Abstract
Surface polypeptide components of the flagellar membrane of Chlamydomonas reinhardi Dang. gametes are identified by their accessibility to in-vivo vectoral labeling by glucose oxidase-coupled lactoperoxidase-dependent 125I iodination. Vectoral labeling is accomplished without observable adverse effects on cell viability or gametic function. Flagella isolated from labeled wild-type cells carry about 3% of the total incorporated label, which is found by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be distributed among 16 identifiable polypeptide bands. The most prominent surface-labeled species migrates in the Mr (relative molecular weight) 350 k region of the gel; each of the remaining iodinated polypeptides, which range in Mr from 25 k to 500 k, carries only a small proportion of incorporated label. To determine which polypeptides are unique to the flagellum and which are contaminants from the cell wall, wild-type profiles were compared with those of mutant strains and of mechanically isolated cell walls. Identification of contaminants was also facilitated by two-dimensional peptide mapping. We conclude that only 11 of the labeled bands are contributed by flagellar polypeptides; the remaining five bands are shown to be contaminants from the cell wall, and additional cell-wall polypeptides are found to co-migrate with flagellar species. A polypeptide designated as a possible membrane tubulin in preliminary studies is shown here to be different from tubulin in its peptide map. The 11 polypeptides assigned as specific flagellar surface components are candidate participants in such biological events as sexual adhesion, flagellar surface motility, and sensory signalling.
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Abbreviations
- Con A:
-
concanavalin A
- PAS:
-
periodate-Schiff
- Mr :
-
relative molecular weight
- SDS-PAGE:
-
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
References
Adair, W.S. (1982) The spaghetti overlay: Simulataneous screening of multiple polyclonal and monoclonal antibodies by immunoautoradiography. Anal. Biochem. 125, 299–306
Adair, W.S., Goodenough, U.W. (1978) Identification of a membrane tubulin in Chlamydomonas flagella. (Abstr.) J. Cell Biol. 78, 54
Adair, W.S., Hwang, C., Goodenough, U.W. (1983) Identification and visualization of the sexual agglutinin from the mating-type plus flagellar membrane of Chlamydomonas. Cell 33, 183–193
Adair, W.S., Monk, B.C., Cohen, R., Goodenough, U.W. (1982) Sexual agglutinins from the Chlamydomonas flagellar membrane: partial purification and characterization. J. Biol. Chem. 257, 4593–4602
Adoutte, A., Ramanthan, R., Lewis, R.M., Dute, R.R., Ling, K., Kung, C., Nelson, D.L. (1980) Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes. J. Cell Biol. 84, 717–738
Bergman, K., Goodenough, U.W., Goodenough, D.A., Jawitz, J., Martin, H. (1975) Gametic differentiation in Chlamydomonas reinhardtii. II. Flagellar membranes and the agglutination reaction. J. Cell Biol 67, 606–622
Bloodgood, R.A. (1977) Motility occuring in association with the surface of the Chlamydomonas flagellum. J. Cell Biol. 75, 983–989
Bloodgood, R.A., Leffler, E.M., Bojczuk, A.T. (1979) Reversible inhibition of Chlamydomonas flagellar surface motility. J. Cell Biol. 82, 664–672
Bordier, C., Crettol-Jarvinen, A. (1979) Peptide mapping of heterogeneous protein samples. J. Biol. Chem. 254, 2565–2567
Catt, J.W., Hills, G.J., Roberts, K. (1978) Glycoproteins from Chlamydomonas reinhardii, and their self-assembly. Planta 131, 165–171
Chen, L.L., Haines, T.H. (1976) The flagellar membrane of Ochromonas danioae. J. Biol. Chem. 251, 1828–1834
Claes, H. (1971) Autolysis of the cell wall from gametes of Chlamydomonas reinhardii. Arch. Mikrobiol. 78, 180–188
Davies, D.R., Plaskitt, A. (1971) Genetical and structural analyses of cell-wall formation in Chlamydomonas reinhardi. Genet. Res. 17, 33–43
Dentler, W.L. (1980) Microtubule-membrane interactions in cilia. I. Isolation and characterization of ciliary membranes from Tetrahymena pyriformis. J. Cell Biol. 84, 364–380
Dute, R., Kung, C. (1978) Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia. J. Cell Biol. 78, 451–464
Fairbanks, G., Steck, T.L., Wallach, D.F.H. (1971) Electrophoretic analysis of the major polypeptides of the human erythrocytemembrane. Biochemistry 10, 2606–2617
Gilula, N.B., Satir, P. (1972) The ciliary necklace: a ciliary membrane specialization. J. Cell Biol. 53, 494–509
Goldman, R., Pollard, T., Rosenbaum, J. (eds) (1976) Cell motility. Cold Spring Harbor Press, Cold Spring Harbor, N. Y.
Goodenough, U.W. (1977) Mating interactions in Chlamydomonas. In: Microbial interactions. Receptors and recognition, Ser. B, vol. 3, pp. 323–350, Reissig, J.L., ed. Chapman & Hall, London
Goodenough, U.W., Adair, W.S., Caligor, E., Forest, C.L., Hoffman, J.L., Mesland, D.A.M., Spath, S. (1980) Membrane-membrane and membrane-ligand interactions in Chlamydomonas mating. In: Membrane-membrane interactions, pp. 131–152, Gilula, N.B., ed. Raven Press, New York
Goodenough, U.W., Jurivich, D. (1978) Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera. J. Cell Biol. 79, 680–693
Goodenough, U.W., St. Clair, H.S. (1975) Bald-2: a mutation affecting the formation of doublet and triplet sets of microtubules in Chlamydomonas reinhardi. J. Cell Biol. 66, 480–491
Gorman, D.S., Levine, R.P. (1965) Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of c. reinhardi. Proc. Natl. Acad. Sci. USA 54, 1665–1669
Henderson, D., Eibl, H., Weber, K. (1979) Structure and biochemistry of mouse hepatic gap junctions. J. Mol. Biol. 132, 193–218
Hertzberg, E.L., Gilula, N.B. (1979) Isolation and characterization of gap junctions from rat liver. J. Biol. Chem. 254, 2138–2147
Heuser, J. (1980) Three-dimensional visualization of coated vesicle formation in fibroblasts. J. Cell Biol. 84, 560–583
Heuser, J.E., Kirschner, M.W. (1980) Filament organization revealed in platinum replicas of freeze-dried cytoskeletons. J. Cell Biol. 86, 212–234
Hoffman, J.L., Goodenough, U.W. (1980) Experimental dissection of flagellar surface motility in Chlamydomonas. J. Cell Biol. 86, 656–665
Hills, G.J., Phillips, J.M., Gay, M.R., Roberts K. (1975) Self-assembly of a plant cell wall in vitro. J. Mol. Biol. 96, 431–441
Hyams, J.S., Borisy, J.G. (1978) Isolated flagellar apparatus of Chlamydomonas: characterization of forward swimming and alteration of wave form and reversal of motion by calcium ions in vitro. J. Cell Sci. 33, 235–253
Hynes, R.O. (1976) Cell surface proteins and malignant transformation. Biochim. Biophys. Acta 458, 73–107
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680–685
Lens, P.F., van den Briel, W., Musgrave, A., van den Ende, H. (1980) Sex-specific glucoproteins in Chlamydomonas flagella. An immunological study. Arch. Microbiol. 126, 77–81
Lewin, R.A. (1952) Studies on the flagella of algae. I. General observations on Chlamydomonas moewusii Gerloff. Biol. Bull. 103, 74–79
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275
Martin, N.C., Goodenough, U.W. (1975) Gametic differentiation in Chlamydomonas reinhardtii. I. Production of gametes and their fine structure. J. Cell Biol. 67, 587–605
Matsudaira, D.T., Burgess, D.R. (1978) SDS microslab linear gradient polyacrylamide gel electrophoresis. Anal. Biochem. 87, 386–396
McLean, R.J., Brown, R.M. (1974) Cell surface differentiation of Chlamydomonas during gametogenesis. I. Mating and concanavalin A agglutinability. Dev. Biol. 36, 279–285
Merkel, S.J., Kaneshiro, E.S., Gruenstein, E.J. (1981) Characterization of the cilia and ciliary membrane proteins of wild-type Paramecium tetraurelia and a pawn mutant. J. Cell Biol. 89, 206–215
Mesland, D.A.M., Hoffman, J.L., Caligor, E., Goodenough, U.W. (1980) Flagellar tip activation stimulated by membrane adhesions in Chlamydomonas gametes. J. Cell Biol. 84, 599–617
Minami, S.A. (1976) Protein synthesis during differentiation of Chlamydomonas. Ph.D. thesis, Harvard University
Musgrave, A., Homan, W., van den Breil, W., Lelie, N., Schol, D., Ero, L., van den Ende, H. (1979) Membrane glycoproteins of Chlamydomonas eugametes flagella. Planta 145, 417–425
O'Farrel, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021
Orr, C.W. (1967) Studies on ascorbic acid. I. Factors influencing the ascorbate-mediated inhibition of catalase. Biochemistry 6, 2995–2999
Ringo, D.L. (1967) Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J. Cell Biol. 33, 543–571
Roberts, K., Gurney-Smith, M., Hills, G.J. (1972) Structure, composition and morphogenesis of the cell wall of Chlamydomonas reinhardi. I. Ultrastructure and preliminary chemical analysis. J. Ultrastruct. Res. 40, 599–613
Rogalski, A.A., Bouck, G.B. (1980) Characterization and localization of a flagellar-specific membrane glycoprotein in Euglena. J. Cell Biol. 86, 424–435
Schmidt, J.A., Eckert, R. (1976) Calcium couples flagellar reversal to photostimulation in Chlamydomonas reinhardtii. Nature (London) 262, 713–715
Segrest, J.P., Jackson, R.L. (1972) Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Methods Enzymol. 28, 54–63
Sleigh, M.A. (1974) (ed.) Cilia and flagella. Academic Press, London New York
Snell, W.J. (1976) Mating in Chlamydomonas: a system for the study of specific cell adhesion. I. Ultrastructural and electrophoretic analysis of flagellar surface components involved in adhesion. J. Cell Biol. 68, 48–69
Snell, W.J. (1982) Study of the release of cell wall degrading enzymes during adhesion of Chlamydomonas gametes. Exp. Cell Res. 138, 109–119
Snell, W.J., Moore, W.S. (1980) Aggregation-dependent turnover of flagellar adhesion molecules in Chlamydomonas gametes. J. Cell Biol. 84, 203–210
Solter, K.M., Gibor, A. (1977) Evidence for role of flagella as sensory transducers in mating of Chlamydomonas reinhardi. Nature (London) 265, 444–445
Stephens, R.E. (1977) Major membrane protein differences in cilia and flagella: evidence for a membrane associated tubulin. Biochemistry 16, 2047–2058
Tuszynski, G.P., Knight, L., Piperno, J.R., Walsh, P.N. (1980) A rapid method for removal of [125I] iodine following iodination of protein solutions. Anal. Biochem. 106, 118–122
Weiss, R.L., Goodenough, D.A., Goodenough, U.W. (1977) Membrane particle arrays associated with the basal body and with contractile vacuole secretion in Chlamydomonas. J. Cell Biol. 72, 133–143
Wiederhold, M.L. (1976) Mechanosensory transduction in “sensory” and “motile” cilia. Annu. Rev. Biophys. Bioeng. 5, 39–62
Wiese, L., Shoemaker, D.W. (1970) On sexual agglutination and mating-type substances (gamones) in isogamous heterothallic Chlamydomonads. II. The effects of concanavalin A on the mating-type reaction. Biol. Bull. 138, 88–95
Witman, G.B., Carlson, K., Berliner, J., Rosenbaum, J.L. (1972) Chlamydomonas flagella I. Isolation and electrophoretic analyses of microtubules. matrix, membranes, and mastigonemes. J. Cell Biol. 54, 507–539
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Monk, B.C., Adair, W.S., Cohen, R.A. et al. Topography of Chlamydomonas: fine structure and polypeptide components of the gametic flagellar membrane surface and the cell wall. Planta 158, 517–533 (1983). https://doi.org/10.1007/BF00397243
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DOI: https://doi.org/10.1007/BF00397243