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. 1994 Oct 1;127(1):129–138. doi: 10.1083/jcb.127.1.129

ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle

PMCID: PMC2120173  PMID: 7929558

Abstract

As part of our ongoing efforts to understand the functional role of vertebrate centractins, we have identified a new member of the actin- related family of proteins in the yeast Saccharomyces cerevisiae using a PCR-based approach. Consistent with the current nomenclature for actin-related proteins in yeast, we propose to denote this locus ACT3. The primary amino acid sequence of Act3p is most similar to canine and human alpha-centractin (73% similarity/54% identity). The sequence of a genomic clone indicates ACT3 lies adjacent to and is transcribed convergently with respect to FUR1 on chromosome VIII. Molecular genetic analysis indicates ACT3 is represented by a single gene from which the corresponding mRNA is expressed at a low level compared to ACT1. Tetrad analysis of heterozygotes harboring a TRP1 replacement of the ACT3- coding region indicates ACT3 is nonessential for growth under normal conditions and at extremes of temperature and osmolarity. However, growth at 14 degrees C indicates a spindle orientation defect similar to phenotypes recently described for yeast harboring mutations in actin, tubulin, or cytoplasmic dynein. Taken together, our data suggest that ACT3 is the S. cerevisiae homologue of vertebrate centractins.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Aniento F., Emans N., Griffiths G., Gruenberg J. Cytoplasmic dynein-dependent vesicular transport from early to late endosomes. J Cell Biol. 1993 Dec;123(6 Pt 1):1373–1387. doi: 10.1083/jcb.123.6.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bomsel M., Parton R., Kuznetsov S. A., Schroer T. A., Gruenberg J. Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells. Cell. 1990 Aug 24;62(4):719–731. doi: 10.1016/0092-8674(90)90117-w. [DOI] [PubMed] [Google Scholar]
  4. Bork P., Sander C., Valencia A. An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7290–7294. doi: 10.1073/pnas.89.16.7290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Clark S. W., Meyer D. I. Long lost cousins of actin. Curr Biol. 1993 Jan;3(1):54–55. doi: 10.1016/0960-9822(93)90151-d. [DOI] [PubMed] [Google Scholar]
  7. Edman J. C., Kovacs J. A., Masur H., Santi D. V., Elwood H. J., Sogin M. L. Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the fungi. Nature. 1988 Aug 11;334(6182):519–522. doi: 10.1038/334519a0. [DOI] [PubMed] [Google Scholar]
  8. Eshel D., Urrestarazu L. A., Vissers S., Jauniaux J. C., van Vliet-Reedijk J. C., Planta R. J., Gibbons I. R. Cytoplasmic dynein is required for normal nuclear segregation in yeast. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11172–11176. doi: 10.1073/pnas.90.23.11172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Flaherty K. M., McKay D. B., Kabsch W., Holmes K. C. Similarity of the three-dimensional structures of actin and the ATPase fragment of a 70-kDa heat shock cognate protein. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):5041–5045. doi: 10.1073/pnas.88.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frankel S., Heintzelman M. B., Artavanis-Tsakonas S., Mooseker M. S. Identification of a divergent actin-related protein in Drosophila. J Mol Biol. 1994 Jan 28;235(4):1351–1356. doi: 10.1006/jmbi.1994.1090. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Gallwitz D., Sures I. Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 May;77(5):2546–2550. doi: 10.1073/pnas.77.5.2546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Garen S. H., Kankel D. R. Golgi and genetic mosaic analyses of visual system mutants in Drosophila melanogaster. Dev Biol. 1983 Apr;96(2):445–466. doi: 10.1016/0012-1606(83)90182-3. [DOI] [PubMed] [Google Scholar]
  14. Gill S. R., Schroer T. A., Szilak I., Steuer E. R., Sheetz M. P., Cleveland D. W. Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein. J Cell Biol. 1991 Dec;115(6):1639–1650. doi: 10.1083/jcb.115.6.1639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Guthrie B. A., Wickner W. Yeast vacuoles fragment when microtubules are disrupted. J Cell Biol. 1988 Jul;107(1):115–120. doi: 10.1083/jcb.107.1.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Harte P. J., Kankel D. R. Genetic analysis of mutations at the Glued locus and interacting loci in Drosophila melanogaster. Genetics. 1982 Jul-Aug;101(3-4):477–501. doi: 10.1093/genetics/101.3-4.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Holton T. A., Graham M. W. A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors. Nucleic Acids Res. 1991 Mar 11;19(5):1156–1156. doi: 10.1093/nar/19.5.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holzbaur E. L., Hammarback J. A., Paschal B. M., Kravit N. G., Pfister K. K., Vallee R. B. Homology of a 150K cytoplasmic dynein-associated polypeptide with the Drosophila gene Glued. Nature. 1991 Jun 13;351(6327):579–583. doi: 10.1038/351579a0. [DOI] [PubMed] [Google Scholar]
  20. Huffaker T. C., Thomas J. H., Botstein D. Diverse effects of beta-tubulin mutations on microtubule formation and function. J Cell Biol. 1988 Jun;106(6):1997–2010. doi: 10.1083/jcb.106.6.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kern L., de Montigny J., Jund R., Lacroute F. The FUR1 gene of Saccharomyces cerevisiae: cloning, structure and expression of wild-type and mutant alleles. Gene. 1990 Apr 16;88(2):149–157. doi: 10.1016/0378-1119(90)90026-n. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Li Y. Y., Yeh E., Hays T., Bloom K. Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):10096–10100. doi: 10.1073/pnas.90.21.10096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McMillan J. N., Tatchell K. The JNM1 gene in the yeast Saccharomyces cerevisiae is required for nuclear migration and spindle orientation during the mitotic cell cycle. J Cell Biol. 1994 Apr;125(1):143–158. doi: 10.1083/jcb.125.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Melki R., Vainberg I. E., Chow R. L., Cowan N. J. Chaperonin-mediated folding of vertebrate actin-related protein and gamma-tubulin. J Cell Biol. 1993 Sep;122(6):1301–1310. doi: 10.1083/jcb.122.6.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meyerowitz E. M., Kankel D. R. A genetic analysis of visual system development in Drosophilia melanogaster. Dev Biol. 1978 Jan;62(1):112–142. doi: 10.1016/0012-1606(78)90096-9. [DOI] [PubMed] [Google Scholar]
  30. Munn A. L., Silveira L., Elgort M., Payne G. S. Viability of clathrin heavy-chain-deficient Saccharomyces cerevisiae is compromised by mutations at numerous loci: implications for the suppression hypothesis. Mol Cell Biol. 1991 Aug;11(8):3868–3878. doi: 10.1128/mcb.11.8.3868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Palmer R. E., Sullivan D. S., Huffaker T., Koshland D. Role of astral microtubules and actin in spindle orientation and migration in the budding yeast, Saccharomyces cerevisiae. J Cell Biol. 1992 Nov;119(3):583–593. doi: 10.1083/jcb.119.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Paschal B. M., Holzbaur E. L., Pfister K. K., Clark S., Meyer D. I., Vallee R. B. Characterization of a 50-kDa polypeptide in cytoplasmic dynein preparations reveals a complex with p150GLUED and a novel actin. J Biol Chem. 1993 Jul 15;268(20):15318–15323. [PubMed] [Google Scholar]
  34. Pierre P., Scheel J., Rickard J. E., Kreis T. E. CLIP-170 links endocytic vesicles to microtubules. Cell. 1992 Sep 18;70(6):887–900. doi: 10.1016/0092-8674(92)90240-d. [DOI] [PubMed] [Google Scholar]
  35. Plamann M., Minke P. F., Tinsley J. H., Bruno K. S. Cytoplasmic dynein and actin-related protein Arp1 are required for normal nuclear distribution in filamentous fungi. J Cell Biol. 1994 Oct;127(1):139–149. doi: 10.1083/jcb.127.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pollard T. D., Cooper J. A. Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem. 1986;55:987–1035. doi: 10.1146/annurev.bi.55.070186.005011. [DOI] [PubMed] [Google Scholar]
  37. Sawin K. E., Scholey J. M. Motor proteins in cell division. Trends Cell Biol. 1991 Nov;1(5):122–129. doi: 10.1016/0962-8924(91)90117-r. [DOI] [PubMed] [Google Scholar]
  38. Schroer T. A., Sheetz M. P. Two activators of microtubule-based vesicle transport. J Cell Biol. 1991 Dec;115(5):1309–1318. doi: 10.1083/jcb.115.5.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schroer T. A., Steuer E. R., Sheetz M. P. Cytoplasmic dynein is a minus end-directed motor for membranous organelles. Cell. 1989 Mar 24;56(6):937–946. doi: 10.1016/0092-8674(89)90627-2. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sullivan D. S., Huffaker T. C. Astral microtubules are not required for anaphase B in Saccharomyces cerevisiae. J Cell Biol. 1992 Oct;119(2):379–388. doi: 10.1083/jcb.119.2.379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Toyn J., Hibbs A. R., Sanz P., Crowe J., Meyer D. I. In vivo and in vitro analysis of ptl1, a yeast ts mutant with a membrane-associated defect in protein translocation. EMBO J. 1988 Dec 20;7(13):4347–4353. doi: 10.1002/j.1460-2075.1988.tb03333.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Vogelstein B. Rapid purification of DNA from agarose gels by centrifugation through a disposable plastic column. Anal Biochem. 1987 Jan;160(1):115–118. doi: 10.1016/0003-2697(87)90620-8. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Wertman K. F., Drubin D. G., Botstein D. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 1992 Oct;132(2):337–350. doi: 10.1093/genetics/132.2.337. [DOI] [PMC free article] [PubMed] [Google Scholar]

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