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. 1996 Jul 1;134(1):117–132. doi: 10.1083/jcb.134.1.117

The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton

PMCID: PMC2120930  PMID: 8698808

Abstract

Arp2p is an essential yeast actin-related protein. Disruption of the corresponding ARP2 gene leads to a terminal phenotype characterized by the presence of a single large bud. Thus, Arp2p may be important for a late stage of the cell cycle (Schwob, E., and R.P. Martin, 1992. Nature (Lond.). 355:179-182). We have localized Arp2p by indirect immunofluorescence. Specific peptide antibodies revealed punctate staining under the plasma membrane, which partially colocalizes with actin. Temperature-sensitive arp2 mutations were created by PCR mutagenesis and selected by an ade2/SUP11 sectoring screen. One temperature-sensitive mutant that was characterized, arp2-H330L, was osmosensitive and had an altered actin cytoskeleton at a nonpermissive temperature, suggesting a role of Arp2p in the actin cytoskeleton. Random budding patterns were observed in both haploid and diploid arp2- H330L mutant cells. Endocytosis, as judged by Lucifer yellow uptake, was severely reduced in the mutant, at all temperatures. In addition, genetic interaction was observed between temperature-sensitive alleles arp2-H330L and cdc10-1. CDC10 is a gene encoding a neck filament- associated protein that is necessary for polarized growth and cytokinesis. Overall, the immunolocalization, mutant phenotypes, and genetic interaction suggest that the Arp2 protein is an essential component of the actin cytoskeleton that is involved in membrane growth and polarity, as well as in endocytosis.

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Selected References

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  1. Adams A. E., Cooper J. A., Drubin D. G. Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast. Mol Biol Cell. 1993 May;4(5):459–468. doi: 10.1091/mbc.4.5.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Amatruda J. F., Gattermeir D. J., Karpova T. S., Cooper J. A. Effects of null mutations and overexpression of capping protein on morphogenesis, actin distribution and polarized secretion in yeast. J Cell Biol. 1992 Dec;119(5):1151–1162. doi: 10.1083/jcb.119.5.1151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Balasubramanian M. K., Hirani B. R., Burke J. D., Gould K. L. The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis. J Cell Biol. 1994 Jun;125(6):1289–1301. doi: 10.1083/jcb.125.6.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blum S., Mueller M., Schmid S. R., Linder P., Trachsel H. Translation in Saccharomyces cerevisiae: initiation factor 4A-dependent cell-free system. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6043–6046. doi: 10.1073/pnas.86.16.6043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonneaud N., Ozier-Kalogeropoulos O., Li G. Y., Labouesse M., Minvielle-Sebastia L., Lacroute F. A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors. Yeast. 1991 Aug-Sep;7(6):609–615. doi: 10.1002/yea.320070609. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Bénédetti H., Raths S., Crausaz F., Riezman H. The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast. Mol Biol Cell. 1994 Sep;5(9):1023–1037. doi: 10.1091/mbc.5.9.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chant J. Cell polarity in yeast. Trends Genet. 1994 Sep;10(9):328–333. doi: 10.1016/0168-9525(94)90036-1. [DOI] [PubMed] [Google Scholar]
  10. Chant J., Herskowitz I. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 1991 Jun 28;65(7):1203–1212. doi: 10.1016/0092-8674(91)90015-q. [DOI] [PubMed] [Google Scholar]
  11. Chant J., Pringle J. R. Budding and cell polarity in Saccharomyces cerevisiae. Curr Opin Genet Dev. 1991 Oct;1(3):342–350. doi: 10.1016/s0959-437x(05)80298-9. [DOI] [PubMed] [Google Scholar]
  12. Clark S. W., Meyer D. I. ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle. J Cell Biol. 1994 Oct;127(1):129–138. doi: 10.1083/jcb.127.1.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clark S. W., Meyer D. I. Centractin is an actin homologue associated with the centrosome. Nature. 1992 Sep 17;359(6392):246–250. doi: 10.1038/359246a0. [DOI] [PubMed] [Google Scholar]
  14. Drubin D. G. Development of cell polarity in budding yeast. Cell. 1991 Jun 28;65(7):1093–1096. doi: 10.1016/0092-8674(91)90001-f. [DOI] [PubMed] [Google Scholar]
  15. Drubin D. G., Jones H. D., Wertman K. F. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol Biol Cell. 1993 Dec;4(12):1277–1294. doi: 10.1091/mbc.4.12.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dulic V., Egerton M., Elguindi I., Raths S., Singer B., Riezman H. Yeast endocytosis assays. Methods Enzymol. 1991;194:697–710. doi: 10.1016/0076-6879(91)94051-d. [DOI] [PubMed] [Google Scholar]
  17. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  18. Flescher E. G., Madden K., Snyder M. Components required for cytokinesis are important for bud site selection in yeast. J Cell Biol. 1993 Jul;122(2):373–386. doi: 10.1083/jcb.122.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Frankel S., Mooseker M. S. The actin-related proteins. Curr Opin Cell Biol. 1996 Feb;8(1):30–37. doi: 10.1016/s0955-0674(96)80045-7. [DOI] [PubMed] [Google Scholar]
  21. Fyrberg C., Fyrberg E. A Drosophila homologue of the Schizosaccharomyces pombe act2 gene. Biochem Genet. 1993 Aug;31(7-8):329–341. [PubMed] [Google Scholar]
  22. Fyrberg C., Ryan L., Kenton M., Fyrberg E. Genes encoding actin-related proteins of Drosophila melanogaster. J Mol Biol. 1994 Aug 19;241(3):498–503. doi: 10.1006/jmbi.1994.1526. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Harata M., Karwan A., Wintersberger U. An essential gene of Saccharomyces cerevisiae coding for an actin-related protein. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8258–8262. doi: 10.1073/pnas.91.17.8258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Herman I. M. Actin isoforms. Curr Opin Cell Biol. 1993 Feb;5(1):48–55. doi: 10.1016/s0955-0674(05)80007-9. [DOI] [PubMed] [Google Scholar]
  26. Hill J., Donald K. A., Griffiths D. E., Donald G. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 1991 Oct 25;19(20):5791–5791. doi: 10.1093/nar/19.20.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Holtzman D. A., Yang S., Drubin D. G. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J Cell Biol. 1993 Aug;122(3):635–644. doi: 10.1083/jcb.122.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kelleher J. F., Atkinson S. J., Pollard T. D. Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. J Cell Biol. 1995 Oct;131(2):385–397. doi: 10.1083/jcb.131.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. 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]
  31. Kübler E., Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 1993 Jul;12(7):2855–2862. doi: 10.1002/j.1460-2075.1993.tb05947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lees-Miller J. P., Helfman D. M., Schroer T. A. A vertebrate actin-related protein is a component of a multisubunit complex involved in microtubule-based vesicle motility. Nature. 1992 Sep 17;359(6392):244–246. doi: 10.1038/359244a0. [DOI] [PubMed] [Google Scholar]
  33. Lees-Miller J. P., Henry G., Helfman D. M. Identification of act2, an essential gene in the fission yeast Schizosaccharomyces pombe that encodes a protein related to actin. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):80–83. doi: 10.1073/pnas.89.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lew D. J., Reed S. I. Morphogenesis in the yeast cell cycle: regulation by Cdc28 and cyclins. J Cell Biol. 1993 Mar;120(6):1305–1320. doi: 10.1083/jcb.120.6.1305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Machesky L. M., Atkinson S. J., Ampe C., Vandekerckhove J., Pollard T. D. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol. 1994 Oct;127(1):107–115. doi: 10.1083/jcb.127.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Magdolen V., Drubin D. G., Mages G., Bandlow W. High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells. FEBS Lett. 1993 Jan 18;316(1):41–47. doi: 10.1016/0014-5793(93)81733-g. [DOI] [PubMed] [Google Scholar]
  37. Matsui Y., Toh-E A. Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1. Mol Cell Biol. 1992 Dec;12(12):5690–5699. doi: 10.1128/mcb.12.12.5690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Michaille J. J., Gouy M., Blanchet S., Duret L. Isolation and characterization of a cDNA encoding a chicken actin-like protein. Gene. 1995 Mar 10;154(2):205–209. doi: 10.1016/0378-1119(94)00865-p. [DOI] [PubMed] [Google Scholar]
  39. Muhua L., Karpova T. S., Cooper J. A. A yeast actin-related protein homologous to that in vertebrate dynactin complex is important for spindle orientation and nuclear migration. Cell. 1994 Aug 26;78(4):669–679. doi: 10.1016/0092-8674(94)90531-2. [DOI] [PubMed] [Google Scholar]
  40. Mulholland J., Preuss D., Moon A., Wong A., Drubin D., Botstein D. Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J Cell Biol. 1994 Apr;125(2):381–391. doi: 10.1083/jcb.125.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mumberg D., Müller R., Funk M. Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res. 1994 Dec 25;22(25):5767–5768. doi: 10.1093/nar/22.25.5767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Munn A. L., Stevenson B. J., Geli M. I., Riezman H. end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol Biol Cell. 1995 Dec;6(12):1721–1742. doi: 10.1091/mbc.6.12.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Murgia I., Maciver S. K., Morandini P. An actin-related protein from Dictyostelium discoideum is developmentally regulated and associated with mitochondria. FEBS Lett. 1995 Mar 6;360(3):235–241. doi: 10.1016/0014-5793(95)00111-l. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  46. Pringle J. R. Staining of bud scars and other cell wall chitin with calcofluor. Methods Enzymol. 1991;194:732–735. doi: 10.1016/0076-6879(91)94055-h. [DOI] [PubMed] [Google Scholar]
  47. Raths S., Rohrer J., Crausaz F., Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. doi: 10.1083/jcb.120.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Riezman H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell. 1985 Apr;40(4):1001–1009. doi: 10.1016/0092-8674(85)90360-5. [DOI] [PubMed] [Google Scholar]
  49. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  50. Schafer D. A., Gill S. R., Cooper J. A., Heuser J. E., Schroer T. A. Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin. J Cell Biol. 1994 Jul;126(2):403–412. doi: 10.1083/jcb.126.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schroer T. A., Fyrberg E., Cooper J. A., Waterston R. H., Helfman D., Pollard T. D., Meyer D. I. Actin-related protein nomenclature and classification. J Cell Biol. 1994 Dec;127(6 Pt 2):1777–1778. doi: 10.1083/jcb.127.6.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schwob E., Martin R. P. New yeast actin-like gene required late in the cell cycle. Nature. 1992 Jan 9;355(6356):179–182. doi: 10.1038/355179a0. [DOI] [PubMed] [Google Scholar]
  53. Snyder M., Gehrung S., Page B. D. Studies concerning the temporal and genetic control of cell polarity in Saccharomyces cerevisiae. J Cell Biol. 1991 Aug;114(3):515–532. doi: 10.1083/jcb.114.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Tanaka T., Shibasaki F., Ishikawa M., Hirano N., Sakai R., Nishida J., Takenawa T., Hirai H. Molecular cloning of bovine actin-like protein, actin2. Biochem Biophys Res Commun. 1992 Sep 16;187(2):1022–1028. doi: 10.1016/0006-291x(92)91299-6. [DOI] [PubMed] [Google Scholar]
  55. Water R. D., Pringle J. R., Kleinsmith L. J. Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae. J Bacteriol. 1980 Dec;144(3):1143–1151. doi: 10.1128/jb.144.3.1143-1151.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weisman L. S., Bacallao R., Wickner W. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol. 1987 Oct;105(4):1539–1547. doi: 10.1083/jcb.105.4.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Welch M. D., Holtzman D. A., Drubin D. G. The yeast actin cytoskeleton. Curr Opin Cell Biol. 1994 Feb;6(1):110–119. doi: 10.1016/0955-0674(94)90124-4. [DOI] [PubMed] [Google Scholar]
  58. Yamochi W., Tanaka K., Nonaka H., Maeda A., Musha T., Takai Y. Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J Cell Biol. 1994 Jun;125(5):1077–1093. doi: 10.1083/jcb.125.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]

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