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. 1996 Dec 2;135(6):1535–1549. doi: 10.1083/jcb.135.6.1535

Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae

PMCID: PMC2133941  PMID: 8978821

Abstract

During cell division, cytoplasmic organelles are not synthesized de novo, rather they are replicated and partitioned between daughter cells. Partitioning of the vacuole in the budding yeast Saccharomyces cerevisiae is coordinated with the cell cycle and involves a dramatic translocation of a portion of the parental organelle from the mother cell into the bud. While the molecular mechanisms that mediate this event are unknown, the vacuole's rapid and directed movements suggest cytoskeleton involvement. To identify cytoskeletal components that function in this process, vacuole inheritance was examined in a collection of actin mutants. Six strains were identified as being defective in vacuole inheritance. Tetrad analysis verified that the defect cosegregates with the mutant actin gene. One strain with a deletion in a myosin-binding region was analyzed further. The vacuole inheritance defect in this strain appears to result from the loss of a specific actin function; the actin cytoskeleton is intact and protein targeting to the vacuole is normal. Consistent with these findings, a mutation in the actin-binding domain of Myo2p, a class V unconventional myosin, abolishes vacuole inheritance. This suggests that Myo2p serves as a molecular motor for vacuole transport along actin filaments. The location of actin and Myo2p relative to the vacuole membrane is consistent with this model. Additional studies suggest that the actin filaments used for vacuole transport are dynamic, and that profilin plays a critical role in regulating their assembly. These results present the first demonstration that specific cytoskeletal proteins function in vacuole inheritance.

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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. Adams A. E., Pringle J. R. Staining of actin with fluorochrome-conjugated phalloidin. Methods Enzymol. 1991;194:729–731. doi: 10.1016/0076-6879(91)94054-g. [DOI] [PubMed] [Google Scholar]
  3. Allen R. D., Weiss D. G., Hayden J. H., Brown D. T., Fujiwake H., Simpson M. Gliding movement of and bidirectional transport along single native microtubules from squid axoplasm: evidence for an active role of microtubules in cytoplasmic transport. J Cell Biol. 1985 May;100(5):1736–1752. doi: 10.1083/jcb.100.5.1736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Ault J. G., Rieder C. L. Centrosome and kinetochore movement during mitosis. Curr Opin Cell Biol. 1994 Feb;6(1):41–49. doi: 10.1016/0955-0674(94)90114-7. [DOI] [PubMed] [Google Scholar]
  6. Bearer E. L., DeGiorgis J. A., Bodner R. A., Kao A. W., Reese T. S. Evidence for myosin motors on organelles in squid axoplasm. Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11252–11256. doi: 10.1073/pnas.90.23.11252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brockerhoff S. E., Stevens R. C., Davis T. N. The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae. J Cell Biol. 1994 Feb;124(3):315–323. doi: 10.1083/jcb.124.3.315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cook R. K., Blake W. T., Rubenstein P. A. Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. J Biol Chem. 1992 May 5;267(13):9430–9436. [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Fedor-Chaiken M., Deschenes R. J., Broach J. R. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell. 1990 Apr 20;61(2):329–340. doi: 10.1016/0092-8674(90)90813-t. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gomes de Mesquita D. S., ten Hoopen R., Woldringh C. L. Vacuolar segregation to the bud of Saccharomyces cerevisiae: an analysis of morphology and timing in the cell cycle. J Gen Microbiol. 1991 Oct;137(10):2447–2454. doi: 10.1099/00221287-137-10-2447. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Haarer B. K., Petzold A., Lillie S. H., Brown S. S. Identification of MYO4, a second class V myosin gene in yeast. J Cell Sci. 1994 Apr;107(Pt 4):1055–1064. doi: 10.1242/jcs.107.4.1055. [DOI] [PubMed] [Google Scholar]
  19. Haas A., Scheglmann D., Lazar T., Gallwitz D., Wickner W. The GTPase Ypt7p of Saccharomyces cerevisiae is required on both partner vacuoles for the homotypic fusion step of vacuole inheritance. EMBO J. 1995 Nov 1;14(21):5258–5270. doi: 10.1002/j.1460-2075.1995.tb00210.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Haas A., Wickner W. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF). EMBO J. 1996 Jul 1;15(13):3296–3305. [PMC free article] [PubMed] [Google Scholar]
  21. Horazdovsky B. F., Emr S. D. The VPS16 gene product associates with a sedimentable protein complex and is essential for vacuolar protein sorting in yeast. J Biol Chem. 1993 Mar 5;268(7):4953–4962. [PubMed] [Google Scholar]
  22. Jansen R. P., Dowzer C., Michaelis C., Galova M., Nasmyth K. Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins. Cell. 1996 Mar 8;84(5):687–697. doi: 10.1016/s0092-8674(00)81047-8. [DOI] [PubMed] [Google Scholar]
  23. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones H. D., Schliwa M., Drubin D. G. Video microscopy of organelle inheritance and motility in budding yeast. Cell Motil Cytoskeleton. 1993;25(2):129–142. doi: 10.1002/cm.970250203. [DOI] [PubMed] [Google Scholar]
  25. Karpova T. S., Tatchell K., Cooper J. A. Actin filaments in yeast are unstable in the absence of capping protein or fimbrin. J Cell Biol. 1995 Dec;131(6 Pt 1):1483–1493. doi: 10.1083/jcb.131.6.1483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Klionsky D. J., Banta L. M., Emr S. D. Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol. 1988 May;8(5):2105–2116. doi: 10.1128/mcb.8.5.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kuznetsov S. A., Langford G. M., Weiss D. G. Actin-dependent organelle movement in squid axoplasm. Nature. 1992 Apr 23;356(6371):722–725. doi: 10.1038/356722a0. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Langford G. M. Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Curr Opin Cell Biol. 1995 Feb;7(1):82–88. doi: 10.1016/0955-0674(95)80048-4. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Liu H., Bretscher A. Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport. J Cell Biol. 1992 Jul;118(2):285–299. doi: 10.1083/jcb.118.2.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McConnell S. J., Stewart L. C., Talin A., Yaffe M. P. Temperature-sensitive yeast mutants defective in mitochondrial inheritance. J Cell Biol. 1990 Sep;111(3):967–976. doi: 10.1083/jcb.111.3.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. 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]
  36. 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]
  37. 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]
  38. 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]
  39. Raymond C. K., Howald-Stevenson I., Vater C. A., Stevens T. H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992 Dec;3(12):1389–1402. doi: 10.1091/mbc.3.12.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Raymond C. K., O'Hara P. J., Eichinger G., Rothman J. H., Stevens T. H. Molecular analysis of the yeast VPS3 gene and the role of its product in vacuolar protein sorting and vacuolar segregation during the cell cycle. J Cell Biol. 1990 Sep;111(3):877–892. doi: 10.1083/jcb.111.3.877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Robinson J. S., Klionsky D. J., Banta L. M., Emr S. D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. doi: 10.1128/mcb.8.11.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schutt C. E., Myslik J. C., Rozycki M. D., Goonesekere N. C., Lindberg U. The structure of crystalline profilin-beta-actin. Nature. 1993 Oct 28;365(6449):810–816. doi: 10.1038/365810a0. [DOI] [PubMed] [Google Scholar]
  43. Shaw J. M., Wickner W. T. vac2: a yeast mutant which distinguishes vacuole segregation from Golgi-to-vacuole protein targeting. EMBO J. 1991 Jul;10(7):1741–1748. doi: 10.1002/j.1460-2075.1991.tb07698.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Simon V. R., Swayne T. C., Pon L. A. Actin-dependent mitochondrial motility in mitotic yeast and cell-free systems: identification of a motor activity on the mitochondrial surface. J Cell Biol. 1995 Jul;130(2):345–354. doi: 10.1083/jcb.130.2.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sutoh K. Identification of myosin-binding sites on the actin sequence. Biochemistry. 1982 Jul 20;21(15):3654–3661. doi: 10.1021/bi00258a020. [DOI] [PubMed] [Google Scholar]
  46. Titus M. A. From fat yeast and nervous mice to brain myosin-V. Cell. 1993 Oct 8;75(1):9–11. [PubMed] [Google Scholar]
  47. Trayer I. P., Trayer H. R., Levine B. A. Evidence that the N-terminal region of A1-light chain of myosin interacts directly with the C-terminal region of actin. A proton magnetic resonance study. Eur J Biochem. 1987 Apr 1;164(1):259–266. doi: 10.1111/j.1432-1033.1987.tb11019.x. [DOI] [PubMed] [Google Scholar]
  48. Vandekerckhove J. S., Kaiser D. A., Pollard T. D. Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin. J Cell Biol. 1989 Aug;109(2):619–626. doi: 10.1083/jcb.109.2.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Vida T. A., Emr S. D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol. 1995 Mar;128(5):779–792. doi: 10.1083/jcb.128.5.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vida T. A., Graham T. R., Emr S. D. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J Cell Biol. 1990 Dec;111(6 Pt 2):2871–2884. doi: 10.1083/jcb.111.6.2871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Wang Y. X., Zhao H., Harding T. M., Gomes de Mesquita D. S., Woldringh C. L., Klionsky D. J., Munn A. L., Weisman L. S. Multiple classes of yeast mutants are defective in vacuole partitioning yet target vacuole proteins correctly. Mol Biol Cell. 1996 Sep;7(9):1375–1389. doi: 10.1091/mbc.7.9.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Warren G., Wickner W. Organelle inheritance. Cell. 1996 Feb 9;84(3):395–400. doi: 10.1016/s0092-8674(00)81284-2. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Weisman L. S., Emr S. D., Wickner W. T. Mutants of Saccharomyces cerevisiae that block intervacuole vesicular traffic and vacuole division and segregation. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1076–1080. doi: 10.1073/pnas.87.3.1076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weisman L. S., Wickner W. Intervacuole exchange in the yeast zygote: a new pathway in organelle communication. Science. 1988 Jul 29;241(4865):589–591. doi: 10.1126/science.3041591. [DOI] [PubMed] [Google Scholar]
  57. Weisman L. S., Wickner W. Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. J Biol Chem. 1992 Jan 5;267(1):618–623. [PubMed] [Google Scholar]
  58. 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]
  59. Xu Z., Wickner W. Thioredoxin is required for vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol. 1996 Mar;132(5):787–794. doi: 10.1083/jcb.132.5.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yaffe M. P. Organelle inheritance in the yeast cell cycle. Trends Cell Biol. 1991 Dec;1(6):160–164. doi: 10.1016/0962-8924(91)90017-4. [DOI] [PubMed] [Google Scholar]

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