Abstract
The principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. We show that these two isoelectric variants of alpha-tubulin are related to one another since posttranslational modification of the cell body precursor form converts it to the axonemal form. During flagellar assembly, precursor alpha-tubulin enters the flagella and is posttranslationally modified within the flagellar matrix fraction prior to or at the time of its addition to the growing axonemal microtubules. Experiments designed to identify the nature of this posttranslational modification have also been conducted. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo and, under these conditions, no other flagellar polypeptides exhibit detectable labeling.
Full Text
The Full Text of this article is available as a PDF (1.1 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Adams G. M., Huang B., Piperno G., Luck D. J. Central-pair microtubular complex of Chlamydomonas flagella: polypeptide composition as revealed by analysis of mutants. J Cell Biol. 1981 Oct;91(1):69–76. doi: 10.1083/jcb.91.1.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Allen C., Borisy G. G. Structural polarity and directional growth of microtubules of Chlamydomonas flagella. J Mol Biol. 1974 Dec 5;90(2):381–402. doi: 10.1016/0022-2836(74)90381-7. [DOI] [PubMed] [Google Scholar]
- Auclair W., Siegel B. W. Cilia regeneration in the sea urchin embryo: evidence for a pool of ciliary proteins. Science. 1966 Nov 18;154(3751):913–915. doi: 10.1126/science.154.3751.913. [DOI] [PubMed] [Google Scholar]
- Barra H. S., Arce C. A., Rodríguez J. A., Caputto R. Some common properties of the protein that incorporates tyrosine as a single unit and the microtubule proteins. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1384–1390. doi: 10.1016/0006-291x(74)90351-9. [DOI] [PubMed] [Google Scholar]
- Binder L. I., Dentler W. L., Rosenbaum J. L. Assembly of chick brain tubulin onto flagellar microtubules from Chlamydomonas and sea urchin sperm. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1122–1126. doi: 10.1073/pnas.72.3.1122. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
- Brown D. L., Rogers K. A. Hydrostatic pressure-induced internalization of flagellar axonemes, disassembly, and reutilization during flagellar regeneration in Polytomella. Exp Cell Res. 1978 Dec;117(2):313–324. doi: 10.1016/0014-4827(78)90145-3. [DOI] [PubMed] [Google Scholar]
- Dentler W. L., Rosenbaum J. L. Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly. J Cell Biol. 1977 Sep;74(3):747–759. doi: 10.1083/jcb.74.3.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Guttman S. D., Gorovsky M. A. Cilia regeneration in starved tetrahymena: an inducible system for studying gene expression and organelle biogenesis. Cell. 1979 Jun;17(2):307–317. doi: 10.1016/0092-8674(79)90156-9. [DOI] [PubMed] [Google Scholar]
- Huang B., Piperno G., Ramanis Z., Luck D. J. Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function. J Cell Biol. 1981 Jan;88(1):80–88. doi: 10.1083/jcb.88.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Huang B., Rifkin M. R., Luck D. J. Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii. J Cell Biol. 1977 Jan;72(1):67–85. doi: 10.1083/jcb.72.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
- Lefebvre P. A., Nordstrom S. A., Moulder J. E., Rosenbaum J. L. Flagellar elongation and shortening in Chlamydomonas. IV. Effects of flagellar detachment, regeneration, and resorption on the induction of flagellar protein synthesis. J Cell Biol. 1978 Jul;78(1):8–27. doi: 10.1083/jcb.78.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lefebvre P. A., Silflow C. D., Wieben E. D., Rosenbaum J. L. Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas flagella. Cell. 1980 Jun;20(2):469–477. doi: 10.1016/0092-8674(80)90633-9. [DOI] [PubMed] [Google Scholar]
- Marcaud L., Hayes D. RNA synthesis in starved deciliated Tetrahymena pyriformis. Eur J Biochem. 1979 Jul;98(1):267–273. doi: 10.1111/j.1432-1033.1979.tb13185.x. [DOI] [PubMed] [Google Scholar]
- McKeithan T. W., Lefebvre P. A., Silflow C. D., Rosenbaum J. L. Multiple forms of tubulin in Polytomella and Chlamydomonas: evidence for a precursor of flagellar alpha-tubulin. J Cell Biol. 1983 Apr;96(4):1056–1063. doi: 10.1083/jcb.96.4.1056. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Merlino G. T., Chamberlain J. P., Kleinsmith L. J. Effects of deciliation of tubulin messenger RNA activity in sea urchin embryos. J Biol Chem. 1978 Oct 10;253(19):7078–7085. [PubMed] [Google Scholar]
- Minami S. A., Collis P. S., Young E. E., Weeks D. P. Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis. Cell. 1981 Apr;24(1):89–95. doi: 10.1016/0092-8674(81)90504-3. [DOI] [PubMed] [Google Scholar]
- Piperno G., Huang B., Luck D. J. Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1977 Apr;74(4):1600–1604. doi: 10.1073/pnas.74.4.1600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piperno G., Huang B., Ramanis Z., Luck D. J. Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components. J Cell Biol. 1981 Jan;88(1):73–79. doi: 10.1083/jcb.88.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Piperno G., Luck D. J. Microtubular proteins of Chlamydomonas reinhardtii. An immunochemical study based on the use of an antibody specific for the beta-tubulin subunit. J Biol Chem. 1977 Jan 10;252(1):383–391. [PubMed] [Google Scholar]
- Randall J., Munden H. R., Prest P. H. The flagellar apparatus as a model organelle for the study of growth and morphopoiesis. With an appendix. Temperature control apparatus used in flagellar regeneration experiments. Proc R Soc Lond B Biol Sci. 1969 Apr 15;173(1030):31–passim. doi: 10.1098/rspb.1969.0034. [DOI] [PubMed] [Google Scholar]
- Raybin D., Flavin M. Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro. J Cell Biol. 1977 May;73(2):492–504. doi: 10.1083/jcb.73.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Remillard S. P., Witman G. B. Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas. J Cell Biol. 1982 Jun;93(3):615–631. doi: 10.1083/jcb.93.3.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rosenbaum J. L., Binder L. I., Granett S., Dentler W. L., Snell W., Sloboda R., Haimo L. Directionality and rate of assembly of chick brain tubulin onto pieces of neurotubules, flagellar axonemes, and basal bodies. Ann N Y Acad Sci. 1975 Jun 30;253:147–177. doi: 10.1111/j.1749-6632.1975.tb19198.x. [DOI] [PubMed] [Google Scholar]
- Rosenbaum J. L., Moulder J. E., Ringo D. L. Flagellar elongation and shortening in Chlamydomonas. The use of cycloheximide and colchicine to study the synthesis and assembly of flagellar proteins. J Cell Biol. 1969 May;41(2):600–619. doi: 10.1083/jcb.41.2.600. [DOI] [PMC free article] [PubMed] [Google Scholar]
- SAGER R., GRANICK S. Nutritional studies with Chlamydomonas reinhardi. Ann N Y Acad Sci. 1953 Oct 14;56(5):831–838. doi: 10.1111/j.1749-6632.1953.tb30261.x. [DOI] [PubMed] [Google Scholar]
- Silflow C. D., Lefebvre P. A., McKeithan T. W., Schloss J. A., Keller L. R., Rosenbaum J. L. Expression of flagellar protein genes during flagellar regeneration in Chlamydomonas. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):157–169. doi: 10.1101/sqb.1982.046.01.019. [DOI] [PubMed] [Google Scholar]
- Silflow C. D., Rosenbaum J. L. Multiple alpha- and beta-tubulin genes in Chlamydomonas and regulation of tubulin mRNA levels after deflagellation. Cell. 1981 Apr;24(1):81–88. doi: 10.1016/0092-8674(81)90503-1. [DOI] [PubMed] [Google Scholar]
- Stephens R. E. Differential protein synthesis and utilization during cilia formation in sea urchin embryos. Dev Biol. 1977 Dec;61(2):311–329. doi: 10.1016/0012-1606(77)90301-3. [DOI] [PubMed] [Google Scholar]
- Weeks D. P., Collis P. S. Induction of microtubule protein synthesis in Chlamydomonas reinhardi during flagellar regeneration. Cell. 1976 Sep;9(1):15–27. doi: 10.1016/0092-8674(76)90048-9. [DOI] [PubMed] [Google Scholar]
- Witman G. B., Carlson K., Berliner J., Rosenbaum J. L. Chlamydomonas flagella. I. Isolation and electrophoretic analysis of microtubules, matrix, membranes, and mastigonemes. J Cell Biol. 1972 Sep;54(3):507–539. doi: 10.1083/jcb.54.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Witman G. B., Plummer J., Sander G. Chlamydomonas flagellar mutants lacking radial spokes and central tubules. Structure, composition, and function of specific axonemal components. J Cell Biol. 1978 Mar;76(3):729–747. doi: 10.1083/jcb.76.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Witman G. B. The site of in vivo assembly of flagellar microtubules. Ann N Y Acad Sci. 1975 Jun 30;253:178–191. doi: 10.1111/j.1749-6632.1975.tb19199.x. [DOI] [PubMed] [Google Scholar]