Abstract
The cell cycle program of polypeptide labeling in syndhronous cultures of wild-type Chlamydomonas reinhardtii was analyzed by pulse-labeling cells with 35SO4 = or [3H]arginine at different cell cycle stages. Nearly 100 labeled membrane and soluble polypeptides were resolved and studied using one-dimensional sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis. The labeling experiments produced the following results. (a) Total 35SO4 = and [3H]arginine incorporation rates varied independently throughout the cell cycle. 35SO4 = incorporation was highest in the mid-light phase, while [3H]arginine incorporation peaked in the dark phase just before cell division. (b) The relative labeling rate for 20 of 100 polypeptides showed significant fluctuations (3-12 fold) during the cell cycle. The remaining polypeptides were labeled at a rate commensurate with total 35SO4 = or [3H]arginine incorporation. The polypeptides that showed significant fluctuations in relative labeling rates served as markers to identify cell cycle stages. (c) The effects of illumination conditions on the apparent cell cycle stage-specific labeling of polypeptides were tested. Shifting light-grown asynchronous cells to the dark had an immediate and pronounced effect on the pattern of polypeptide labeling, but shifting dark-phase syndhronous cells to the light had little effect. The apparent cell cycle variations in the labeling of ribulose 1,5-biphosphate (RUBP)-carboxylase were strongly influenced by illumination effects. (d) Pulse-chase experiments with light-grown asynchronous cells revealed little turnover or inter- conversion of labeled polypeptides within one cell generation, meaning that major polypeptides, whether labeled in a stage-specific manner or not, do not appear transiently in the cell cycle of actively dividing, light-grown cells. The cell cycle program of labeling was used to analyze effects of a temperature-sensitive cycle blocked (cb) mutant. A synchronous culture of ts10001 was shifted to restrictive temperature before its block point to prevent it from dividing. The mutant continued its cell cycle program of polypeptide labeling for over a cell generation, despite its inability to divide.
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- Armstrong J. J., Moll B., Surzycki S. J., Levine R. P. Genetic transcription and translation specifying chloroplast components in Chlamydomonas reinhardi. Biochemistry. 1971 Feb 16;10(4):692–701. doi: 10.1021/bi00780a022. [DOI] [PubMed] [Google Scholar]
- Beck D. P., Levine R. P. Synthesis of chloroplast membrane polypeptides during synchronous growth of Chlamydomonas reinhardtii. J Cell Biol. 1974 Dec;63(3):759–772. doi: 10.1083/jcb.63.3.759. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Blair G. E., Ellis R. J. Protein synthesis in chloroplasts. I. Light-driven synthesis of the large subunit of fraction I protein by isolated pea chloroplasts. Biochim Biophys Acta. 1973 Aug 24;319(2):223–234. doi: 10.1016/0005-2787(73)90013-0. [DOI] [PubMed] [Google Scholar]
- Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
- Bourguignon L. Y., Palade G. E. Incorporation of polypeptides into thylakoid membranes of Chlamydomonas reinhardtii. Cyclic variations. J Cell Biol. 1976 May;69(2):327–344. doi: 10.1083/jcb.69.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chiang K. S., Sueoka N. Replication of chloroplast DNA in Chlamydomonas reinhardi during vegetative cell cycle: its mode and regulation. Proc Natl Acad Sci U S A. 1967 May;57(5):1506–1513. doi: 10.1073/pnas.57.5.1506. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoober J. K. A major polypeptide of chloroplast membranes of Chlamydomonas reinhardi. Evidence for synthesis in the cytoplasm as a soluble component. J Cell Biol. 1972 Jan;52(1):84–96. doi: 10.1083/jcb.52.1.84. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hoober J. K. Sites of synthesis of chloroplast membrane polypeptides in Chlamydomonas reinhardi y-1. J Biol Chem. 1970 Sep 10;245(17):4327–4334. [PubMed] [Google Scholar]
- Howell S. H., Naliboff J. A. Conditional mutants in Chlamydomonas reinhardtii blocked in the vegetative cell cycle. I. An analysis of cell cycle block points. J Cell Biol. 1973 Jun;57(3):760–772. doi: 10.1083/jcb.57.3.760. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Iwanij V., Chua N. H., Siekevitz P. Synthesis and turnover of ribulose biphosphate carboxylase and of its subunits during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol. 1975 Mar;64(3):572–585. doi: 10.1083/jcb.64.3.572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones R. F., Kates J. R., Keller S. J. Protein turnover and macromolecular synthesis during growth and gametic differentiation in Chlamydomonas reinhardtii. Biochim Biophys Acta. 1968 May 21;157(3):589–598. doi: 10.1016/0005-2787(68)90156-1. [DOI] [PubMed] [Google Scholar]
- Kates J. R., Jones R. F. Periodic increases in enzyme activity in synchronized cultures of Chlamydomonas reinhardtii. Biochim Biophys Acta. 1967 Aug 22;145(1):153–158. doi: 10.1016/0005-2787(67)90664-8. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [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]