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. 1996 Oct;144(2):495–510. doi: 10.1093/genetics/144.2.495

Sec3 Mutations Are Synthetically Lethal with Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection

B K Haarer 1, A Corbett 1, Y Kweon 1, A S Petzold 1, P Silver 1, S S Brown 1
PMCID: PMC1207545  PMID: 8889515

Abstract

Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids.

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Selected References

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  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. Amberg D. C., Basart E., Botstein D. Defining protein interactions with yeast actin in vivo. Nat Struct Biol. 1995 Jan;2(1):28–35. doi: 10.1038/nsb0195-28. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bauer F., Urdaci M., Aigle M., Crouzet M. Alteration of a yeast SH3 protein leads to conditional viability with defects in cytoskeletal and budding patterns. Mol Cell Biol. 1993 Aug;13(8):5070–5084. doi: 10.1128/mcb.13.8.5070. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bender A., Pringle J. R. Use of a screen for synthetic lethal and multicopy suppressee mutants to identify two new genes involved in morphogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Mar;11(3):1295–1305. doi: 10.1128/mcb.11.3.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bretscher A., Drees B., Harsay E., Schott D., Wang T. What are the basic functions of microfilaments? Insights from studies in budding yeast. J Cell Biol. 1994 Aug;126(4):821–825. doi: 10.1083/jcb.126.4.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. 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]
  10. Chant J., Mischke M., Mitchell E., Herskowitz I., Pringle J. R. Role of Bud3p in producing the axial budding pattern of yeast. J Cell Biol. 1995 May;129(3):767–778. doi: 10.1083/jcb.129.3.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chant J., Pringle J. R. Patterns of bud-site selection in the yeast Saccharomyces cerevisiae. J Cell Biol. 1995 May;129(3):751–765. doi: 10.1083/jcb.129.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chowdhury S., Smith K. W., Gustin M. C. Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J Cell Biol. 1992 Aug;118(3):561–571. doi: 10.1083/jcb.118.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Cossart P. Actin-based bacterial motility. Curr Opin Cell Biol. 1995 Feb;7(1):94–101. doi: 10.1016/0955-0674(95)80050-6. [DOI] [PubMed] [Google Scholar]
  14. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Flick J. S., Thorner J. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Sep;13(9):5861–5876. doi: 10.1128/mcb.13.9.5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Freeman N. L., Chen Z., Horenstein J., Weber A., Field J. An actin monomer binding activity localizes to the carboxyl-terminal half of the Saccharomyces cerevisiae cyclase-associated protein. J Biol Chem. 1995 Mar 10;270(10):5680–5685. doi: 10.1074/jbc.270.10.5680. [DOI] [PubMed] [Google Scholar]
  17. Fujita A., Oka C., Arikawa Y., Katagai T., Tonouchi A., Kuhara S., Misumi Y. A yeast gene necessary for bud-site selection encodes a protein similar to insulin-degrading enzymes. Nature. 1994 Dec 8;372(6506):567–570. doi: 10.1038/372567a0. [DOI] [PubMed] [Google Scholar]
  18. Gerst J. E., Ferguson K., Vojtek A., Wigler M., Field J. CAP is a bifunctional component of the Saccharomyces cerevisiae adenylyl cyclase complex. Mol Cell Biol. 1991 Mar;11(3):1248–1257. doi: 10.1128/mcb.11.3.1248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gerst J. E., Rodgers L., Riggs M., Wigler M. SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4338–4342. doi: 10.1073/pnas.89.10.4338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Govindan B., Bowser R., Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. doi: 10.1083/jcb.128.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Grundmann U., Nerlich C., Rein T., Lottspeich F., Küpper H. A. Isolation of cDNA coding for the placental protein 15 (PP15). Nucleic Acids Res. 1988 May 25;16(10):4721–4721. doi: 10.1093/nar/16.10.4721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Haarer B. K., Lillie S. H., Adams A. E., Magdolen V., Bandlow W., Brown S. S. Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells. J Cell Biol. 1990 Jan;110(1):105–114. doi: 10.1083/jcb.110.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Haarer B. K., Petzold A. S., Brown S. S. Mutational analysis of yeast profilin. Mol Cell Biol. 1993 Dec;13(12):7864–7873. doi: 10.1128/mcb.13.12.7864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lew D. J., Reed S. I. A cell cycle checkpoint monitors cell morphogenesis in budding yeast. J Cell Biol. 1995 May;129(3):739–749. doi: 10.1083/jcb.129.3.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lillie S. H., Brown S. S. Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J Cell Biol. 1994 May;125(4):825–842. doi: 10.1083/jcb.125.4.825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lillie S. H., Brown S. S. Suppression of a myosin defect by a kinesin-related gene. Nature. 1992 Mar 26;356(6367):358–361. doi: 10.1038/356358a0. [DOI] [PubMed] [Google Scholar]
  29. Lillie S. H., Pringle J. R. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol. 1980 Sep;143(3):1384–1394. doi: 10.1128/jb.143.3.1384-1394.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ma H., Kunes S., Schatz P. J., Botstein D. Plasmid construction by homologous recombination in yeast. Gene. 1987;58(2-3):201–216. doi: 10.1016/0378-1119(87)90376-3. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Moore M. S., Blobel G. Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10212–10216. doi: 10.1073/pnas.91.21.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Na S., Hincapie M., McCusker J. H., Haber J. E. MOP2 (SLA2) affects the abundance of the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae. J Biol Chem. 1995 Mar 24;270(12):6815–6823. doi: 10.1074/jbc.270.12.6815. [DOI] [PubMed] [Google Scholar]
  36. Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
  37. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  38. Protopopov V., Govindan B., Novick P., Gerst J. E. Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae. Cell. 1993 Sep 10;74(5):855–861. doi: 10.1016/0092-8674(93)90465-3. [DOI] [PubMed] [Google Scholar]
  39. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  40. Sohn R. H., Goldschmidt-Clermont P. J. Profilin: at the crossroads of signal transduction and the actin cytoskeleton. Bioessays. 1994 Jul;16(7):465–472. doi: 10.1002/bies.950160705. [DOI] [PubMed] [Google Scholar]
  41. Vojtek A., Haarer B., Field J., Gerst J., Pollard T. D., Brown S., Wigler M. Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae. Cell. 1991 Aug 9;66(3):497–505. doi: 10.1016/0092-8674(81)90013-1. [DOI] [PubMed] [Google Scholar]

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