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. 1980 Dec;144(3):1143–1151. doi: 10.1128/jb.144.3.1143-1151.1980

Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae.

R D Water, J R Pringle, L J Kleinsmith
PMCID: PMC294781  PMID: 7002908

Abstract

We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams J., Hansche P. E. Population studies in microorganisms. I. Evolution of diploidy in Saccharomyces cerevisiae. Genetics. 1974 Feb;76(2):327–338. doi: 10.1093/genetics/76.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen E. D., Sussman A. S. Presence of an actin-like protein in mycelium of Neurospora crassa. J Bacteriol. 1978 Aug;135(2):713–716. doi: 10.1128/jb.135.2.713-716.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benveniste K., Wilczek J., Ruggieri A., Stern R. Translation of collagen messenger RNA in a cell-free system derived from wheat germ. Biochemistry. 1976 Feb 24;15(4):830–835. doi: 10.1021/bi00649a016. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Byers B., Goetsch L. A highly ordered ring of membrane-associated filaments in budding yeast. J Cell Biol. 1976 Jun;69(3):717–721. doi: 10.1083/jcb.69.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clarke M., Spudich J. A. Nonmuscle contractile proteins: the role of actin and myosin in cell motility and shape determination. Annu Rev Biochem. 1977;46:797–822. doi: 10.1146/annurev.bi.46.070177.004053. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  9. Furthmayr H., Timpl R. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem. 1971 Jun;41(2):510–516. doi: 10.1016/0003-2697(71)90173-4. [DOI] [PubMed] [Google Scholar]
  10. Gallis B. M., McDonnell J. P., Hopper J. E., Young E. T. Translation of poly(riboadenylic acid)-enriched messenger RNAs from the yeast, Saccharomyces cerevisiae, in heterologous cell-free systems. Biochemistry. 1975 Mar 11;14(5):1038–1046. doi: 10.1021/bi00676a024. [DOI] [PubMed] [Google Scholar]
  11. Garrels J. I., Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976 Dec;9(4 Pt 2):793–805. doi: 10.1016/0092-8674(76)90142-2. [DOI] [PubMed] [Google Scholar]
  12. Gozes I., Schmitt H., Littauer U. Z. Translation in vitro of rat brain messenger RNA coding for tubulin and actin. Proc Natl Acad Sci U S A. 1975 Feb;72(2):701–705. doi: 10.1073/pnas.72.2.701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hartwell L. H. Cell division from a genetic perspective. J Cell Biol. 1978 Jun;77(3):627–637. doi: 10.1083/jcb.77.3.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hartwell L. H., Mortimer R. K., Culotti J., Culotti M. Genetic Control of the Cell Division Cycle in Yeast: V. Genetic Analysis of cdc Mutants. Genetics. 1973 Jun;74(2):267–286. doi: 10.1093/genetics/74.2.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartwell L. H. Saccharomyces cerevisiae cell cycle. Bacteriol Rev. 1974 Jun;38(2):164–198. doi: 10.1128/br.38.2.164-198.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hinnen A., Hicks J. B., Fink G. R. Transformation of yeast. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1929–1933. doi: 10.1073/pnas.75.4.1929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Holland M. J., Holland J. P. Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry. 1978 Nov 14;17(23):4900–4907. doi: 10.1021/bi00616a007. [DOI] [PubMed] [Google Scholar]
  18. Hunter T., Garrels J. I. Characterization of the mRNAs for alpha-, beta- and gamma-actin. Cell. 1977 Nov;12(3):767–781. doi: 10.1016/0092-8674(77)90276-8. [DOI] [PubMed] [Google Scholar]
  19. Koteliansky V. E., Glukhova M. A., Bejanian M. V., Surguchov A. P., Smirnov V. N. Isolation and characterization of actin-like protein from yeast Saccharomyces cerevisiae. FEBS Lett. 1979 Jun 1;102(1):55–58. doi: 10.1016/0014-5793(79)80927-8. [DOI] [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. 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]
  22. Lazarides E., Lindberg U. Actin is the naturally occurring inhibitor of deoxyribonuclease I. Proc Natl Acad Sci U S A. 1974 Dec;71(12):4742–4746. doi: 10.1073/pnas.71.12.4742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. LeStourgeon W. M., Rusch H. P. Localization of nucleolar and chromatin residual acidic protein changes during differentiation in Physarum polycephalum. Arch Biochem Biophys. 1973 Mar;155(1):144–158. doi: 10.1016/s0003-9861(73)80017-7. [DOI] [PubMed] [Google Scholar]
  24. Luduena R. F., Woodward D. O. Alpha- and beta-tubulin: separation and partial sequence analysis. Ann N Y Acad Sci. 1975 Jun 30;253:272–283. doi: 10.1111/j.1749-6632.1975.tb19206.x. [DOI] [PubMed] [Google Scholar]
  25. Mortimer R. K., Hawthorne D. C. Genetic mapping in yeast. Methods Cell Biol. 1975;11:221–233. doi: 10.1016/s0091-679x(08)60325-8. [DOI] [PubMed] [Google Scholar]
  26. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  27. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  28. Pringle J. R. Methods for avoiding proteolytic artefacts in studies of enzymes and other proteins from yeasts. Methods Cell Biol. 1975;12:149–184. doi: 10.1016/s0091-679x(08)60956-5. [DOI] [PubMed] [Google Scholar]
  29. Roberts B. E., Paterson B. M. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell-free system from commercial wheat germ. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2330–2334. doi: 10.1073/pnas.70.8.2330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger J. W. Presence of actin during chromosomal movement. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2451–2455. doi: 10.1073/pnas.72.6.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  32. Storti R. V., Rich A. Chick cytoplasmic actin and muscle actin have different structural genes. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2346–2350. doi: 10.1073/pnas.73.7.2346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tse T. P., Taylor J. M. Translation of albumin messenger RNA in a cell-free protein-synthesizing system derived from wheat germ. J Biol Chem. 1977 Feb 25;252(4):1272–1278. [PubMed] [Google Scholar]
  34. Tung J. S., Knight C. A. Effect of charge on the determination of molecular weight of proteins by gel electrophoresis in SDS. Biochem Biophys Res Commun. 1971 Mar 19;42(6):1117–1121. doi: 10.1016/0006-291x(71)90020-9. [DOI] [PubMed] [Google Scholar]
  35. Vandekerckhove J., Weber K. The amino acid sequence of Physarum actin. Nature. 1978 Dec 14;276(5689):720–721. doi: 10.1038/276720a0. [DOI] [PubMed] [Google Scholar]
  36. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  37. Wilkinson L. E., Pringle J. R. Transient G1 arrest of S. cerevisiae cells of mating type alpha by a factor produced by cells of mating type a. Exp Cell Res. 1974 Nov;89(1):175–187. doi: 10.1016/0014-4827(74)90200-6. [DOI] [PubMed] [Google Scholar]

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