Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1992 Jun 1;117(5):1067–1076. doi: 10.1083/jcb.117.5.1067

Purification, characterization, and immunofluorescence localization of Saccharomyces cerevisiae capping protein

PMCID: PMC2289475  PMID: 1315784

Abstract

Capping protein binds the barbed ends of actin filaments and nucleates actin filament assembly in vitro. We purified capping protein from Saccharomyces cervisiae. One of the two subunits is the product of the CAP2 gene, which we previously identified as the gene encoding the beta subunit of capping protein based on its sequence similarity to capping protein beta subunits in chicken and Dictyostelium (Amatruda, J. F., J. F. Cannon, K. Tatchell, C. Hug, and J. A. Cooper. 1990. Nature (Lond.) 344:352-354). Yeast capping protein has activity in critical concentration and low-shear viscometry assays consistent with barbed- end capping activity. Like chicken capping protein, yeast capping protein is inhibited by PIP2. By immunofluorescence microscopy yeast capping protein colocalizes with cortical actin spots at the site of bud emergence and at the tips of growing buds and shmoos. In contrast, capping protein does not colocalize with actin cables or with actin rings at the site of cytokinesis.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amatruda J. F., Cannon J. F., Tatchell K., Hug C., Cooper J. A. Disruption of the actin cytoskeleton in yeast capping protein mutants. Nature. 1990 Mar 22;344(6264):352–354. doi: 10.1038/344352a0. [DOI] [PubMed] [Google Scholar]
  3. Ankenbauer T., Kleinschmidt J. A., Walsh M. J., Weiner O. H., Franke W. W. Identification of a widespread nuclear actin binding protein. Nature. 1989 Dec 14;342(6251):822–825. doi: 10.1038/342822a0. [DOI] [PubMed] [Google Scholar]
  4. Babul J., Stellwagen E. Measurement of protein concentration with interferences optics. Anal Biochem. 1969 Apr 4;28(1):216–221. doi: 10.1016/0003-2697(69)90172-9. [DOI] [PubMed] [Google Scholar]
  5. Caldwell J. E., Heiss S. G., Mermall V., Cooper J. A. Effects of CapZ, an actin capping protein of muscle, on the polymerization of actin. Biochemistry. 1989 Oct 17;28(21):8506–8514. doi: 10.1021/bi00447a036. [DOI] [PubMed] [Google Scholar]
  6. Caldwell J. E., Waddle J. A., Cooper J. A., Hollands J. A., Casella S. J., Casella J. F. cDNAs encoding the beta subunit of cap Z, the actin-capping protein of the Z line of muscle. J Biol Chem. 1989 Jul 25;264(21):12648–12652. [PubMed] [Google Scholar]
  7. Casella J. F., Craig S. W., Maack D. J., Brown A. E. Cap Z(36/32), a barbed end actin-capping protein, is a component of the Z-line of skeletal muscle. J Cell Biol. 1987 Jul;105(1):371–379. doi: 10.1083/jcb.105.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Casella J. F., Maack D. J., Lin S. Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. J Biol Chem. 1986 Aug 15;261(23):10915–10921. [PubMed] [Google Scholar]
  9. Cooper J. A., Blum J. D., Pollard T. D. Acanthamoeba castellanii capping protein: properties, mechanism of action, immunologic cross-reactivity, and localization. J Cell Biol. 1984 Jul;99(1 Pt 1):217–225. doi: 10.1083/jcb.99.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cooper J. A., Walker S. B., Pollard T. D. Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil. 1983 Apr;4(2):253–262. doi: 10.1007/BF00712034. [DOI] [PubMed] [Google Scholar]
  11. Cross F., Hartwell L. H., Jackson C., Konopka J. B. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. doi: 10.1146/annurev.cb.04.110188.002241. [DOI] [PubMed] [Google Scholar]
  12. Ey P. L., Ashman L. K. The use of alkaline phosphatase-conjugated anti-immunoglobulin with immunoblots for determining the specificity of monoclonal antibodies to protein mixtures. Methods Enzymol. 1986;121:497–509. doi: 10.1016/0076-6879(86)21050-2. [DOI] [PubMed] [Google Scholar]
  13. Ford S. K., Pringle J. R. Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC11 gene product and the timing of events at the budding site. Dev Genet. 1991;12(4):281–292. doi: 10.1002/dvg.1020120405. [DOI] [PubMed] [Google Scholar]
  14. Haarer B. K., Pringle J. R. Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck. Mol Cell Biol. 1987 Oct;7(10):3678–3687. doi: 10.1128/mcb.7.10.3678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartmann H., Noegel A. A., Eckerskorn C., Rapp S., Schleicher M. Ca2+-independent F-actin capping proteins. Cap 32/34, a capping protein from Dictyostelium discoideum, does not share sequence homologies with known actin-binding proteins. J Biol Chem. 1989 Jul 25;264(21):12639–12647. [PubMed] [Google Scholar]
  16. Heiss S. G., Cooper J. A. Regulation of CapZ, an actin capping protein of chicken muscle, by anionic phospholipids. Biochemistry. 1991 Sep 10;30(36):8753–8758. doi: 10.1021/bi00100a006. [DOI] [PubMed] [Google Scholar]
  17. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  18. Kilimann M. W., Isenberg G. Actin filament capping protein from bovine brain. EMBO J. 1982;1(7):889–894. doi: 10.1002/j.1460-2075.1982.tb01265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kim H. B., Haarer B. K., Pringle J. R. Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site. J Cell Biol. 1991 Feb;112(4):535–544. doi: 10.1083/jcb.112.4.535. [DOI] [PMC free article] [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. Nakajima H., Hirata A., Ogawa Y., Yonehara T., Yoda K., Yamasaki M. A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol. 1991 Apr;113(2):245–260. doi: 10.1083/jcb.113.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  24. Pollard T. D., Cooper J. A. Methods to characterize actin filament networks. Methods Enzymol. 1982;85(Pt B):211–233. doi: 10.1016/0076-6879(82)85022-2. [DOI] [PubMed] [Google Scholar]
  25. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  26. Redding K., Holcomb C., Fuller R. S. Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae. J Cell Biol. 1991 May;113(3):527–538. doi: 10.1083/jcb.113.3.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roberts C. J., Raymond C. K., Yamashiro C. T., Stevens T. H. Methods for studying the yeast vacuole. Methods Enzymol. 1991;194:644–661. doi: 10.1016/0076-6879(91)94047-g. [DOI] [PubMed] [Google Scholar]
  28. Schleicher M., Gerisch G., Isenberg G. New actin-binding proteins from Dictyostelium discoideum. EMBO J. 1984 Sep;3(9):2095–2100. doi: 10.1002/j.1460-2075.1984.tb02096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sloat B. F., Pringle J. R. A mutant of yeast defective in cellular morphogenesis. Science. 1978 Jun 9;200(4346):1171–1173. doi: 10.1126/science.349694. [DOI] [PubMed] [Google Scholar]
  31. Sprague G. F., Jr Assay of yeast mating reaction. Methods Enzymol. 1991;194:77–93. doi: 10.1016/0076-6879(91)94008-z. [DOI] [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yansura D. G. Expression as trpE fusion. Methods Enzymol. 1990;185:161–166. doi: 10.1016/0076-6879(90)85016-h. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES