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. 1997 Aug;8(8):1481–1499. doi: 10.1091/mbc.8.8.1481

Yeast actin cytoskeleton mutants accumulate a new class of Golgi-derived secretary vesicle.

J Mulholland 1, A Wesp 1, H Riezman 1, D Botstein 1
PMCID: PMC276171  PMID: 9285820

Abstract

Many yeast actin cytoskeleton mutants accumulate large secretory vesicles and exhibit phenotypes consistent with defects in polarized growth. This, together with actin's polarized organization, has suggested a role for the actin cytoskeleton in the vectorial transport of late secretory vesicles to the plasma membrane. By using ultrastructural and biochemical analysis, we have characterized defects manifested by mutations in the SLA2 gene (also known as the END4 gene), previously found to affect both the organization of the actin cytoskeleton and endocytosis in yeast. Defects in cell wall morphology, accumulated vesicles, and protein secretion kinetics were found in sla2 mutants similar to defects found in act1 mutants. Vesicles that accumulate in the sla2 and act1 mutants are immunoreactive with antibodies directed against the small GTPase Ypt1p but not with antibodies directed against the homologous Sec4p found on classical "late" secretory vesicles. In contrast, the late-acting secretory mutants sec1-1 and sec6-4 are shown to accumulate anti-Sec4p-positive secretory vesicles as well as vesicles that are immunoreactive with antibodies directed against Ypt1p. The late sec mutant sec4-8 is also shown to accumulate Ypt1p-containing vesicles and to exhibit defects in actin cytoskeleton organization. These results indicate the existence of at least two classes of morphologically similar, late secretory vesicles (associated with Ypt1p+ and Sec4p+, respectively), one of which appears to accumulate when the actin cytoskeleton is disorganized.

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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 A. E., Botstein D., Drubin D. G. A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation. Science. 1989 Jan 13;243(4888):231–233. doi: 10.1126/science.2643162. [DOI] [PubMed] [Google Scholar]
  2. Adams A. E., Botstein D., Drubin D. G. Requirement of yeast fimbrin for actin organization and morphogenesis in vivo. Nature. 1991 Dec 5;354(6352):404–408. doi: 10.1038/354404a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Baba M., Baba N., Ohsumi Y., Kanaya K., Osumi M. Three-dimensional analysis of morphogenesis induced by mating pheromone alpha factor in Saccharomyces cerevisiae. J Cell Sci. 1989 Oct;94(Pt 2):207–216. doi: 10.1242/jcs.94.2.207. [DOI] [PubMed] [Google Scholar]
  5. Bacon R. A., Salminen A., Ruohola H., Novick P., Ferro-Novick S. The GTP-binding protein Ypt1 is required for transport in vitro: the Golgi apparatus is defective in ypt1 mutants. J Cell Biol. 1989 Sep;109(3):1015–1022. doi: 10.1083/jcb.109.3.1015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chant J. Septin scaffolds and cleavage planes in Saccharomyces. Cell. 1996 Jan 26;84(2):187–190. doi: 10.1016/s0092-8674(00)80972-1. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drubin D. G., Nelson W. J. Origins of cell polarity. Cell. 1996 Feb 9;84(3):335–344. doi: 10.1016/s0092-8674(00)81278-7. [DOI] [PubMed] [Google Scholar]
  10. Dunn B., Stearns T., Botstein D. Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4. Nature. 1993 Apr 8;362(6420):563–565. doi: 10.1038/362563a0. [DOI] [PubMed] [Google Scholar]
  11. Esmon B., Novick P., Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. doi: 10.1016/0092-8674(81)90063-5. [DOI] [PubMed] [Google Scholar]
  12. Franzusoff A., Schekman R. Functional compartments of the yeast Golgi apparatus are defined by the sec7 mutation. EMBO J. 1989 Sep;8(9):2695–2702. doi: 10.1002/j.1460-2075.1989.tb08410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Graham T. R., Emr S. D. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol. 1991 Jul;114(2):207–218. doi: 10.1083/jcb.114.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haarer B. K., Corbett A., Kweon Y., Petzold A. S., Silver P., Brown S. S. SEC3 mutations are synthetically lethal with profilin mutations and cause defects in diploid-specific bud-site selection. Genetics. 1996 Oct;144(2):495–510. doi: 10.1093/genetics/144.2.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harsay E., Bretscher A. Parallel secretory pathways to the cell surface in yeast. J Cell Biol. 1995 Oct;131(2):297–310. doi: 10.1083/jcb.131.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Jedd G., Richardson C., Litt R., Segev N. The Ypt1 GTPase is essential for the first two steps of the yeast secretory pathway. J Cell Biol. 1995 Nov;131(3):583–590. doi: 10.1083/jcb.131.3.583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. 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]
  25. Munn A. L., Riezman H. Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes. J Cell Biol. 1994 Oct;127(2):373–386. doi: 10.1083/jcb.127.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. 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]
  30. 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]
  31. Preuss D., Mulholland J., Franzusoff A., Segev N., Botstein D. Characterization of the Saccharomyces Golgi complex through the cell cycle by immunoelectron microscopy. Mol Biol Cell. 1992 Jul;3(7):789–803. doi: 10.1091/mbc.3.7.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Read E. B., Okamura H. H., Drubin D. G. Actin- and tubulin-dependent functions during Saccharomyces cerevisiae mating projection formation. Mol Biol Cell. 1992 Apr;3(4):429–444. doi: 10.1091/mbc.3.4.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  36. Schimmöller F., Singer-Krüger B., Schröder S., Krüger U., Barlowe C., Riezman H. The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO J. 1995 Apr 3;14(7):1329–1339. doi: 10.1002/j.1460-2075.1995.tb07119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Schmitt H. D., Puzicha M., Gallwitz D. Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell. 1988 May 20;53(4):635–647. doi: 10.1016/0092-8674(88)90579-x. [DOI] [PubMed] [Google Scholar]
  38. Segev N., Botstein D. The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. Mol Cell Biol. 1987 Jul;7(7):2367–2377. doi: 10.1128/mcb.7.7.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Walworth N. C., Brennwald P., Kabcenell A. K., Garrett M., Novick P. Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae. Mol Cell Biol. 1992 May;12(5):2017–2028. doi: 10.1128/mcb.12.5.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Wright R., Rine J. Transmission electron microscopy and immunocytochemical studies of yeast: analysis of HMG-CoA reductase overproduction by electron microscopy. Methods Cell Biol. 1989;31:473–512. doi: 10.1016/s0091-679x(08)61624-6. [DOI] [PubMed] [Google Scholar]

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