar region contains the recognition site for signal peptidase, the ER lumenal enzyme that catalyzes the cleavage of the signal peptide from the preprotein (Evans et al., 1986; Gilmore, 1991; Rapoport et al., 1996). Signal peptidase acts as the signal peptide emerges from the translocation channel on the lumenal side of the ER. The cleavage site recognized by signal peptidase is defined by amino acids immediately following the hydrophobic core with small side chains residing at positions -1 and -3 relative to the cleavage site (von Heijne, 1990; Izard and Kendall, 1994). The variability in the length and amino acid composition of signal peptides was elegantly detailed using a genetic selection to differentiate functional from aberrant signal sequences (Kaiser et al., 1987). Yeast grown in media lacking glucose express the secreted form of invertase, an enzyme that hydrolyzes sucrose and contains a cleavable signal sequence. A plasmid was then constructed in which nucleotides encoding the endogenous invertase signal sequence were replaced by random fragments of human DNA. Only plasmids containing a functional signal peptide fused to invertase allowed cells to secrete the enzyme and grow on sucrose. DNA sequencing of 23 of the plasmids residing in the transformants indicated that the functional signal peptides ranged from 17 to 52 amino acids and often contained charged residues interspersed throughout the sequence. One of the few characteristics of these signal peptides was that hydrophobic residues were enriched -2.6-fold and that charged residues were depleted -2.8-fold relative to sequences unable to support invertase secretion (Kaiser et af., 1987). This result corroborated a previous statistical analysis in which the most important characteristic of a signal peptide was shown to be its overall hydrophobicity (von Heijne, 1985). A surprising conclusion from this study was that -20% of the random sequences functioned as bona fide signal peptides, suggesting that the machinery recognizing signal sequences is highly promiscuous. Signal peptides exist predominantly in solution as random coils, sometimes containing some p-sheet content, but adopt an a-helical conformation in nonpolar solvents or when presented with a hydrophobic surface (Gierasch, 1989; Izard and Kendall, 1994). Because signal peptides contain a hydrophobic core and have been shown to implant into membrane-like surfaces (Shinnar and Kaiser, 1984; Briggs et al., 1985; Batenburg et al., 1988; Killian et al., 1990; Hoyt and Gierasch, 1991; McKnight et al., 1991; Martoglio et af., 1995), signal peptides were suggested to initiate import of proteins across lipid bilayers, bypassing the requirement for a membrane-associated translocation machine (von Heijne and Blomberg, 1979; Inouye and Halehoua, 1980; Wickner, 1979, 1980; Engleman and Steitz, 1981).

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Although this model is appealing, biochemical experiments using ERderived microsomes indicate that signal peptides interact with many proteins during translocation. For example, signal peptides bind to the SRP soon after their synthesis (Walter and Lingappa, 1986; Walter and Johnson, 1994), an event that slows down protein translation and helps target the ribosome to the E R membrane, at which point translation reinitiates (see Section 111,AJ). The membrane environment in which the signal peptide resides was first probed by Gilmore and Blobel (1985), who showed that a short preprotein containing its signal peptide could be extracted from the E R membrane with an aqueous perturbant, an unlikely event if the signal peptide was embedded in the lipid bilayer. Crowley et al. (1993) then tagged the signal peptide of a preprotein with a fluorescent ligand and observed that the signal peptide exists exclusively in an aqueous environment, verifying the results of Gilmore and Blobel (1985) and suggesting that the signal peptide is contained within a hydrophilic channel. The proteins with which a signal peptide associate have been examined in a number of cross-linking experiments. Robinson et al. (1987) first determined that a signal peptide may contact an integral membrane protein in mammalian ER microsomes. Although one report indicated that the signal peptide from a mammalian preprotein can be cross-linked to both proteins and lipids (Martoglio et al., 1995), others demonstrated that signal peptides only cross-link to specific proteinaceous components of the translocation complex, including (i) a factor originally called signal sequence receptor (SSR) (Wiedmann et al., 1987; Krieg et al., 1989; Thrift et al., 1991) that also interacts with mature regions of preproteins and is consequently now known as translocon-associated protein (TRAP) (Hartmann et al., 1993); translocating chain-associating membrane protein (TRAM), a component required for the import of some preproteins into the mammalian E R (Gorlich et al., 1992a; Gorlich and Rapoport, 1993; Voigt et al., 1996) but whose precise role is mysterious; and (iii) the translocation channel in the E R membrane, Sec6lp, first identified genetically in yeast (Deshaies and Schekman, 1987; Stirling et al., 1992) and later apparent in the mammalian E R using chemical and photo-cross-linking techniques (High et al., 1991, 1993; Kellaris et al., 1991; Thrift et al., 1991; Gorlich et al., 1992b; Mothes et al., 1994; Jungnickel and Rapoport, 1995; Nicchitta et al., 1995). The interaction of the signal peptide with components of the translocation machinery might also regulate protein import into the ER. Protein translocation occurs either cotranslationally, in which the ribosome remains associated with the nascent chain while protein import proceeds, or posttranslationally, in which the completely synthesized preprotein is released from the ribosome and then translocates into the ER (see Fig. 2). Recent evidence suggests that different signal peptides may target preproteins to subcom-

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-6%-

ribosome

cytosol

ER lumen co-translational translocation

post-translational translocation FIG. 2 Preproteins can be translocated into the ER either co- or posttranslationally. During cotranslational translocation, the ribosome-preprotein complex is targeted to the ER membrane before translation proceeds and the nascent chain is driven directly into the ER lumen. During posttranslational translocation, the proprotein is completely synthesized on and released from a cytosolic ribosome before it is translocated.

plexes of the translocation machinery in yeast that are committed to either the co- or posttranslational pathways (Feldheim and Schekman, 1994;Panzner et af., 1995; Ng et af., 1996). It was also observed that the translocation complex itself can discriminate between a functional and nonfunctional signal peptide (Jungnickel and Rapoport, 1995), and that pure signal peptides open a channel in reconstituted bacterial membranes (Simon and Blobel, 1992). Together, these results demonstrate that signal peptides are not simply lipophilic molecules, but rather contact many proteinaceous components and play an active role during protein translocation into the ER.

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111. Preprotein Targeting to the ER A. Cotranslational Protein Translocation

1. Signal Recognition Particle a Identification of Signal Recognition Particle As indicated previously, signal peptides interact with the SRP to target the nascent polypeptideribosome complex to the E R membrane, at which time protein translocation initiates. The discovery of SRP derived from the early observation that salt-washed, dog pancreas microsomes were unable to translocate a wheat germ-translated secretory protein (Warren and Dobberstein, 1978). Because the salt-washed extract restored translocation in this in vitro system (Warren and Dobberstein, 1978), the extract was fractionated to purify the active component, SRP, an 11s complex containing six equimolar polypeptides with relative molecular masses of 72,68,54,19,14, and 9 kDa (Walter and Blobel, 1980), and a single 7s RNA (“7SL” or “SRP RNA”; Walter and Blobel, 1982). Pure SRP arrested the translation of a secreted but not a cytosolic protein in a membrane-free wheat germ extract and bound loosely to ribosomes translating the cytosolic protein but tightly to ribosomes expressing the secreted protein (Walter et al., 1981). Because translation arrest exposes only the amino-terminal signal peptide of the secreted protein to the cytosol, it was proposed that SRP recognizes signal peptides to bind selectively to ribosomes translating secretory proteins, inhibiting further protein synthesis (Walter and Blobel, 1981a). SRP-mediated translation arrest was alleviated when microsomes were added to the reaction (Walter and Blobel, 1981b), suggesting that a membrane-associated factor dislocates SRP from the ribosome so that translation and translocation may proceed. Later evidence indicated that during translation in reticulocyte lysate, SRP effects only a kinetic delay in translation and not arrest (Wolin and Walter, 1989), a discrepancy arising from the use of heterologous components (i.e., dog pancreas microsomes and wheat germ extract) in the in vitro assay. Regardless, it is now clear that SRP scans translating ribosomes for emerging signal peptides (Ogg and Walter, 1995), selectively retards the translation of secreted proteins, and targets the stalled ribosome-nascent chain complex to the membrane, where it interacts with a membrane component that releases SRP. Only after SRP is released does cotranslational protein translocation proceed (Gilmore and Blobel, 1983). b. MolecularDissection of SRP The secondary structure of the 7SL RNA in SRP is highly conserved between most organisms and contains four domains (Ullu et al., 1982; Poritz et al., 1988; Althoff et al., 1994). These

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domains include a double stem-loop structure whose composition varies and a central double-helical core that bifurcates into two separate stemloop domains. Interestingly, the secondary structure of the SRP RNA from the yeast S. cerevisiae appears quite different than that from human, plant, and even other yeast species (Felici et al., 1989; Hann and Walter, 1991; Althoff et al., 1994; Walter and Johnson, 1994). It has been proposed that the 7SL RNA provides the structural framework of SRP and may mediate the interaction of the particle with the ribosome (Walter and Johnson, 1994). The interaction of the polypeptides in SRP with 7SL RNA has been studied extensively (see below). To perform a structure-function analysis of SRP, the protein components in the particle have been biochemically dissected. It was first determined that SRP could be disassembled into subcomplexes and individual components using anion-exchange resins and by adjusting the salt and magnesium concentrations (Walter and Blobel, 1983; Siegel and Walter, 1985; Scoulica et al., 1987).The isolation of the genes encoding the polypeptides in mammalian SRP (Lingelbach et al., 1988; Bernstein et al., 1989; Romisch et al., 1989; Strub and Walter, 1990; Herz et al., 1990) permitted the expression of SRP subunits by in vitro transcription/translation reactions (Lingelbach et al., 1988; Romisch et al., 1989, 1990; Strub and Walter, 1990; Liitcke et al., 1993; Hauser et al., 1995). Thus, the activities of the individual components or subcomplexes could be assayed for specific SRP-dependent activities, namely, signal sequence binding (Siegel and Walter, 1988a; Zopf et al., 1990, 1993; High and Dobberstein, 1991; Lutcke et al., 1992; Hauser et al., 1995), membrane targeting (Siegel and Walter, 1988a), and translation arrest (Siegel and Walter, 1985, 1986; Strub and Walter, 1990). The interaction of SRP proteins with each other and with the 7SL RNA has also been investigated extensively by in vitro binding assays (Lingelbach et al., 1988; Romisch et al., 1990; Strub et al., 1991; Zopf et al., 1990; Liitcke et al., 1993; Hauser et al., 1995) and by nuclease and free radical protection experiments (Siegel and Walter, 1988b; Strub et al., 1991). The picture that emerges from these studies is that the 9 and 14 kDa subunits (SRP9 and SPR14, respectively) form a tight complex, as do the 68 (SRP68) and 72 kDa (SRP72) proteins (Siegel and Walter, 1988c; Walter and Johnson, 1994). The SRP9/SRP14 and SRP68/SRP72 complexes occupy unique positions on the 7SL RNA. In contrast, SRP54, the subunit of 54 kDa, and SRP19 (the 19 kDa subunit) exist as monomers and bind to distinct regions on the 7SL RNA adjacent to SRP68/SRP72. SRP19 is required, however, for SRP54 interaction with SRP RNA. Combining the results of the biochemical assays and mapping experiments described previously indicates that the following functional domains are present in SRP. The domain containing the SRP9/14 complex arrests translation, whereas the domain with the SRP68/72 subunits is required for

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membrane targeting. When a cytosolic extract is incubated with ribosomes presenting photoactivatable probes on signal peptides, only SRP54 crosslinks to the signal peptide (Krieg er al., 1986; Kurzchalia et al., 1986). Crosslinking between fragments of pure SRP54 and signal peptides (Zopf et al., 1990, 1993), or proteolytic cleavage of cross-linked products (High and Dobberstein, 1991; Lutcke et al., 1992), indicates that a specific domain in SRP54 recognizes signal peptides. This signal peptide-binding, methioninerich domain of SRP54, known as the M domain, has been proposed to form an amphipathic a-helix that might accommodate the hydrophobic core of signal peptides (Bernstein et al., 1989). It has recently been suggested that the SRP54-SRP RNA complex functions as the core machinery of SRP because this partial complex is sufficient to support the translocation of a translation-arrested preprotein (Hauser et al., 1995). That distinct regions of SRP fulfill such unique functional roles indicates that large conformational changes must be relayed across the particle for coordinated action. The ultimate structure-function dissection of SRP will certainly depend on X-ray crystallographic analyses of particle subdomains and the continued genetic dissection of yeast SRP (see below).

c. Role of GTP during SRP Action The cloning of the gene encoding SRP54 revealed the presence of a GTP binding site (Bernstein et al., 1989; Romisch et al., 1989), now known as the G domain. SRP54 containing a mutation in the G domain was defective for ER membrane and signal peptide binding (Bacher et al., 1996). To understand how GTP and signal peptide binding to SRP54 are coupled, Miller et al. (1993) examined the effect of signal peptides on SRP54-nucleotide complex formation. It was discovered that signal peptides inhibit GTP binding to SRP54 and stabilize SRP54 in a nucleotide-free state. Because the dissociation of a signal peptide from SRP54 required GTP binding (Connolly and Gilmore, 1989), Miller et al. (1993) proposed that an ER-associated protein might act as a guanine nucleotide loading protein, causing the release of a signal peptide to the translocation complex in the E R membrane. It was recently proposed that a component in the ribosome, and not an ER-associated factor, stimulates GTP binding to SRP (Bacher et al., 1996). Miller et al. (1993) may have failed to observe physiological GTP loading of SRP because pure signal sequences, instead of ribosome-tethered nascent chains, were used. A new model for the GTP/SRP cycle arising from the work of Bacher et al. (1996) is depicted in Fig. 3 (Rapoport et al., 1996). After the signal peptide is released, GTP hydrolysis frees SRP from the membrane-associated SRP receptor (Connolly et al., 1991), permitting SRP to recycle for another round of preprotein targeting. Because this GTPase activity of SRP54 is catalyzed by the SRP receptor (Miller et al., 1993; see Section IV,B,l), GTP is required both to trigger the release of a ligand

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FIG. 3 The SRP cycle, as first presented by Bacher ef al. (1996). SRP (@) bound to GDP interacts with the emerging preprotein and the ribosome catalyzes GTP/GDP exchange on SRP. The SRP-GTP-ribosome complex is then targeted to the ER membrane and SRP binds to the SRP receptor (SR; =), releasing the preprotein to the translocation complex and triggering GTP hydrolysis. The GDP-bound form of SRP is freed and may recycle.

(i.e., the signal peptide) and to recycle the complex when the reaction is complete. This cycle ensures the productive delivery of a nascent preprotein to the ER membrane. 2. Nascent Polypeptide Associated Complex

Is SRP the lone factor capable of targeting preproteins to the ER membrane? Although SRP positively selects preproteins containing signal peptides, there might be another complex that acts antagonistically to SRP or that recognizes preproteins that ultimately reside in the cytosol. Wiedmann

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and co-workers sought to answer these questions, prompted further by the quandary that if ribosomes have an inherent affinity for the E R membrane (Borgese et al., 1974), then why is SRP also required to usher ribosomes to the E R (Lauring et al., 1995a)? To identify novel preprotein-associated factors that might act before SRP, a photoactivatable cross-linking technique that previously led to the identification of SRP54 as the signal sequence-binding subunit of SRP (Krieg et al., 1986; Kurzchalia et al., 1986) was used, and it has been utilized extensively to identify proteins that interact with translating and translocating polypeptides (Martoglio and Dobberstein, 1996; Brunner, 1996). An mRNA encoding either a secreted or cytosolic polypeptide, but lacking a termination codon, is translated under conditions in which the available lysyl-tRNA contains a photoactivatable cross-linker conjugated to the E amino group of the lysine. Thus, any lysine present in the protein product can be cross-linked covalently to an adjacent chemical bond, and the absence of a stop codon precludes the release of the nascent chain from the ribosome. Wiedmann et al. (1994) discovered that short nascent chains of both secreted and cytosolic proteins cross-linked to a dimeric protein complex they called nascent polypeptide associated complex (NAC). NAC did not associate with proteins that were translated beyond a certain size, and the complex was extracted from ribosomes by salt, demonstrating that NAC was not composed of ribosomal proteins. As found previously (Krieg et al., 1986; Kurzchalia et al., 1986), nascent chains from secretory proteins were also cross-linked to SRP54. NAC was then purified based on its ability to cross-link to the nascent chain of a cytosolic protein (Wiedmann et al., 1994), revealing the NAC complex to be composed of novel gene products with molecular weights of 33 and 21 kDa. Nascent chain cross-linking studies in cytosolic extracts depleted for NAC exposed a most intriguing result: Cross-links between SRP54 and both secreted and cytosolic proteins were discovered. Furthermore, when NAC-depleted cytosol was assayed for preprotein translocation activity, it was observed that even SRP-bound cytosolic proteins could be imported into ER microsomes. When NAC was added back to the extract, cross-links between SRP54 and the cytosolic proteins were absent, and the aberrant translocation of the cytosolic protein was prevented. The conclusions from these studies were that (i) NAC and SRP compete for preproteins at early stages during their synthesis, and (ii) in the absence of NAC, SRP can bind to and target cytosolic proteins to the E R membrane for translocation (Neupert and Lill, 1994; Wickner, 1995; Lauring et al., 1995a). This suggests that the binding of SRP to the signal peptide is by default and occurs only if NAC has failed to interact first with the nascent chain of a secreted protein. How then are preproteins targeted to the E R membrane? Both Lauring et al. 1995b,c) and Jungnickel and Rapoport (1995) have now shown that

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in NAC-depleted extracts, the affinity of the ribosome for the protein translocation channel, Sec6lp, may be sufficient to initiate protein translocation; therefore, SRP-mediated translation arrest allows enough time for the ribosome to find Sec6lp in the E R membrane, suggesting that the SRP receptor may not be required for ribosome-nascent chain binding to the E R membrane but might serve only to recycle SRP. This hypothesis is controversial, however, because the identity of the ribosome receptor in the E R membrane and whether SRP still positively selects secreted preproteins for E R targeting in vivo remain unknown. It is also still far from clear what regulates the association of NAC with the ribosome, how NAC and SRP compete for preproteins, whether NAC interacts with all cytosolic proteins, and how the ribosome is targeted to the ER. It has been suggested that NAC might recruit molecular chaperones to a nascent protein to prevent aggregation and ensure efficient folding (Hartl, 1996). Because studies on NAC have been performed exclusively in in vitro systems in which the time allowed for E R targeting and translocation may be much greater than that encountered in vivo, other factors, including SRP and the SRP receptor, might be required for efficient targeting in the cell. Also, the formation of NAC-depleted extracts requires harsh salt washes that might expose components of the ribosome that do not normally have an affinity for the membrane. Future in vivo studies, perhaps with the yeast NAC family (Shi et al., 1995), will be critical to further define NAC’s role during the selection of preproteins for the cytosol or for the secretory pathway. What is clear, though, is that NAC is a newly identified component of the ER targeting machinery that interacts early during the synthesis of secreted proteins, shielding the polypeptide from solvent until it elongates to a critical length (-30 amino acids; Wang et al., 1995), at which time SRP displaces NAC and arrests translation.

3. Ribosome Receptor It has generally been assumed that a ribosome receptor resides in the E R membrane. This conclusion is based on the work of Borgese et al. (1974), who showed that an E R membrane protein mediates saturable, saltdependent ribosome binding in vitro. Later studies identified the E R membrane-associated ribophorins (Kriebich et al. 1978) as the putative ribosome receptor, a result later disputed from biochemical data (Hortsch et al., 1986; Savitz and Meyer, 1990). [Ribophorins were ultimately found to be components of the oligosaccharyl transferase (Kelleher et al., 1992), an enzyme complex that attaches the core glycosylation moiety to translocating nascent chains.] Nonetheless, the search for the “true” ribosome receptor continued in earnest because it has remained unknown (i) whether the affinity of SRP for the SRP receptor is sufficient to target and dock the

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ribosome at the ER membrane, and (ii) how and why the ribosome remains attached at the ER membrane once SRP has become dislodged during cotranslational translocation. In extracts depleted of NAC, the ability of ribosome-tethered cytosolic proteins to be translocated into the ER, albeit at a low efficiency (see above; Weidmann et al., 1994), and that of preproteins to be targeted and translocated in the absence of SRP (Lauring et al., 1995b,c; Jungnickel and Rapoport, 1995) have only further stimulated the drive to identify the ribosome receptor in the ER membrane. A strong candidate for this receptor was identified by Savitz and Meyer (1990), who assumed that such a receptor should contain a large cytosolic ribosome-binding domain. By gently proteolyzing ER-derived microsomes, these investigators identified a soluble 160-kDa protein fragment that inhibited ribosome binding in vitro. Proteoliposomes reconstituted with the fulllength pure protein, known as p180, supported ribosome binding and exhibited a Kd identical to that obtained using native microsomes (-1.5 X lo-* M; Borgese et al., 1974). Further evidence that p180 was the ribosome receptor derived from the observation that detergent-solubilized extracts specifically depleted of p180 were unable to cotranslationally translocate a preprotein or bind ribosomes when reconstituted into liposomes (Savitz and Meyer, 1993); however, readdition of pure p180 restored preprotein translocation and ribosome binding activity. Furthermore, anti-pl80 monoclonal antibodies inhibited translocation and ribosome binding (Savitz and Meyer, 1993). The isolation of the gene encoding p180 revealed that the protein contains an astounding 54 repeats of a basic 10-amino acid repeat as its amino terminus, giving p180 a predicted p l of -10 (Wanker et al., 1995). Finally, yeast microsomes prepared from a strain heterologously expressing p180 exhibited an increased capacity to bind ribosomes as long as the amino-terminal basic region remained intact (Wanker et al., 1995), supporting the conclusion that p180 is a ribosome receptor in the mammalian ER. Results contradicting the supposition that p180 is the receptor were simultaneously obtained by Nunnari et al. (1991), Collins and Gilmore (1991), and Sugano and colleagues (Tazawa et al., 1991; Ichimura et al., 1992). These groups either observed ribosome binding in reconstituted protein fractions lacking p180 (Nunnari et al., 1991; Collins and Gilmore, 1991) or identified a novel protein, p34, as the ribosome receptor (Tazawa et al., 1991; Ichimura et al., 1992). While the function of p180 was being debated in the literature, Rapoport and colleagues took a direct approach to purify the mammalian ribosome receptor. Gorlich et al. (1992b) fractionated detergent-solubilized microsomes to isolate E R membrane protein-ribosome complexes. Puromycininduced release of the ribosome revealed the presence of an integral E R membrane protein homologous to Sec6lp, the yeast translocation channel

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(Deshaies and Schekman, 1987; Stirling et al., 1992; Musch et al., 1992; Sanders et al., 1992). The mammalian Sec6lp is -50% identical to the yeast protein and, like the yeast homolog associates with translocating nascent chains (Gorlich et al., 1992b). The ribosome-Sec6lp complex was sensitive to high salt but was unaffected by the absence or presence of a nascent chain in this case. Purified Sec6lp bound to ribosomes with a Kd in the nanomolar range and selective proteolysis of Sec6lp abrogated ribosome binding (Kalies et al., 1994). The yeast Sec6lp homolog could also be purified as a ribosome-binding protein, although the stoichiometry of associated ribosomes was lower than that found with mammalian Sec6lp (Panzner et al., 1995). This result might stem from the fact that yeast posttranslationally translocate some preproteins (see Section 111,B). The identity of Sec6lp as a ribosome receptor was also consistent with the results of Crowley et al. (1993), who showed that as nascent chains translocate into an aqueous channel, the ribosome forms a tight seal with the E R membrane. Thus, during cotranslational protein translocation, the growing polypeptide chain may pass directly into the channel, Secblp, and then into the E R lumen. This model is most easily envisaged if the ribosome directly contacts Sec6lp. It is vital to state that there is likely to be more than one ribosome receptor in the E R membrane. This conclusion is based on the observation that many ribosomes studding the rough E R are removed by low concentrations of salt, whereas a second class of ribosomes remains stably bound (Wolin and Walter, 1993).The salt-resistant ribosomes may then be stripped by puromycin and higher concentrations of salt, conditions that displace ribosomes from Sec61p (Kalies et al., 1994). Also, ribosome binding to the E R membrane appears to take place in at least two stages, an initial one that is Sec6lp independent and a later one that requires Sec6lp (Murphy et al., 1997).

4. Does the Ribosome “Push” Preproteins into the ER Lumen? If the ribosome directly contacts the translocation channel, Sec6lp, during cotranslational protein translocation, then the ribosome might direct or push the preprotein into the lumen of the ER. Indeed, Gorlich and Rapoport (1993) provided strong evidence to support this model. Reconstituted proteoliposomes lacking lumenal components and containing only the SRP receptor and the Sec6lp complex (see Section IV,B) supported the cotranslational translocation of a preprotein, suggesting that the ribosome generated the force to import the preprotein. On the contrary, Nicchitta and Blobel (1993) showed that E R microsomes depleted of lumenal components were unable to complete the cotranslational translocation of a preprotein.

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However, readdition of “reticuloplasm,” or lumenal contents, restored translocation. Mutations in two E R lumenal factors also prevent the cotranslational translocation of preproteins into yeast microsomes (Brodsky et al., 1995). The simplest conclusion from these studies is that although the ribosome might be able to drive a preprotein into the E R lumen by coupling translation to translocation, full translocation activity probably requires specific proteins in the ER, most likely hps70 molecular chaperones. These chaperones might “gate” the translocation channel, Sec6lp, a requirement that might not be necessary in reconstituted systems (Gorlich and Rapoport, 1993). This point is discussed further in Section V.

6. Posttranslational Protein Translocation Preproteins can be translocated into the mammalian and yeast E R both during and after translation (Fig. 2). Although the vast majority of preproteins are translocated into the mammalian E R cotranslationally (Walter and Johnson, 1994), there are many examples of small peptides that may 4~ imported into mammalian microsomes posttranslationally (Watts et al., 1983; Zimmermann and Mollay, 1986; Schlenstedt and Zimmermann, 1987; Wiech et al., 1987; Schlenstedt et al., 1990, 1992; Klappa et al., 1991). These molecules translocate into the ER without the aid of SRP, most likely because they are too short to interact with SRP when they emerge from the ribosome (Walter and Blobel, 1981a). Furthermore, disruption of the genes encoding the SRP components in yeast is not lethal but results in the accumulation of some preproteins in the cytosol and a reduced rate of growth (Felici et al., 1989; Amaya and Nakano, 1991; Hann and Walter, 1991; Stirling and Hewitt, 1992;Brown eta/., 1994). Thus, the cotranslational translocation pathway represents only one route for protein import into the ER. Posttranslational translocation, the alternate route that handles both short peptides in mammals and has been shown to operate both in vivo and in vitro in yeast (Hansen et al., 1986; Rothblatt and Meyer, 1986; Waters and Blobel, 1986; Hansen and Walter, 1988; Rothblatt et al., 1989), is SRP independent and is described below. 1. The Protein Folding Problem

Folded proteins are unable to translocate across membranes (Brodsky and Schekman, 1994) most likely because the diameter of the translocation pore restricts the size of proteins that pass through it. Thus, it is imperative that preproteins are at least partially unfolded as they cross the E R membrane. Probably the most vital role that SRP plays during cotranslational translocation is to prevent a secreted protein from folding in the cytosol;

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by coupling translation to translocation, nascent proteins transit directly into the lumen of the ER and only then begin to fold (Gething and Sambrook, 1992). In contrast, posttranslationally translocated proteins are fully synthesized before being targeted to the ER and could fold in the cytosol (Fig. 2). Cytosolic molecular chaperones prevent premature protein folding, thereby facilitating the targeting of posttranslationally translocated proteins to the ER membrane. 2. Molecular Chaperones

Molecular chaperones are defined as molecules that prevent protein aggregation and may aid protein folding by maintaining polypeptides in productive folding pathways (Gething and Sambrook, 1992; Morimoto e? al., 1994; Hartl, 1996). Many chaperones were first identified as heat shock proteins (hsps), factors whose synthesis is induced by cellular stress, a condition in which proteins can denature in vivo and expose hydrophobic domains. To prevent denatured proteins from aggregating, molecular chaperones shield hydrophobic patches of amino acids from the solvent (Gething et al., 1995). Pelham (1986) first suggested that chaperones might also be required during protein translocation into the ER because hydrophobic domains are similarly exposed. As discussed previously, this problem is particularly relevant during posttranslational translocation. It is now apparent from both biochemical and genetic studies that cytosolic molecular chaperones homologous to the bacterial DnaK and DnaJ proteins are required for posttranslational translocation (Brodsky and Schekman, 1994). Although DnaK and DnaJ were first identified because they are required for phage A DNA replication, these factors are also necessary for bacterial growth and cell division at elevated temperatures (Georgopoulos et al., 1994), for protein translocation across the bacterial plasma membrane (Wild et al., 1992), and during protein folding and protein degradation (Hartl, 1996). Together, these functions define DnaK and DnaJ as molecular chaperones. a. In Vitro Analysis of Posttranslational Protein Translocation Assays for posttranslational protein translocation utilize a radiolabeled secretory protein that contains a cleavable signal peptide and/or a core glycosylation acceptor site, translocation-competent ER-derived microsomes, a source of metabolic energy, and cytosol. The appearance of an ER-processed precursor (i.e., signal peptide-cleaved and/or core glycosylated) by SDSPAGE indicates that translocation has occurred and may be verified by demonstrating that the ER-modified preprotein is protected from added protease, whereas untranslocated species are degraded. Such assays have been developed using both mammalian (Wiech and Zimmermann, 1993)

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and yeast microsomes (Hansen et al., 1986; Rothblatt and Meyer, 1986; Waters and Blobel, 1986).

b. Hsp7Os To determine which components in the cytosol are required for posttranslational translocation into mammalian microsomes, Zimmermann and colleagues (1988) showed that the import of a bacterial extracttranslated viral coat protein into dog pancreas microsomes was stimulated by mammalian 70-kDa heat shock proteins (hsp70s) and ATP. Hsp7Os bind and release peptides concomitant with ATP hydrolysis and thus prevent protein aggregation (Gething et al., 1995). To show that hsp70s are required to retain a precursor in an unfolded conformation before import, Klappa et al. (1991) demonstrated that another in vitro translated preprotein could be posttranslationally translocated in the absence of cytosolic factors when added to the import reaction directly from denaturant. Import of both substrates was SRP independent, demonstrating that posttranslational translocation into the mammalian E R does not require SRP (Zimmermann et al., 1988; Klappa et al., 1991). The requirement for hsp70s during posttranslational protein translocation has been studied more fully in yeast because both genetic and biochemical methods are readily available. To determine the effect of hsp70s on protein translocation in vivo, Deshaies et al. (1988) used a strain in which the genes encoding the major cytosolic hsp70s were disrupted but which was viable because one cytosolic hsp70, Ssalp, was encoded for on a galactoseregulated plasmid (Werner-Washburne et al., 1987). When cells were grown in glucose to prevent transcription of SSA1, the cellular levels of Ssalp fell, and ER-targeted preproteins accumulated in the cytosol. To demonstrate directly that cytosolic Ssalp is required for protein translocation, purified Ssalp was shown to restore translocation competence to a wheat germsynthesized preprotein (Chirico et al., 1988; Deshaies et al., 1988; Brodsky et al., 1993), a result made possible because the hsp70 in wheat germ lysate is unable to support preprotein translocation into yeast microsomes (Waters et al., 1986). It was also discovered that trace amounts of cytosol stimulated translocation, suggesting that another factor(s) in the cytosol might be required. Chirico et al. (1988) then showed that the Ssalp requirement for posttranslational protein translocation in vitro could be bypassed if a preprotein was denatured in urea before being added to the translocation reaction. Thus, cytosolic hsp70s facilitate protein translocation by retaining preproteins in a nonnative conformation, confirming the hypothesis presented previously by Pelham (1986). Because hsp70s sustain proteins in a nonnative conformation, it was anticipated that any hsp70 homolog might functionally substitute for Ssalp in the in vitro reaction. However, it was discovered that Ssalp, but neither a bacterial nor an ER lumenal hsp70, was able to support preprotein translo-

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cation (Brodsky et al., 1993), suggesting either that cytosolic hsp70s play a more elaborate role during posttranslational translocation or that they interact specificallywith other cytosolicfactors during import. In accordance with these results, Wiech et al. (1993) showed that a lumenal hsp70 could not substitute functionally for a cytosolic hsp70 when posttranslational translocation was measured in the presence of mammalian microsomes. Bush and Meyer (1996) have proposed that posttranslational translocation into the yeast ER may also proceed via an Ssalp- and SRP-independent mechanism.This conclusion is based on the observation that Ssalp-depleted extracts were translocation competent in vitro [contrary to those results presented previously (Deshaies er al., 1988;Chirico et al., 19881but defective for refolding a chemically denatured enzyme. The validity of this hypothesis awaits further proof, perhaps through the identification of mutants specifically defective for this putative pathway. c. Ydjlp The ATPase activity of the bacterial hsp70, DnaK, is enhanced

by the DnaJ molecular chaperone. DnaJ specifically stimulates the ATPase activity of DnaK while a third chaperone, GrpE, facilitates the exchange of ADP for ATP on DnaK (Liberek et al., 1991). Optimal protein folding and A phage DNA synthesis in vitro occurs only when all three proteins are present (Zylicz et al., 1989; Langer et al., 1992; Schroder et al., 1993; Hartl, 1996). Because small amounts of yeast cytosol were required for maximal Ssalp-mediated translocation activity (Chirico et al., 1988; Deshaies et al., 1988), it was suggested that a cytosolic GrpE and/or DnaJ homolog might also be required for posttranslational protein translocation. A cytosolic DnaJ homolog in yeast, Ydjlp, was discovered independently by Caplan and Douglas (1991) and Atencio and Yaffe (1992) and is -32% identical to DnaJ. The ATPase activity of Ssalp was stimulated by Ydjlp (Cyr et al., 1992; Ziegelhoffer et al., 1995), as observed previously for the bacterial DnaK and DnaJ proteins (Lieberek et al., 1991), and the ability of Ssalp to bind to a permanently unfolded polypeptide (Cyr el al., 1992) and to a yeast preprotein (Chirico, 1992) was abolished in the presence of Ydjlp and ATP. It has recently been shown that Ssalp forms large oligomers in the presence of Ydjlp and ATP (King et al., 1995), a result whose in vivo significance is mysterious. Together, these results demonstrate that a DnaK-DnaJ interacting pair, Ssalp and Ydjlp, exists in the yeast cytosol. To prove that Ydjlp is required for posttranslational translocation, Caplan et al. (1992a) created a temperature-sensitive ydjI mutant stain that accumulates an untranslocated ER-targeted preprotein in the cytosol at the nonpermissive temperature. Because Ydjlp is prenylated and resides on the cytoplasmic face of the ER membrane (Caplan and Douglas, 1991; Atencio and Yaffe, 1992; Caplan et al., 1992b), it is possible that Ssalp targets unfolded preproteins to the ER where, upon interaction with Ydjlp,

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the preprotein is released and translocates posttranslationally into the E R lumen. The inability of the ydjl temperature-sensitive protein to stimulate the ATPase activity of Ssalp (Caplan et af., 1992a), an event required for protein release (Cyr et af., 1992; Chirico, 1992), and the fact that many chaperones in the mammalian cytosol, including hsp70s, interact with nascent polypeptides during translation (Beckmann et al., 1990; Frydman et af., 1994; Frydman and Hartl, 1996; Rassow and Pfanner, 1996) favor this model. The inability of some DnaJ homologs to substitute fully for Ydjlp (Caplan and Douglas, 1991; Levy et af.,1995) and of other DnaK homologs to substitute for Ssalp (Brodsky et al., 1993; Cyr and Douglas, 1994) also support this model because the release of preproteins to the translocation machine should require the specific interaction of DnaK-DnaJ pairs. Genetic evidence for the specific interaction between Ssalp and Ydjlp was obtained by Becker et al. (1996). Because protein translocation into both yeast (Chirico et af., 1988) and mammalian microsomes (Nicchitta and Blobel, 1990; Nicchitta et al., 1991; Wiech et af., 1993) is inhibited at an early stage by a sulfhydryl-modifying reagent, N-ethylmaleimide (NEM), an obscure NEM-sensitive cytosolic factor was also suggested to be required for translocation. However, Liu et al. (1996) determined that nucleotide-free Ssalp is modified and inactivated by NEM. Nonetheless? an in vitro posttranslational translocation assay using pure cytosolic components and a preprotein has not been achieved. One possibility is that a eukaryotic GrpE homolog in the cytosol, which has not been identified, might be required.

C. Coordination between the Co- and Posttranslational Pathways in Vivo As discussed previously, disruption of the genes encoding the subunits of SRP in the yeast S. cerevisiae is not lethal (Felici et al., 1989; Amaya and Nakano, 1991; Hann and Walter, 1991; Stirling and Hewitt, 1992; Brown et af., 1994). O n the contrary, depletion of the corresponding gene products arrests cell growth and division (Hann and Walter, 1991; Ogg and Walter, 1995). It was suggested that yeast could adapt to the absence of the cotranslational pathway, but when the proteins required for this pathway were rapidly depleted, the cells were unable to adapt. One could imagine that the adaptive response might induce the synthesis of factors required for the posttranslational pathway. Indeed, this was shown by Arnold and Wittrup (1994), who observed that the steady-state levels of both Ydjlp and Ssalp increase during SRP54 depletion. This conclusion must be interpreted with caution, however, because the depletion of SRP54 and resulting accumula-

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tion of some preproteins in the cytosol could trigger a cellular stress response. Ogg et al. (1995) also obtained evidence consistent with the model that disruption of the cotranslational pathway activates the SRP-independent, or posttranslational, pathway in yeast. Mutations in SEC6.5, the gene encoding an SRPl9-related protein in yeast, destabilize yeast SRP (Hann et al., 1992; Stirling and Hewitt, 1992), but the sec6.5 translocation defect and temperature sensitivity could be suppressed by low levels of cycloheximide. Thus, if preproteins are translated more slowly, then the reduced levels of SRP in the sec65 cells may still be sufficient for translation arrest and/or targeting. Cycloheximide could not suppress the growth defects in strains containing a disruption in the SRP.54 or SEC6.5 genes (Ogg et al., 1995), suggesting that the SRP-independent pathway had already supplanted the SRP-dependent pathway. Interestingly, because another inhibitor of protein synthesis, anisomycin, does not suppress the temperature sensitivity or protein translocation defect in the sec6.5 strain, Ogg etal. (1995) suggested that SRP interacts with ribosomes only at a specific stage during protein synthesis, i.e., that stage at which cycloheximide, but not anisomycin, acts. The signal sequence dictates whether a preprotein translocates either co- or posttranslationally in yeast (Rothblatt et al., 1989; Feldheim and Schekman, 1994; Ng et al., 1996), and the translocation defects for a given preprotein vary depending on whether components in the co- or posttranslational translocation pathways are absent or mutated (Rothblatt et al., 1989; Hann and Walter, 1991; Feldheim and Schekman, 1994; Ogg and Walter, 1995; Ng et al., 1996). In vivo analyses of the co- and posttranslational pathways in yeast may be difficult to interpret because the accumulation of one class of preproteins might block preproteins using the alternate pathway if a common component in the translocation machinery is required. Such a result is evident when preprotein accumulation is measured in yeast containing a mutation in SEC61 (Rothblatt et al., 1989; Ng et al., 1996), the gene encoding the translocation channel (Stirling et al., 1992; Musch et al., 1992; Sanders et al., 1992). Here, defects in the import of both co- and posttranslationally translocated preproteins are evident, indicating that the pore handles preproteins delivered by both routes. One solution to this problem is to measure preprotein translocation in vitro in which the import of a single substrate is assayed. Although it is clear that yeast survive in the absence of SRP, only a few preproteins translocate posttranslationally into yeast microsomes (Rothblatt et al., 1987; Hansen and Walter, 1988; Brodsky et al., 1995). This result implies either that yeast SRP is not absolutely required for cotranslational translocation or that the in vitro assay might be lacking components for the efficient posttranslational translocation of some substrates. Although a functional assay for purified yeast SRP (Brown et al., 1994) has not yet been developed,

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yeast cytosol depleted for SRP cannot support the cotranslational translocation of a preprotein in vitro (Ng et al., 1996). Finally, it is still formally possible that yeast SRP plays a role distinct from that in the mammalian cytosol. In fact, an SRP54 homolog in the stroma of the plant chloroplast acts posttranslationally to direct a protein complex into the thylakoid membrane (Li et al., 1995). Such a role for SRP in yeast would certainly confound the interpretation of the data presented previously. Overall, more work is required on the genetics and biochemistry of yeast SRP in preprotein targeting, and this has recently begun in earnest (Brown et af., 1994; Ng et al., 1996).

IV. ER Membrane Translocation Machinery A. Yeast Translocation Complex 1. Genetic Selection for Translocation-DefectiveMutants

To isolate mutations in yeast that are defective for preprotein translocation into the ER, Schekman and colleagues (Deshaies and Schekman, 1987; Rothblatt et al., 1989; Corsi and Schekman, 1996) initiated a selection based on the previously successful isolation of secretion mutants in Escherichia coli (Oliver and Beckwith, 1981) that led to the identification of the membrane components of the bacterial translocation complex (Wickner et al., 1991). Yeast unable to grow in the absence of uracil (due to a mutation in the URA4 gene) were transformed with a plasmid encoding Ura4p fused behind the signal sequence of the yeast-mating pheromone precursor, prepro-a factor (ppaF; Fig. 4). Cells containing a functional translocation machine could import the ppaF-Ura4p fusion protein into the ER, which then becomes secreted to the media, leaving wild-type cells phenotypically ura-. On the other hand, if cells contained a partially defective translocation machine such that some of the fusion protein was unable to transit into the ER, the cells would be ura+ and could grow on media lacking uracil. Temperature-sensitive mutants were subsequently isolated and their corresponding wild-type genes were cloned. Because protein translocation is an essential process (Deshiaes and Schekman, 1987, 1989; Rothblatt et al., 1989; Sadler et al., 1989), this selection required that mutations in the translocation complex did not completely block preprotein import into the ER. As a result of this restriction, not every component in the translocation machine was isolated. 2. Characterization of Components in the Sec Complex The genes corresponding to three temperature-sensitive mutants isolated by Schekman and colleagues were designed SEC61, SEC62, and SEC63,

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A

B

wild type

see mutant

FIG. 4 The scheme by which the first translocation mutants were isolated in yeast. In wildtype cells (A), a signal sequence-tagged His4 protein will be directed into the lumen of the ER and secreted, leaving his4 mutant cells unable to grow when a substrate converted into histidine by Hislp is present (Deshaies and Schekman, 1987). If a partial defect is present in a constituent of the translocation machinery, however, the fusion protein will remain cytosolic and the cells will grow in the presence of the substrate (B).

each of which is essential (Deshaies and Schekman, 1987, 1989; Rothblatt et al., 1989; Sadler et al., 1989). Variations on this selection scheme, by using other prototrophic markers or membrane proteins fusions, either have yielded one of these genes (Toyn et al., 1988; Stirling et al., 1992; Ng etal., 1996) or have isolated new components in the translocation machinery (Green et al., 1992; Stirling et al., 1992). Multicopy suppressor analyses (Kurihara and Silver, 1993; Esnault et al., 1993) and biochemical methods

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(Deshaies et al., 1991; Feldheim et al., 1993; Feldheim and Schekman, 1994; Panzner et al., 1995) were also used to identify novel members of the yeast translocation complex. Chemical cross-linking and purification of the translocation complex from detergent-solubilized extracts indicated that many of these components physically associate with one another (Deshaies et al., 1991; Brodsky and Schekman, 1993; Esnault et al., 1994; Panzner et al., 1995), and specific pairs of the temperature-sensitive mutants were synthetically lethal (Rothblatt et al., 1989; Kurihara and Silver, 1993), a phenomenon in which the growth of a haploid cell containing two temperature-sensitive mutations in combination is more restrictive than in cells containing either one of the two mutations. In other cases, synthetic lethal interactions indicated that the respective gene products physically associate (Brodsky and Schekman, 1994). Overall, preprotein translocation in yeast requires a variety of integral membrane and membrane-associated factors that together comprise the translocation machine (Fig. 5; Brodsky and Schekman, 1994). This was most conclusively demonstrated by Panzner et al., (1995), who purified this complete Sec protein complex in toto from detergent-solubilized yeast microsomes and measured its translocation activity in vitro. In the initial attempt to identify which SEC gene product encodes the translocation pore, Miisch et al. (1992) and Sanders et al. (1992) incubated microsomes with preproteins that were only partially translocated and were thus assumed to be stuck in the pore. After cross-linking, the membranes were solubilized and specific Sec proteins were immunoprecipitated to determine which factor associated with the arrested precursor protein. Both groups discovered that Sec6lp was the translocation pore.

Cytosol Sec63p

BiPlKaRp

Lumen FIG. 5 The components of the yeast translocation complex. The DnaJ homologous region of Sec63p is highlighted. The cytosolic chaperones Ssalp and Ydjlp, although required for the translocation of some precursors, have not been isolated as components of the complex and are therefore absent.

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This result was somewhat anticipated because Sec6lp is an integral membrane protein containing 10 membrane-spanning segments (Stirling et al., 1992; Rapoport, 1992; Wilkinson et al., 1996) and thus has the capacity to form an aqueous pore in the membrane. Homologs of yeast Sec6lp also function as translocation pores in the mammalian ER and in the bacterial plasma membrane (Rapoport, 1992; Rapoport et al., 1996), and two small proteins associated with Sec6lp (Sbhlp and Ssslp), whose functions are unknown, are conserved from yeast to mammals (Hartmann et al., 1994; Panzner et al., 1995). The gene encoding Ssslp was first isolated as a multicopy suppressor of a sec6Z temperature-sensitive mutant, and a cross-linked complex between Sec6lp and Ssslp was observed (Esnault et al., 1993, 1994). Also, the mammalian Ssslp homolog can functionally replace SSSZ in yeast (Hartmann et al., 1994). These results demonstrate that the Sec6lp complex, containing Sec6lp, Sbhlp, and Ssslp, has been conserved, as might be expected for an essential component of the translocation machine. Interestingly, the yeast ER contains homologs of Sbhlp and Sec6lp known respectively as Sbh2p and Sshlp (Finke et al., 1996). Although neither Sbh2p nor Sshlp are essential, strains deleted for both SBHZ and SBH2 exhibit restricted growth at elevated temperatures and accumulate some untranslocated secretory proteins, and strains containing both a temperature-sensitive sec61 mutation and deleted for SSHZ show synthetic effects. It has been proposed that Sshlp may exclusively mediate cotranslational translocation because it fails to associate with the Sec62p/63pcomplex (see below) but binds ribosomes (Finke et al., 1996). In the absence of ATP, a condition under which preproteins bind to the ER membrane but do not translocate (Sanz and Meyer, 1989), an arrested preprotein also cross-linked to Sec62p (Miisch et al., 1992; Lyman and Schekman, 1997), suggesting that Sec62p may be a signal peptide-binding protein. Interestingly, microsomes prepared from sec62 strains exhibited posttranslational but not cotranslational translocation defects (Deshaies et al., 1989), indicating that if Sec62p does recognize signal peptides, it may only associate with preproteins that do not have SRP bound to their signal sequences. Sec62p traverses the membrane twice with both the amino and carboxy termini facing the cytosol (Deshaies and Schekman, 1990). Although a Drosophila homolog of Sec62p has recently been isolated that is able to rescue the sec62 temperature-sensitive defect, a mammalian homolog has not been identified (Noel and Cartwright, 1994). Because pairwise temperature-sensitive mutations in SEC6Z, SEC62, and SEC63 are synthetically lethal (Rothblatt et al., 1989), and because SEC63 is a multicopy suppressor of a toxic Sec62p-invertase chimera (Deshaies and Schekman, 1990), it was anticipated that Sec6lp, Sec62p, and Sec63p form a complex in the ER membrane. This hypothesis was confirmed by Deshaies et al. (1991) by chemically cross-linking Triton X-100-solubilized

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microsomal proteins and adding antibodies to either Sec62p or Sec63p. Two novel proteins coimmunoprecipitated with the resulting Sec61p/Sec62p/ Sec63p complex and are now know as Sec7lp and Sec72p (see below). The predicted amino acid sequence and topology of Sec63p indicated that a lumenal domain of the protein was homologous to the DnaJ molecular chaperone from E. cofi (Sadler et af., 1989; Feldheim et af., 1992). Because DnaJ forms a functional complex with the DnaK chaperone (see Section III,B,2), a DnaK homolog in the yeast E R was expected to interact with Sec63p. A candidate for this interacting factor is BiP (heavy chain binding protein). BiP, a lumenal DnaWhsc70 homolog, was first discovered because it binds to incompletely folded proteins in the mammalian E R (Haas and Wabl, 1983). A yeast homolog of BiP, Kar2p, was later identified independently by two groups (Normington et af., 1989; Rose et al., 1989) and is -50% identical to DnaK and -65% identical to the cytosolic hsp70 in yeast, Ssalp (see Section III,B,2). A role for BiP during protein translocation in yeast was first demonstrated by Vogel et a f . (1990), who showed that strains containing temperature-sensitive mutations in kar2, the gene encoding yeast BiP, accumulate preproteins in the cytosol at the nonpermissive temperature. Cells depleted for BiP also accumulated preproteins (Vogel et al., 1990; Nguyen et af., 1991). Finally, microsomes prepared from strains containing these temperature-sensitive kar2 mutations display temperaturesensitive co- and posttranslational translocation defects in vitro (Sanders et af., 1992; Brodsky et af., 1995). Another hsc70 in the E R lumen, known as Lhslp/Ssilp/Cerlp, was also recently identified (Craven et al., 1996; Baxter et al., 1996; Hamilton and Flynn, 1996). Although the gene encoding this chaperone is not essential, cells deleted for the gene display some translocation defects and exhibit synthetic lethal interactions when combined with specific kar2 mutations. It is unknown, however, whether Lhslp/Ssilp/Cerlp’s action is directly or indirectly coupled to the translocation machinery. To demonstrate that Sec63p and BiP form a functional DnaK-DnaJ pair in the yeast ER, a Sec63p-BiP complex from octylglucoside-solubilized microsomes was purified that restored protein translocation activity to proteoliposomes prepared from a strain containing the sec63-1 mutation (Brodsky and Schekman, 1993). Scidmore et af.(1993) obtained genetic confirmation that Sec63p and BiP interact by determining that dominant suppressors of the sec63-I temperature-sensitive mutant map to kar2. Corsi and Schekman (1997) have shown that the Sec63p-BiP pair is maintained through the direct interaction between BiP and the DnaJ domain of Sec63p. Because the Sec63p-BiP complex was labile if purified in the presence of ATPyS (Brodsky and Schekman, 1993), and because Ydjlp, Ssalp, and ATP are necessary for protein translocation (Section III,B,2), ATP-

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dependent DnaK-DnaJ pairs exist on both sides of the ER membrane. A mechanistic model for the function of these chaperones during protein translocation is described in Section V,B. The Sec63p-BiP complex also contains two additional factors (Brodsky and Schekman, 1993) that were previously observed as cross-linked members of a translocation complex containing Sec6lp, Sec62p, and Sec63p (Deshaies et al., 1991). Disruptions in the genes encoding these proteins, known originally as SEC66 and SEC67 but now called SEC71 and SEC72 (Green et al., 1992), indicated that they were not essential for growth (Feldheim et al., 1993; Kurihara and Silver, 1993; Feldheim and Schekman, 1994;Fang and Green, 1994). Nonetheless, sec71 and sec72 strains accumulate some preproteins in the cytoplasm. Because the differing sensitivities of preproteins to the SEC72 mutation lie in their signal peptides (Feldheim and Schekman, 1994), it was suggested that Sec7lp and Sec72p might comprise the signal peptide recognition complex. Lyman and Schekman (1997) have now obtained data showing that Sec62p, Sec7lp, and Sec72p interact with preproteins in the absence of ATP, and that the disruption of this interaction can be dissolved by ATP and BiP. Thus, BiP and ATP are directly involved in the process by which signal peptides are released from this receptor complex and delivered to the translocation pore. These data are consistent with a model in which BiP, like G protein-coupled hormone receptors, might sense the presence of a ligand (i.e., the signal sequence) on the opposite face of the membrane and thus become activated to drive the next step in translocation-the transfer of the preprotein to and opening of the translocation pore (Brodsky, 1996). Recent evidence from Ng and Walter (1996) indicates that each member of the Sec63p-BiP complex (Sec63p, BiP, Sec7lp, and Sec72p) is required for nuclear membrane fusion, or karyogamy, during mating in yeast. This observation solves a long-standing puzzle regarding why BiP, a lumenal ER protein, is necessary for a nuclear membrane phenomenon (Rose et al., 1989).Because the nuclear envelope and the ER membrane are continuous, the Sec63p-BiP complex might be part of a “fusogen” machine that triggers homotypic nuclear-ER membrane fusion during mating in yeast (Latterich and Schekman, 1994). Consequently, this complex may not only be required for protein translocation but also might serve as a recognition motif that uses BiP-catalyzed ATP hydrolysis to fuse two nuclei during mating. An in vitro assay that measures nuclear envelope-ER membrane fusion was developed and will certainly facilitate a molecular description of this process (Latterich and Schekman, 1994). Although the components of the translocation complex have been defined, it is mysterious how they drive the insertion of a preprotein into Sec6lp, the translocation pore. Because it has been found that some subcomplexes of the translocation machine participate in only the co- or post-

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translational pathway (Deshaies and Schekman, 1989; Panzner et al., 1995; Ng et al., 1996), whereas other subcomplexes are required for both pathways (Brodsky et al., 1995), different preproteins might be targeted to a common translocation pore, Sec6lp, through unique components in the translocation machinery. To better define how these components in the translocation machine act, the protein translocation reaction has been reconstituted in vesicles prepared from detergent-solubilized yeast microsomes (Brodsky etal., 1993; Panzner et al., 1995). The isolation of subcomplexes of the translocation machine (Brodsky and Schekman, 1993;Panzner et al., 1995), in conjunction with assays that measure partial reactions during translocation (e.g., signal peptide binding or the association of a signal peptide with the translocation pore), will certainly facilitate the future dissection of this ornate protein complex.

B. Translocation Machine in the Mammalian ER Cotranslationally translocated preproteins interact with many E R components during their import into the mammalian ER. Although most of these factors were identified through cross-linking studies, others were isolated using biochemical complementation assays (Rapoport et al., 1996). Reconstituted vesicles obtained from detergent-solubilized mammalian microsomes have served to elucidate which of these molecules are unnecessary, required, or stimulatory for protein translocation in vitro (Nicchitta and Blobel, 1990; Nicchitta et al., 1991; Migliaccio et al., 1992; Gorlich and Rapoport, 1993). Although the isolation of the proteins and corresponding genes encoding these factors has been accomplished, many of their specific functions during translocation remain unknown.

1. SRP Receptor The SRP receptor (SR) is a heterodimeric protein complex consisting of a membrane-associated 69-kDa a subunit (SRa)and an integral membrane 30-kD p subunit (SRP; Tajima et al., 1986). Because proteolyzed microsomal membranes are unable to support cotranslational protein translocation and release SRP-mediated translation arrest in vitro (Walter et al., 1979), SR was first characterized by assaying for a membrane component that restored protein translation to arrested ribosomes. Gilmore and Blobel (1982a,b) discovered that this activity derived from either a membrane-associated factor or a 60-kDa soluble protein fragment obtained after partial proteolysis of microsomal membranes. Meyer et al. (1982) independently purified the 60-kDa fragment, to which antibodies were previously raised (Meyer et

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al., 1980), and determined that this polypeptide released the SRP-mediated translation block and ultimately derived from a -72-kDa membrane protein (Hortsch et al., 1985). The -72-kDa protein became known as either SR (SRP receptor; Gilmore et al., 1982b) or docking protein (Meyer et al., 1982) and is identical to SRa. To demonstrate that SRP and SR physically interact, Gilmore et al. (1982b) purified the a subunit of SR from detergent-solubilized membrane extracts using an SRP affinity column. Consistent with this result, Meyer et al. (1982) showed that antibodies against the a subunit prevented the SR-mediated release of translation arrest. Tajima et al. (1986) identified the P subunit of SR using SRP affinity column chromatography, the presence of which was previously obscured because a proteolytic fragment of SRa and an abundant E R membrane protein comigrated with SRP on denaturing polyacrylamide gels. The isolation of the genes encoding the a (Lauffer et al., 1985; Connolly and Gilmore, 1989) and P (Miller et al., 1995) subunits of SR revealed that they contain consensus sites for GTP binding. The purified SR subunits bind GTP in vitro (Miller et al., 1995), and a yeast SRa homolog has been isolated that is only 32% identical to the mammalian protein but whose sequence similarity is significantly higher in the conserved GTP-binding domain (Ogg et al., 1992). Mutations engineered into this site in mammalian SRa either impair or completely inactivate protein translocation and GTP binding (Rapiejko and Gilmore, 1992). Why SRP and both subunits of SR bind GTP is unknown, but it has recently been demonstrated that the bacterial SRP and SR homologs are able to reciprocally activate each other’s GTPase activities (Powers and Walter, 1995). One model (Miller et al., 1995) suggests that SRP stimulates the GTPase activity of SR, ensuring that only properly targeted preproteins become associated with the translocation machinery. Thus, the GTP/SRP cycle might increase the fidelity of translocation by introducing a timed, proofreading step, similar to the role that elongation factors and GTP play during protein translation (Thompson, 1988). 2. Translocon-Associated Protein After the release of the signal peptide from SRP, the preprotein interacts with the E R membrane-associated translocation machinery. Initial attempts to identify a signal peptide-binding protein in the E R membrane utilized the method described in Section 111,A,2.Wiedmann et al. (1987) synthesized a preprotein in the presence of SRP to arrest translation and selectively present a photoactivable cross-linker-bearing signal peptide to ER-derived mammalian microsomes. Photolysis-induced cross-linking yielded a preprotein-membrane protein complex. This membranous factor, a glycoprotein

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with a relative molecular mass of 34 kDa, was named SSR. Antibodies prepared against purified SSR inhibited protein translocation in an in uitro assay (Harmann et al., 1989), suggesting that SSR was essential for translocation. Surprisingly, Migliaccio et al. (1992) discovered that reconstituted vesicles lacking SSR were translocation competent, and it was found that both signal peptides and mature regions of preproteins cross-linked to SSR (Krieg et al., 1989; Gorlich et al., 1992b). These results demonstrated that the antibody inhibition observed by Hartmann et al. (1989) was probably due to a nonspecific antibody-mediated block of the translocation machinery, and that SSR may simply reside near the translocation machine. Similar observations were obtained for the ribophorins, components of the oligosacchary1transferase enzyme complex (Kelleher et al., 1992); antibodies against ribophorins also inhibited ribosome binding and protein import into microsomes (Yu et al., 1990), although the ribophorins are clearly not part of the translocation machinery. SSR is now known to be one subunit in a tetrameric complex that has been renamed TRAP (Hartmann et al., 1993). Although the role of TRAP during protein translocation in unknown, its proximity to preproteins suggests that it might play a role in the import of only specific proteins, that it might chaperone the insertion of membrane proteins into the lipid bilayer (Hartmann et al., 1993), or that it might be required for the retention of E R membrane proteins (Wada et al., 1991). 3. The Sec6lp Complex

Recent evidence suggests that the signal sequence of a preprotein interacts with Sec6lp, and not SSR/TRAP, after it is released from SRP (Rapoport et al., 1996). As discussed previously, Sec6lp is the translocation pore in the E R membrane identified initially from a genetic selection in yeast (Deshaies and Schekman, 1987; Stirling et al., 1992), functionally characterized using translocation-arrested precursor proteins (Milsch et al., 1992; Sanders et al., 1992), and later observed in the mammalian E R as a ribosome-associated membrane protein (Gorlich et al., 1992b). The mammalian Sec61 protein (also called Sec6la) copurifies with two additional proteins known as Sec61P (homologous to the yeast Sbhl protein) and Sec6l-y (homologous to the yeast Sssl protein) (Gorlich and Rapoport, 1993; Hartmann et al., 1994). Reconstituted vesicles containing only the mammalian Sec6lp complex and SR are able to cotranslationally translocate some preproteins (Gorlich and Rapoport, 1993; Voigt et al., 1996), emphasizing the central role of Sec6lp in the translocation reaction. Jungnickel and Rapoport (1995) determined that wild-type signal sequences bind tightly to Sec6lp in reconstituted vesicles, whereas translocation-defective

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mutated signal sequences can be extracted from the membrane with relatively low concentrations of salt, a result verified by Belin et al. (1996). Thus, Sec6lp binds to ribosomes, proofreads signal sequences, and opens to allow preprotein entry into the lumen of the ER. Because Secl6 plays an essential role at many stages of protein import, it was expected that preproteins would be found associated with it at all times during their translocation into the ER. Indeed, this is the case. Mothes et al. (1994) observed that Sec6lp contacts a soluble preprotein throughout its transit into the ER, although Nicchitta et al. (1995) observed that the efficiency of cross-linkingto Sec6lp at later stages during protein translocation was markedly reduced, a result these investigators interpreted as an indication that “topological alterations” exist between Sec6lp and the nascent chain. Such alterations may indicate that hydrophobic portions of the preprotein may be diffusing laterally from the Sec6lp channel and into the lipid bilayer (Martoglio et al., 1995). It is also clear that Sec6lp does not interact equally with each portion of the signal sequence (Martoglio and Dobberstein, 1996). The aminoterminal hydrophilic region of the signal peptide does not contact Sec6lp, whereas the central hydrophobic domain and the carboxy-terminal hydrophilic domains do associate with Sec6lp (High et al., 1993; Mothes et al., 1994). The central hydrophobic domain also contacts lipids and TRAM, another membrane protein in close proximity to a translocating polypeptide (see Section IV,B,4). The initial events during the cotranslational translocation of a preprotein are as follows (Rapoport et al., 1996): The interaction of the nascent chainribosome complex with the Sec6lp complex triggers the oligomerization of Sec6lp into a tetramer (Hanein et al., 1996), as observed using highresolution electron microscopic imaging. The signal peptide resides initially in an aqueous environment, occluded within Sec6lp (Crowley et al., 1994; Mothes et al., 1994). After SRP is displaced and translation resumes, the affinity of the ribosome for Sec6lp increases (Wolin and Walter, 1993; Jungnickel and Rapoport, 1995). When the nascent chain reaches a length of -70 amino acids, a gate opens in the translocation channel, allowing the preprotein to enter the lumen of the ER (Crowley et al., 1994), and the signal peptide is cleaved by the lumenal enzyme signal peptidase. The tight association between the ribosome and Sec6lp may be loosened, however, if translational pausing occurs (Hegde and Lingappa, 1996), an event common to preproteins containing “pause-transfer’’ sequences. The physiological relevance of these sequences might be to allow enough time for specific domains to fold in the lumen before translation proceeds. Nonetheless, a tight junction between the ribosome and Sec6lp is required during cotranslational translocation because cross-links between elongating nascent chains and Sec6lp diminish if the large and small subunits of the

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ribosome are dissociated (Nicchitta et al., 1995). Longer segments of the nascent chain extending into the lumen continue to contact Sec6lp (Mothes et al., 1994) as proteins in the E R begin to associate with the elongating nascent chain. The translocation of nascent chains containing transmembrane-spanning segments of integral membrane proteins presents a special problem because these domains must be transferred from Sec6lp or from a Sec6lp-lipid interface in which they initially reside (High et al., 1991; Martoglio et al., 1995; Do et al., 1996) to the lipid bilayer. The dissociation of the Sec6lp tetramer and/or action of other integral E R membrane proteins might facilitate this transfer, and it is thought that proteins translocate through a central 40-60A pore formed at the junction of the Sec6lp tetrameric complex (Hanein et al., 1996; Hamman et al., 1997). The small subunits associated with Sec6lp, Sec61P, and Sec6l-y (Sbhlp and Sssslp, respectively in yeast) have been proposed to facilitate membrane protein insertion (Hartmann et al., 1994). In fact, it has recently been shown that Sec61P also contacts transmembrane segments of translocating membrane proteins (Laird and High, 1997), and that transmembrane segments of preproteins pass into the lipid bilayer before translation is complete (Mothes et al., 1997). Although this process has only recently begun to be dissected, a molecular description of membrane protein translocation will certainly indicate that Sec6lp is unlike most ion channels or pores and is highly regulated and dynamic (Andrews and Johnson, 1996). 4. Translocating Chain-Associating Membrane Protein In addition to Sec6lp, a second mammalian E R membrane protein cross-links to translocating polypeptides and is required for the translocation of some preproteins (Walter, 1992; Rapoport et al., 1996). This factor, known as TRAM, is a 36-kDa polytopic glycoprotein that was purified by concanavalin A and S-sepharose column chromatography based on its ability, when reconstituted into phospholipid vesicles, to cross-link to the amino terminus of a preprotein (Gorlich et al., 1992a). Reconstituted vesicles depleted of TRAM were defective for the translocation of many, but not all, preproteins examined (Gorlich et al., 1992a; Voigt et al., 1996). Although TRAM-dependent translocation was dictated by a preprotein’s signal sequence, no single characteristic of this sequence was found to be critical for TRAMdependence (Voigt et al., 1996). The requirement for TRAM during the translocation of a subset of preproteins was elucidated by a series of in vitro cross-linking experiments using truncated preproteins and protease protection assays. Cross-links between TRAM and amino acids preceding the hydrophobic core of the signal sequence have been uncovered (High et al., 1993; Mothes et al., 1994). Because this region of the signal sequence is most likely the

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first encountered by the E R membrane, an early role for TRAM during protein translocation was suggested. Jungnickel and Rapoport (1995) showed, however, that TRAM was not required to discriminate between functional and aberrant signal sequences, indicating that TRAM was required after the initial E R targeting event. To further define the time at which TRAM acts during translocation, a protease protection assay was used by Voigt et al. (1996) to indicate at which step preproteins become shielded within the translocation complex. In the absence of TRAM, TRAM-dependent preproteins were trapped at a step identical to that at which preproteins containing nonfunctional signal sequences were found. Thus, TRAM is required once the preprotein has been received at the ER membrane but before it becomes inserted into Sec6lp. A critical event during this window of time is the insertion of the signal peptide as a loop, an event that precedes the import of the mature region of a preprotein and suggests that TRAM might serve to orient the signal sequence (Mothes er al., 1994). This hypothesis is supported by the observation that TRAM associates with the amino terminus of the signal peptide (High et al., 1993; Mothes et al., 1994). Therefore, once TRAM binds to and retains this domain on the cytosolic face of the E R membrane, the hydrophobic core of the signal peptide might spontaneously insert into the lipid bilayer or the Sec6lp-lipid interface (Martoglio et al., 1995) and form a hairpin, dragging the amino terminus of the mature portion of the preprotein into the translocation complex (Fig. 1B). However, the orientation of a membrane-spanning domain may also depend on the amino acid sequences adjacent to the transmembrane domain (Spies, 1995). A putative role for TRAM during membrane protein biogenesis was elegantly detailed in a study by Do et al. (1996) in which a photoreactive probe was engineered into the middle of a transmembrane domain. By varying the length of the translated portion of the preprotein following this transmembrane domain, its interaction with components in the translocation complex could be followed. Although Sec6lp contacted this domain for only a limited time during translocation, cross-links with TRAM were observed from the time that the transmembrane domain emerged from the ribosome until translation was complete (Do et al., 1996). Surprisingly, puromycin-induced disruption of cotranslational translocation inhibited the interaction of the membrane-spanning domain with SecGlp, but not always with TRAM. By varying the length of the preprotein and examining crosslinked complexes with TRAM in the presence and absence of puromycin, it was determined that the transmembrane domain interacted differentially with TRAM during translocation, suggesting that the TRAM-membranespanning segment complex is dynamic. One model is that TRAM associates with hydrophobic translocating segments and facilitates their insertion into the lipid bilayer, perhaps by altering the structure of Sec6lp. This scenario

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is consistent with the observation that TRAM might play a role in signal peptide topogenesis, and that signal peptides also interact with lipids, Sec6lp, and TRAM (Martoglio and Dobberstein, 1996; Johnson, 1997).

V. Lumenal Components Required for Protein Translocation

A. Co- and Posttrranslational Protein Translocation As discussed in Section III,A,4, preproteins can cotranslationally translocate into reconstituted vesicles devoid of lumenal contents (Gorlich and Rapoport, 1993; Tyedmers et al., 1996). The simplest interpretation of this result is that the energy required for cotranslational translocation derives exclusively from the ribosome “pushing” or feeding the preprotein through Sec6lp and into the lumen. By comparison, during posttranslational translocation, translation is uncoupled from translocation and another force must drive protein import. As a result of work on the mechanisms of posttranslational protein translocation into the yeast E R and mitochondria, the picture that has emerged is that the preprotein is driven into the organelle by compartmentalized factors (Brodsky, 1996). In the next sections, the roles that ATP and lumenal components play during protein translocation into the E R will be discussed in more detail.

6.BiP/Kar2p The requirement for hydrolyzable ATP during posttranslational protein translocation was apparent from studies in which this process was first assayed using ER-derived yeast microsomes and a radiolabeled preprotein (Hansen et al., 1986; Rothblatt and Meyer, 1986; Waters and Blobel, 1986). Although ATP was required for cytosolic hsp70s to retain preproteins in a translocation-competent conformation (see Section 111,B,2; Deshaies et al., 1988; Chirico et al., 1988), it was difficult to imagine how these cytosolic chaperones could drive preprotein import. The answer to this dilemma derived initially from the work of Rose and colleagues. Cells containing temperature-sensitive mutations in the BiP-encoding KAR2 gene were unable to grow at the nonpermissive temperature and accumulated untranslocated preproteins (see Section IV,A,2). Direct evidence that this lumenal hsp70 is essential during posttranslational protein translocation was obtained by Sanders et al. (1992), who found that BiP associated with translocating preproteins, and by Brodsky et al. (1993), who showed that BiP was

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specifically required for translocation in reconstituted vesicles. In addition, microsomes derived from kar2 mutant cells were defective for posttranslational protein translocation (Sanders et af., 1992; Brodsky et al., 1995), and BiP/Kar2p is required to complete translocation, most likely by pulling the preprotein into the lumen (Lyman and Schekman, 1995). Three models have been proposed to explain how BiP might drive posttranslational protein import into the ER lumen (Fig. 6). The first model depicts BiP as a reversible “glue” that prevents retrograde transport of the preprotein from the ER and to the cytosol (Fig. 6A; Simon et al., 1992). Such a role for lumenal hsp70s is required because the translocation channels in the mammalian ER (Garcia et al., 1988; Ooi and Weiss, 1992; Nicchitta and Blobel, 1993), the mitochondrial outer membrane (Ungermann et af., 1994), and the bacterial plasma membrane (Schiebel et af., 1991) appear to be passive once opened and because Sec6lp can reexport some polypeptides back to the cytosol if they are targeted for degradation (Wiertz et al., 1996; Brodsky and McCracken, 1997). Thus, a polypeptide chain might be able to slide bidirectionally across the membrane unless a lumenal factor “locks” onto the preprotein. Once such a factor, such as BiP, binds to the preprotein, retrograde transport is prevented, and only forward transport is allowed. Because Brownian or random thermal motion might serve as the ultimate source of this bidirectional movement, this model has been termed the “Brownian Ratchet” (Simon et af., 1992). Evidence supporting the Brownian Ratchet model has been obtained by Neupert and co-workers (e.g., Ungermann et al., 1994) from studies on the mechanism of protein import into the mitochondria. The second model depicts the hsp70 as a motor, grabbing and pulling the importing chain during successive rounds of ATP binding and hydrolysis (Fig. 6B; Glick, 1995). This model arose from the observation that during preprotein translocation into isolated mitochondria folded domains of proteins may unravel as they are pulled through the translocation channel (Glick, 1995), an event unlikely to occur spontaneously. Inherent in this “motor model” for hsp70 function is that to generate force, the hsp70 must be anchored to an immobile substrate, such as a membrane protein. Indeed, Sec63p serves this role for BiP (see Section IV,A,2; Brodsky and Schekman, 1993;Scidmore et af.,1993) and an inner membrane-associated factor known as Tim44 binds to the mitochondrial hsp70 and is required for preprotein translocation into this organelle (Schneider et af., 1994; Rassow et al., 1994; Kronidou et af., 1994). Although definitive proof for this model has been contested for mitochondrial translocation, Lyman and Schekman (1995) have observed that BiP is required to complete preprotein translocation into the yeast ER, an event that depends on the integrity of the Sec63p-BiP complex. Although this result suggests that BiP might actively grab and pull preproteins into the yeast ER, definitive proof for the motor model

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awaits a defined, in vitro system in which an hsp70-generated force may be observed. A final model (Fig. 6C) to explain the action of BiP during posttranslational protein translocation into the yeast E R envisions that BiP responds to the binding of a preprotein on the cytosolic face of the E R and then conformationally activates the translocation complex (Brodsky, 1996). This mechanism is analogous to the action of G protein-coupled receptors during hormone binding and subsequent activation of the G protein, a process in which the GTP-bound G a subunit acts as an effector. In this scenario, a preprotein binding to its receptor, composed of the Sec62, Sec71, and Sec72 proteins (Musch et al., 1992; Feldheim and Schekman, 1994; Lyman and Schekman, 1997), activates the neighboring Sec63 protein to stimulate ATP/ ADP exchange on BiP. This exchange would release BiP from the membrane (Brodsky and Schekman, 1993). BiP may then be available to bind to translocating precursor proteins and effect import, facilitate protein folding, or interact with another component of the translocation machinery. Because it has been proposed that a lumenal hsp70 might be the “gate” for the translocation channel (Crowley et al., 1994), releasing BiP from the translocation complex might open this gate and permit entry of a preprotein into the lumen. Scjlp, a soluble ER lumenal DnaJ homolog that interacts with BiP, might catalyze ATP hydrolysis on the membrane-released population of BiP (Schlenstedt et al., 1995). One prediction from this last model is that mutations in BiP may prevent early, cytosolic steps during the translocation of a preprotein. Thus, if BiP is unable to open the pore or trigger the release of a preprotein from the receptor complex to Sec6lp, then preproteins should be trapped at early stages of translocation. Three pieces of evidence support this hypothesis. First, in the absence of ATP, a preprotein does not interact with the translocation channel and remains associated with the receptor complex (Musch et al., 1992; Lyman and Schekman, 1995). Because BiP is the only known ATP-requiring protein in the translocation complex and ATP hydrolysis is required for BiP function (Brodsky et al., 1996), BiP might be necessary to facilitate the transfer of the preprotein to the channel, as data from Lyman and Schekman (1997) have suggested. Second Sanders et al. (1992) characterized mutations in the gene encoding BiP, KAR2, that prevented the association of a preprotein with Sec6lp, indicating that BiP must be required for early events during translocation that occur on the opposite face of the E R membrane. Zimmermann and colleagues (Dierks et al., 1996) have also obtained biochemical data consistent with this model. Third, mutations in BiP prevented the import of both a post- and cotranslationally translocated substrate into ER-derived microsomes (Brodsky et al., 1995). Because the energy required to drive cotranslational translocation might derive from the ribosome (Gorlich and Rapoport, 1993), BiP could be

Ratchet A

/---

I

Motor B

FIG, 6 Three models depicting how hsp70s might drive posttranslational protein translocation into the yeast ER (see text for details). (A) The molecular ratchet model (Simon ef al., 1992), (B) the force generating motor model (Glick, 1995). and ( C ) the ligand-coupled receptor model (Brodsky, 1996).

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C

ADP

ATP

FIG. 6 Continued

necessary to activate the translocation complex, a process that would be necessary for both co- and posttranslationally translocated preproteins.

C. A Role for BiP in Translocation into the Mammalian ER? Although mutations in BiP abrogated cotranslational translocation into yeast microsomes (Brodsky et af., 1995), Gorlich and Rapoport (1993) reported that cotranslational translocation into proteoliposomes containing pure mammalian proteins occurred in the absence of lumenal factors. One way to explain this discrepancy is that the yeast and mammalian translocation complexes have diverged. Another view holds that translocation into the mammalian ER is indeed stimulated by lumenal chaperones but that the translocation complex in the reconstituted system is already in an “activated,” translocation-component state, thus bypassing the requirement for additional factors. For example, specific detergents used during reconstitution could open the translocation

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pore, an event that might normally be initiated by lumenal chaperones (Crowley et af.,1994; Brodsky, 1996). Alternatively, the preproteins examined in such defined systems may not be representative of the full range of translocating substrates encountered by the ER in viva In support of these latter possibilities, Nicchitta and Blobel (1993) showed that mammalian microsomes emptied of their lumenal contents were translocation deficient, but that readdition of “reticuloplasm” restored preprotein translocation. Because these investigators demonstrated further that reticuloplasmfree vesicles were capable of binding preproteins and cleaving their signal sequences but were unable to fully import the preprotein into the lumen, the role of the lumenal factors must be to complete translocation (Nicchitta and Blobel, 1993). Although BiP was a major component in the reticuloplasm, definitive proof that BiP is necessary and sufficient for translocation in this system is lacking. Earlier reports from Freedman and colleagues (Bulleid and Freedman, 1988; Paver ef af., 1989) that microsomes depleted of protein disulfide isomerase (PDI), an abundant lumenal protein required for disulfide bond rearrangement, were translocation competent indicate that not every chaperone in the lumen plays the same role as BiP. Although BiP was also apparently depleted from these microsomes (Bulleid and Freedman, 1988; Paver et af., 1989),catalytic quantities of the protein might have remained in the depleted vesicles. In fact, Nicchitta has found that PDI is preferentially extracted before BiP from microsomes treated using a protocol similar to that performed by Freedman and co-workers (Nicchitta and Blobel, 1993; C. Nicchitta, personal communication). Overall, a resolution to the question of BiP’s role, if any, during preprotein translocation into the mammalian ER awaits studies examining a broader range of preproteins, alternative reconstituted systems, and partial import reactions that may better define when and how various members of the translocation machine act during preprotein import. Also, although the lack of a genetic analysis of translocation into the mammalian ER has hampered further progress on this front, dominant-negative mutants in mammalian BiP have been shown to cause aberrant ER phenotypes in tissue culture cells (Hendershot et af., 1995). Perhaps the design of site-directed, dominant mutations in the primary components of the mammalian translocation machine will provide enlightening results.

VI. Concluding Remarks Protein transport across biological membranes is energetically unfavorable. However, as described in this review, the membrane of the ER contains

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an elaborate machine that facilitates this process. Cytosolic and lumenal proteins also appear to be required during translocation. The isolation and characterization of the components involved in translocation has utilized techniques ranging from genetic analyses to biophysical methods, and studies in this field have further elucidated the roles of molecular chaperones, of GTP-binding proteins, of proteinaceous pores, and of multiprotein complexes. Undoubtedly, the continued characterization of protein translocation will not only lead to a molecular mechanism for this process but also likely elucidate the functions of a diverse spectrum of protein families.

Acknowledgments I thank Susie Lyman, Chris Nicchitta, and Karin Romisch for their patient and thorough review of this chapter and the National Science Foundation and American Cancer Society for supporting research on protein translocation in my laboratory.

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Wolin, S. L., and Walter, P. (1993). Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes. J . Cell Biol. 121, 121 1-1219. Yu, Y., Sabatini, D. D., and Kreibich, G. (1990). Antiribophorin antibodies inhibit targeting to the ER membrane of ribosomes containing nascent secretory polypeptides. J. Cell Biol. 111,1335-1342. Ziegelhoffer, T., Lopez-Buesa, P., and Craig, E. A. (1995). The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydjlp and peptide substrates. J. B i d . Chem. 270,10412-10419. Zimmermann, R., and Mollay, C. (1986). Import of honeybee prepromelitin into the endoplasmic reticulum. J. Biol. Chem. 261, 12889-12986. Zimmermann, R., Sagstetter, M., Lewis, M. J., and Pelham, H. R. B. (1988). Seventy-kilodalton heat shock proteins and an additional component from reticulocyte lysate stimulate import of M13 procoat protein into microsomes. E M B O J, 7, 2875-2880. Zopf, D., Bernstein, H. D., Johnson, A. E., and Walter, P. (1990). The methionine rich domain of the 54 kD protein subunit of the signal recognition particle contains an RNA binding site and can be crosslinked to a signal sequence. E M B O J 9,4511-4517. Zopf, D., Bernstein, H. D., and Walter, P. (1993). GTPase domain of the 54kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding. J. Cell Biol. 120, 1113-1121. Zylicz, M., Ang, D., Liberek, K.,and Georgopoulos, C. (1989). Initiation of A DNA replication with purified host and bacteriophage encoded proteins: The role of the dnaK, d n d , and grpE heat shock proteins. E M B O J. 8, 1601-1608.

A

Antigens autoantigens, see Autoantigens role in autoimmune diseases host antigens, release, 151-152 superantigens, 154-155 Antioxidants, in therapy for autoimmune diseases, 193-195 Apoplastic transport, via plasma membrane transporters biochemical studies, 49-51 low-affinity transport, 54 sucrose efflux into apoplast, 54 SUT1-mediated transport studies, 53-54 transporter gene identification, 51 transporter protein structure, 51-53 transporter regulation, 55-56 transporter targeting to plasma membrane, 54-55 Apoplasts, sucrose efflux into, 54 Apoptosis, T lymphocytes, 141-143 Arabidopsis, multiple tubulin genes, 216 Arabidopsis thaliana, companion cells, 44 Arthropods, multiple tubulin genes, 217-218 Aspergillus nidaluns, multiple tubulin genes, 215 Autoantigens immununological tolerance to, 185-186 T cell recognition chronicity and phenomenon of determinant spreading, 182 dominant and cryptic epitopes in autoantigens, 179-182 in human autoimmune disease, 182-184

Acetylation, tubulin, 247-248 Actin, interaction with lipid membranes actin-binding shuttle proteins, 91-94 actin capping proteins, 94-95 actin-lipid interactions, 84-85 actin nucleating proteins, 85-91 actin severing proteins, 95 annexin, 97-98 caldesmon, 97-98 focal contact proteins, 95-96 myosins, 97 phosphoinositide regulation, 98-99 synapsin, 97-98 Actin-associated proteins, interaction with lipid membranes actin-binding shuttle proteins, 91-94 actin capping proteins, 94-95 actin-lipid interactions, 84-85 actin nucleating proteins, 85-91 actin severing proteins, 95 annexin, 97-98 caldesmon, 97-98 focal contact proteins, 95-96 myosins, 97 phosphoinositide regulation, 98-99 synapsin, 97-98 a-Actinin, interaction with lipid membranes, 88-89 Ag+-NOR, numbers and distribution in chromosomes, 16-18 Algae, green, glucose uptake, 42 Amplification, oncogenes in cells with monosomies, 24-27 Annexin, interaction with lipid membranes, 97-98

329

330 Autoimmune diseases development, contributing factors autoimmunity and infection, 150-151 bystander damage, 153 host antigen release, 151-152 molecular mimicry, 153-154 superantigens, 154-155 effect of diet, 159-162 effect of neuroendocrine system, 163- 166 genetic factors, 149-150 role of stress proteins, 156-159 human, role of autoantigens, 182-184 immune mechanisms effector mechanisms in tissue destruction, 147-148 lymphocyte activation, role of costimulation, 135-138 lymphocyte effector functions, role of cytokines, 138-141 lymphocyte trafficking, 133-135 peripheral immunological tolerance, danger model, 143-147 T lymphocyte death and apoptosis, 141-143 intervention antagonist modulation of autoimmune response, 187-188 antioxidants in therapy, 193-195 cytokine modulation of autoimmune response, 188-191 future therapies, 195-198 gene therapy, 193 immunological tolerance to autoantigens, 185-186 therapy directed at T cell receptor, 191-192 models insulin-dependent diabetes mellitus, 174-176 multiple sclerosis models, 167-173 other models of autoimmunity, 176-179 spectrum, 128-131 Autoimmunity antagonist modulation, 187-188 cytokine modulation, 188-191 models, 176-179 organ specificity, 131-132 role in autoimmune diseases, 150-151 Autosomes retention in leukemic and tumor cells, 19-21 whole, and fragments, extracopying, 28

INDEX

B Binding constants, cytoskeleton-lipid interactions, 84 BiP protein role in mammalian ER translocation, 313-314 role in protein translocation, 309-313 Brugia, multiple tubulin genes, 217

C Caenorhabditis elegans, multiple tubulin genes, 217 Caldesmon, interaction with lipid membranes, 97-98 Capping proteins, interaction with lipid membranes, 94-95 Cell culture, karyotypic variability Ag+-NOR numbers and distribution, 16-18 chromosome changes, 13-16 chromosome set balance of leukemic and tumor cells, 18-19 loss of sex chromosome, 16 retention of disomy on autosomes, 19-21 Cell death, T lymphocytes, 141-143 Cell lines with autosomal monosomies oncogene amplification, 24-27 polyploidization on chromosomes, 22-24 whole autosome and fragment extracopying, 28 CHO, multiple tubulin genes, 219 karyotypic evolution, 9-13 purity and stability, 2-3 reconstructed karyotype, 9 Chaenocephalus aceratus, multiple tubulin genes, 222 Chaperones, molecular, role in posttranslational protein translocation heat shock protein 70, 293-294 in virro analysis, 292-293 Ydjlp protein, 294-295 Chromosomes Ag+-NOR numbers and distribution, 16-18 breakpoint localization, 6-9 metaphase identical marker, 4-6

331

INDEX

normal samples, preparation, 4 number, analysis, 4 preparation, 3 staining, 3-4 monosomy on, cell polyploidization, 22-24 numbers, normal and marker, 6 set balance, in leukemic and tumor cells, 18-19 sex, see Sex Chromosomes Colletrichum gloeosporiodes, multiple tubulin genes, 215 Companion cells, in phloem, 44 Computer, assisted structure predictions, cytoskeleton-lipids, 81 Cytogenetics, in analysis of cultured cells chromosome breakpoint localization, 6-9 chromosome number analysis, 4 chromosome sample preparation, 4 identical marker chromosomes, 4-6 karyotypic analysis of metaphase plates, 4 metaphase chromosome preparation, 3 metaphase chromosome staining, 3-4 normal and marker chromosome numbers, 6 reconstructed karyotype of cell lines, 9 Cytokines modulation of autoimmune response, 188-191 role in lymphocyte effector functions, 138-141 Cytoskeleton-lipid interactions binding constants, 84 computer-assisted structure predictions, 81 differential scanning calorimetry, 78 film balance technique, 78 hydrophobic photolabeling, 79-81 miscellaneous techniques, 84 neutron-reflection, 82-84

D Destabilization, karyotypic, factors, 2-3 Determinant spreading, chronicity and phenomenon, 182 Detyrosination-tyrosination cycle, tubulin, 248-249 Diabetes mellitus, insulin-dependent, models, 174-176

Diet, effect on autoimmune disease development, 159-162 Differential scanning calorimetry, in analysis of cytoskeleton-lipid interactions, 78 Disomy, retention on autosomes, 19-21 Dissection, molecular, signal recognition particle, 283-285 Drosophila hydei, multiple tubulin genes, 218 Drosophila melanogaster, multiple tubulin genes, 217 Dynamin I, interaction with lipid membranes, 105-107 Dynein, interaction with lipid membranes, 104-105

E Endoplasmic reticulum lumenal components for protein translocation BiP-Kar2p, 309-313 BiP role in mammalian ER translocation, 313-314 components for protein translocation, 309 membrane translocation machinery Sec6lp complex, 305-307 SRP receptor, 303-304 translocating chain-associated membrane protein, 307-309 translocon-associated protein, 304-305 yeast translocation complex, 297-303 preprotein targeting co- and posttranslational pathway coordination in vivo, 295-297 cotranslational translocation nascent polypeptide-associated complex, 286-288 ribosome receptor, 288-290 role of ribosome, 290-291 signal recognition particle, 283-286 posttranslational translocation molecular chaperones, 292-295 protein folding problem, 291-292 Epitopes, dominant and cryptic, in autoantigens, T cell recognition, 179-182 ER, see Endoplasmic reticulum

332

INDEX

Evolution, karyotypic, cell lines, 9-13 Ezrin, interaction with lipid membranes, 96

F Filamin, interaction with lipid membranes, 90-91 Film balance technique, in analysis of cytoskeleton-lipid interactions, 78 Fungi, multiple tubulin genes, 215 Fusarium moniliforme, multiple tubulin genes, 215

H Haemonchus, multiple tubulin genes, 217 Heat shock proteins hsp70, role in posttranslational protein translocation, 293-294 role in autoimmune disease, 157 Heliothis virescens, multiple tubulin genes, 218 Hisactophilin, interaction with lipid membranes, 92-93

I G Genes role in autoimmune disease, 149-150 in therapeutic approach to autoimmune diseases, 193 transporter, identification, 51 tubulin, multiple isotypes adaptive isotype with no specific function, 224-225 arthropods, 217-218 behavior in vitro, 229, 235-241 echinoderms, 218 evolution, 242-245 functional significance, 222-223 fungi, 215 isotype properties, 208-209 model of no functional significance, 223-224 molluscs, 217 nematodes, 217 plants, 215-216 protists, 209, 215 specific function, 225-228 structure-function correlations, 228-229 y-tubulin, 241-242 vertebrates, 218-222 Glucose, uptake in green algae, 42 Glycine, multiple tubulin genes, 216 Gobionorofhen gibberifrons, multiple tubulin genes, 222 GTP, role during signal recognition peptide action, 285-286

Immune mechanisms, in autoimmune diseases effector mechanisms in tissue destruction, 147-148 lymphocyte activation, role of costimulation, 135-138 lymphocyte effector functions, role of cytokines, 138-141 lymphocyte trafficking, 133-135 peripheral immunological tolerance, danger model, 143-147 T lymphocyte death and apoptosis, 141-143 Immunological tolerance to autoantigens, 185-186 peripheral, danger model, 143-147 Infection, role in autoimmune diseases, 150-151 Insulin-dependent diabetes mellitus, models, 174-176 Intermediate filament proteins, interaction with lipid membranes in situ interactions, 110-111 in vitro interactions. 107-110

K Kar2p protein, role in protein translocation, 309-313 Karyot ypes evolution, cell lines, 9-13 reconstructed, cell lines, 9 Karyotypic analysis, metaphase plates, 4

INDEX

333

Karyotypic variability, cells in culture Ag'-NOR numbers and distribution, 16-18 chromosome changes, nonrandom character, 13-16 chromosome set balance of leukemic and tumor cells, 18-19 loss of sex chromosome, 16 retention of disomy on autosomes, 19-21

L Leishmania mexicana, multiple tubulin genes, 215 Leukemia cells autosomes, retention of disomy, 19-21 with monosomy, oncogene amplification, 25-26 and tumor cells, chromosome set balance, 18-19 Lipid-cytoskeleton interactions binding constants, 84 computer-assisted structure predictions, 81 differential scanning calorimetry, 78 film balance technique, 78 hydrophobic photolabeling, 79-81 miscellaneous techniques, 84 neutron-reflection, 82-84 posttranslational modifications, 75-77 Lipid membranes, interactions with actin-binding shuttle proteins, 91-94 with actin capping proteins, 94-95 actin-lipid interactions, 84-85 with actin nucleating proteins, 85-91 with actin severing proteins, 95 with annexin, 97-98 with caldesmon, 97-98 with dynamin I, 105-107 with dynein, 104-105 with focal contact proteins, 95-96 with intermediate filament proteins in siru interactions, 110-111 in virro interactions, 107-110 with MAP-2, 102-104 with microtubules, 99-102 with myosins, 97 phosphoinositide regulation, 98-99 with synapsin, 97-98

with Tau protein, 102-104 with tubulin, 99-102 Lumen, ER components for protein translocation BiP-KaRp, 309-313 BiP role in mammalian ER translocation, 313-314 co- and posttranslational translocation, 309 ribosome role in preprotein targeting, 290-291 Lymphocytes, in autoimmune disease activation, role of costimulation, 135-138 effector functions, role of cytokines, 138-141 trafficking, 133-135

M MAP-2, interaction with lipid membranes, 102-104 MARCKS, interaction with lipid membranes, 93-94 Markers, chromosome chromosome breakpoint localization during formation, 6-9 numbers, 6 Membrane proteins, translocating chainassociated, 307-309 Membranes ER, translocation machinery Sec6lp complex, 305-307 SRP receptor, 303-304 translocating chain-associated membrane protein, 307-309 translocon-associated protein, 304-305 yeast translocation complex, 297-303 lipid, see Lipid membranes tubulin, 252 Metabolites, transport, sucrose as, 45-46 Metalloproteinase, role in effector mechanisms in tissue destruction, 147-148 Metaphase, chromosomes preparation, 3 staining, 3-4 Microtubules, interaction with lipid membranes, 99-102

INDEX

Models active phloem loading in plants, 59-61 autoimmune diseases insulin-dependent diabetes mellitus, 174-176 multiple sclerosis models, 167-173 other models of autoimmunity, 176-179 danger, peripheral immunological tolerance, 143-147 functional significance of tubulin isotypes adaptive isotype with no specific function, 224-225 no significance, 223-224 specific function, 225-228 Molluscs, multiple tubulin genes, 217 Monosomy, autosomal, affected cell lines oncogene amplification, 24-27 polyploidization on several chromosomes, 22-24 whole autosome and fragment extracopying, 28 Multiple sclerosis, models, 167-173 Myosin, interaction with lipid membranes, 97

N Naegleria gruberi, multiple tubulin genes, 215 Nascent polypeptide-associated complex, in preprotein targeting to ER, 286-288 Nematodes, multiple tubulin genes, 217 Neuroendocrine system, role in autoimmune disease development, 163-166 Neutron reflection, in analysis of cytoskeleton-lipid interactions, 82-84 Norothenia coriiceps, multiple tubulin genes, 222

0 Oncogenes, amplification in cells with monosomies, 24-27 Organs, specificity in autoimmunity, 131-132

P Paracentrotus lividus, multiple tubulin genes, 218 Patella, multiple tubulin genes, 217 Phloem active loading in plants, models, 59-61 anatomy and function, 43-45 SUTl localization, 56-59 velocity of translocation, 42-43 Phosphoinositides, regulation, 98-99 Phosphorylation, tubulin, 246-247 Photolabeling, hydrophobic, in analysis of cytoskeleton-lipid interactions, 79-81 Physarum polycephalum, multiple tubulin genes, 215 Plants active phloem loading, models, 59-61 long-distance transport in, 43-45 multiple tubulin genes, 215-216 velocity of translocation, 42-43 Plasma membrane transporters, role in apoplastic transport biochemical studies, 49-51 low-affinity transport, 54 sucrose efflux into apoplast, 54 SUT1-mediated transport studies, 53-54 transporter gene identification, 51 transporter protein structure, 51-53 transporter regulation, 55-56 transporter targeting to plasma membrane, 54-55 Plasmodesmata, role in symplastic transport, 46-49 Plasmodium fakiparum, multiple tubulin genes, 209,215 Plates, metaphase, karyotypic analysis, 4 Polyglutamylation, tubulin, 249-251 Polyglycylation, tubulin, 251 Polyploidization, cells with monosomy on chromosomes, 22-24 Ponticulin, interaction with lipid membranes, 88 Preprotein targeting, to ER co- and posttranslational pathway coordination in vivo, 295-297 cotranslational translocation nascent polypeptide-associated complex, 286-288 ribosome receptor, 288-290

INDEX

335

role of ribosome, 290-291 signal recognition particle, 283-286 posttranslational translocation molecular chaperones, 292-295 protein folding problem, 291-292 Profilin, interaction with lipid membranes, 91-92 Protein folding, in posttranslational protein translocation, 291-292 Protists, multiple tubulin genes, 215 Prunus amygdalus, multiple tubulin genes, 216

R Rat, multiple tubulin genes in brain, 219 Ribosome receptor, role in preprotein targeting to ER, 288-290 Ribosomes, role in preprotein targeting to ER, 290-291 RNA, messenger, SUT1, production location, 57

S Saccharomyces, multiple tubulin genes, 215 Schizosaccharomyces, multiple tubulin genes, 215 Sec6lp complex, role in translocation machine in mammalian ER, 305-307 Sec complex, components, characterization, 297-303 Severing proteins, interaction with lipid membranes, 95 Sex chromosomes, loss, 16 Sieve elements, in phloem, 44 Signal peptides, role in translocation, 279-282 Signal recognition particle identification, 283 molecular dissection, 283-285 role of GTP during action, 285-286 Signal recognition particle receptor, role in translocation machine in mammalian ER, 303-304 Spectrin, interaction with lipid membranes, 89-90 SRP, see Signal recognition particle

Stabilization, karyotypic destabilization, factors, 2-3 Staining, metaphase chromosomes, 3-4 Stress proteins, role in autoimmune disease, 156-159 Stronglyocentrotus purpuratus, multiple tubulin genes, 218 Sucrose efflux into apoplast, 54 as transport metabolite, 45-46 Sucrose transporters, SUTl functions in vivo, 61-63 localization in phloem, 56-59 mRNA, production, 57 transport mediated by, studies, 53-54 Sugar transport apoplastic transport via plasma membrane transporters biochemical studies, 49-51 efflux of sucrose into apoplast, 54 low-affinity transport, 54 SUT1-mediated transport, 53-54 transporter gene identification, 51 transporter protein structure, 51-53 transporter regulation, 55-56 transporter targeting to plasma membranes, 54-55 models for active phloem loading, 59-61 SUTl in vivo function, 61-63 SUTl localization in phloem, 56-59 symplastic transport via plasmodesmata, 46-49 Superantigens, role in autoimmune disease, 154-155 Symplastic transport, via plasmodesmata, 46-49 Synapsin, interaction with lipid membranes, 97-98

T Talin, interaction with lipid membranes, 85-88 Targeting, see Preprotein targeting Tau protein, interaction with lipid membranes, 102-104 T cell receptor, therapy directed at, 191-192

336 T cells death and apoptosis, 141-143 recognition of autoantigens chronicity and phenomenon of determinant spreading, 182 dominant and cryptic epitopes in autoantigens, 179-182 in human autoimmune disease, 182-1 84 Therapy, for autoimmune diseases antioxidants in, 193-195 directed at T cell receptor, 191-192 future therapies, 195-198 gene therapy, 193 Tissues, destruction, effector mechanisms, 147-148 T lymphocytes, see T cells Tolerance, see Immunological tolerance Torpedo marmorata, multiple tubulin genes, 222 Translation posttranslational lipid modifications, 75-77 posttranslational modifications, tubulin acetylation, 247-248 membrane tubulin, 252 other modifications, 251-252 phosphorylation, 246-247 polyglutamylation, 249-251 polyglycylation, 251 b2-tubulin. 249 tyrosination-detyrosination cycle, 248-249 Translocation cotranslational, in preprotein targeting to ER nascent polypeptide-associated complex, 286-288 ribosome receptor, 288-290 role of ribosome, 290-291 signal recognition particle, 283-286 ER membrane machinery Sec6lp complex, 305-307 SRP receptor, 303-304 translocating chain-associated membrane protein, 307-309 translocon-associated protein, 304-305 yeast translocation complex, 297-303 in preprotein targeting to ER co- and posttranslational pathway coordination in vivo, 295-297

INDEX

posttranslational translocation molecular chaperones, 292-295 protein folding problem, 291-292 role of ER lumenal components BiP-Kadp, 309-313 BiP role in mammalian ER translocation, 313-314 components for protein translocation, 309 role of signal peptides, 279-282 steps, 278 velocity in phloem, 42-43 Translocon-associated protein, role in translocation machine in mammalian ER, 304-305 Transport apoplastic, see Apoplastic transport long-distance, in plants, 43-45 sucrose as metabolite, 45-46 sugar, see Sugar transport symplastic, via plasmodesmata, 46-49 Transporters encoding genes, identification, 51 plasma membrane, see Plasma membrane transporters regulation, 55-56 structure, 51-53 sucrose, see Sucrose transporters targeting to plasma membrane, 54-55 Trichostrongylus, multiple tubulin genes, 217 Tubulin interactions, with lipid membranes, 99-102 isotype expression adaptive isotype with no specific function, 224-225 arthropods, 217-218 behavior in vitro, 229, 235-241 echinoderms, 218 evolution, 242-245 functional significance, 222-223 fungi, 215 model of no functional significance, 223-224 molluscs, 217 nematodes, 217 plants, 215-216 protists, 209, 215 specific function, 225-228

INDEX

structure-function correlations, 228-229 y-tubulin, 241-242 vertebrates, 218-222 posttranslational modifications acetylation, 247-248 membrane tubulin, 252 other modifications, 251-252 phosphorylation, 246-247 polyglutamylation, 249-251 polyglycylation, 251 A2-tubulin, 249 tyrosination-detyrosination cycle, 248-249 a-Tubulin, encoding gene as tubulin isotype adaptive isotype with no specific function, 224-225 behavior in vifro, 229, 235-241 evolution, 242-245 functional significance, 222-223 model of no functional significance, 223-224 properties, 208-209 specific function, 225-228 structure-function correlations, 228-229 0-Tubulin, encoding gene as tubulin isotype adaptive isotype with no specific function, 224-225 behavior in vifro, 229, 235-241 evolution, 242-245 functional significance, 222-223 model of no functional significance, 223-224 properties, 208-209 specific function, 225-228 structure-function correlations, 228-229 y-Tubulin evolution. 242-245 as tubulin isotype, 241-242 A2-Tubulin, as a-tubulin form, 249 Tumor cells

337 autosomes, retention of disomy, 19-21 chromosomes, Ag'-NOR numbers and distribution, 16-18 and leukemic, chromosome set balance, 18-19 with monosomy, oncogene amplification, 24-27 P19 carcinoma, multiple tubulin genes, 220-221 Tyrosination-detyrosination cycle, tubulin, 248-249

v Variability, see Karyotypic variability Vertebrates, multiple tubulin genes, 218-222 Vicia faba, companion cells, 44 Vinculin, interaction with lipid membranes, 95-96

X Xenopus laevis, multiple tubulin genes, 221

Y Ydjlp protein, role in posttranslational protein translocation, 294-295 Yeast, translocation complex genetic selection for mutants, 297 Sec complex component characterization, 297-303

z Zinnia elegans, multiple tubulin genes, 216

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