Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Aug 2;130(4):835–845. doi: 10.1083/jcb.130.4.835

A truncated form of the Pho80 cyclin redirects the Pho85 kinase to disrupt vacuole inheritance in S. cerevisiae

PMCID: PMC2199970  PMID: 7642701

Abstract

Partitioning of the vacuole during cell division in Saccharomyces cerevisiae begins during early S phase and ends in late G2 phase before the yeast nucleus migrates into the bud neck. We have isolated and characterized a new mutant, vac5-1, which is defective in vacuole segregation. Cells with the vac5-1 mutation can form large buds without vacuoles. The VAC5 gene was cloned and is identical to PHO80. PHO80 encodes a cyclin which acts in a complex with a cdc-like kinase, PHO85, as a negative regulator of two transcription factors (PHO2 and PHO4) that govern the expression of metabolic phosphatases. The vacuole inheritance defect in vac5-1 cells is dependent on the presence of the Pho85 kinase and its targets Pho4p and Pho2p. As with other alleles of PHO80, phosphatase levels are elevated in vac5-1 mutants. A suppressor, the COOH-terminal half of the Gal11 transcription factor, rescues the vac5-1 phenotype of defective vacuole inheritance without altering the vac5-1 phenotype of elevated phosphatase levels. In addition, neither maximal nor minimal levels of expression of the inducible "PHO" system phosphatases causes a vacuole inheritance defect. Though vac5-1 is recessive, pho80 delta or pho85 delta strains do not show a defect in vacuole inheritance, suggesting that vac5-1 is not a complete loss-of- function allele. Sequence analysis shows that the vac5-1 allele encodes a truncated form of the Pho80 cyclin and overexpression of vac5-1 in pho80 delta cells causes a vacuole inheritance defect. We conclude that the vac5-1 allele directs the Pho85 kinase to regulate, via transcription factors Pho4 and Pho2, genes that affect vacuole inheritance but which are not known to be under normal PHO pathway control.

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arndt K. T., Styles C., Fink G. R. Multiple global regulators control HIS4 transcription in yeast. Science. 1987 Aug 21;237(4817):874–880. doi: 10.1126/science.3303332. [DOI] [PubMed] [Google Scholar]
  2. Berben G., Legrain M., Hilger F. Studies on the structure, expression and function of the yeast regulatory gene PHO2. Gene. 1988 Jun 30;66(2):307–312. doi: 10.1016/0378-1119(88)90367-8. [DOI] [PubMed] [Google Scholar]
  3. Bergman L. W. A DNA fragment containing the upstream activator sequence determines nucleosome positioning of the transcriptionally repressed PHO5 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1986 Jul;6(7):2298–2304. doi: 10.1128/mcb.6.7.2298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berlin V., Styles C. A., Fink G. R. BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin. J Cell Biol. 1990 Dec;111(6 Pt 1):2573–2586. doi: 10.1083/jcb.111.6.2573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braus G., Mösch H. U., Vogel K., Hinnen A., Hütter R. Interpathway regulation of the TRP4 gene of yeast. EMBO J. 1989 Mar;8(3):939–945. doi: 10.1002/j.1460-2075.1989.tb03455.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brazas R. M., Stillman D. J. Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein. Mol Cell Biol. 1993 Sep;13(9):5524–5537. doi: 10.1128/mcb.13.9.5524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bun-Ya M., Nishimura M., Harashima S., Oshima Y. The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol. 1991 Jun;11(6):3229–3238. doi: 10.1128/mcb.11.6.3229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Burgess S. M., Delannoy M., Jensen R. E. MMM1 encodes a mitochondrial outer membrane protein essential for establishing and maintaining the structure of yeast mitochondria. J Cell Biol. 1994 Sep;126(6):1375–1391. doi: 10.1083/jcb.126.6.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  10. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Conradt B., Shaw J., Vida T., Emr S., Wickner W. In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol. 1992 Dec;119(6):1469–1479. doi: 10.1083/jcb.119.6.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Espinoza F. H., Ogas J., Herskowitz I., Morgan D. O. Cell cycle control by a complex of the cyclin HCS26 (PCL1) and the kinase PHO85. Science. 1994 Nov 25;266(5189):1388–1391. doi: 10.1126/science.7973730. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Haas A., Conradt B., Wickner W. G-protein ligands inhibit in vitro reactions of vacuole inheritance. J Cell Biol. 1994 Jul;126(1):87–97. doi: 10.1083/jcb.126.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hadwiger J. A., Wittenberg C., Richardson H. E., de Barros Lopes M., Reed S. I. A family of cyclin homologs that control the G1 phase in yeast. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6255–6259. doi: 10.1073/pnas.86.16.6255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hayashi N., Oshima Y. Specific cis-acting sequence for PHO8 expression interacts with PHO4 protein, a positive regulatory factor, in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Feb;11(2):785–794. doi: 10.1128/mcb.11.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hirst K., Fisher F., McAndrew P. C., Goding C. R. The transcription factor, the Cdk, its cyclin and their regulator: directing the transcriptional response to a nutritional signal. EMBO J. 1994 Nov 15;13(22):5410–5420. doi: 10.1002/j.1460-2075.1994.tb06876.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaffman A., Herskowitz I., Tjian R., O'Shea E. K. Phosphorylation of the transcription factor PHO4 by a cyclin-CDK complex, PHO80-PHO85. Science. 1994 Feb 25;263(5150):1153–1156. doi: 10.1126/science.8108735. [DOI] [PubMed] [Google Scholar]
  22. Kaplan K. B., Swedlow J. R., Varmus H. E., Morgan D. O. Association of p60c-src with endosomal membranes in mammalian fibroblasts. J Cell Biol. 1992 Jul;118(2):321–333. doi: 10.1083/jcb.118.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kim Y. J., Björklund S., Li Y., Sayre M. H., Kornberg R. D. A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II. Cell. 1994 May 20;77(4):599–608. doi: 10.1016/0092-8674(94)90221-6. [DOI] [PubMed] [Google Scholar]
  24. Klionsky D. J., Herman P. K., Emr S. D. The fungal vacuole: composition, function, and biogenesis. Microbiol Rev. 1990 Sep;54(3):266–292. doi: 10.1128/mr.54.3.266-292.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lemire J. M., Willcocks T., Halvorson H. O., Bostian K. A. Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1985 Aug;5(8):2131–2141. doi: 10.1128/mcb.5.8.2131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lipsky N. G., Pagano R. E. A vital stain for the Golgi apparatus. Science. 1985 May 10;228(4700):745–747. doi: 10.1126/science.2581316. [DOI] [PubMed] [Google Scholar]
  27. Long R. M., Mylin L. M., Hopper J. E. GAL11 (SPT13), a transcriptional regulator of diverse yeast genes, affects the phosphorylation state of GAL4, a highly specific transcriptional activator. Mol Cell Biol. 1991 Apr;11(4):2311–2314. doi: 10.1128/mcb.11.4.2311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lucocq J. M., Pryde J. G., Berger E. G., Warren G. A mitotic form of the Golgi apparatus in HeLa cells. J Cell Biol. 1987 Apr;104(4):865–874. doi: 10.1083/jcb.104.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Madden S. L., Creasy C. L., Srinivas V., Fawcett W., Bergman L. W. Structure and expression of the PHO80 gene of Saccharomyces cerevisiae. Nucleic Acids Res. 1988 Mar 25;16(6):2625–2637. doi: 10.1093/nar/16.6.2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Madden S. L., Johnson D. L., Bergman L. W. Molecular and expression analysis of the negative regulators involved in the transcriptional regulation of acid phosphatase production in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Nov;10(11):5950–5957. doi: 10.1128/mcb.10.11.5950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McConnell S. J., Yaffe M. P. Intermediate filament formation by a yeast protein essential for organelle inheritance. Science. 1993 Apr 30;260(5108):687–689. doi: 10.1126/science.8480179. [DOI] [PubMed] [Google Scholar]
  32. McConnell S. J., Yaffe M. P. Nuclear and mitochondrial inheritance in yeast depends on novel cytoplasmic structures defined by the MDM1 protein. J Cell Biol. 1992 Jul;118(2):385–395. doi: 10.1083/jcb.118.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Measday V., Moore L., Ogas J., Tyers M., Andrews B. The PCL2 (ORFD)-PHO85 cyclin-dependent kinase complex: a cell cycle regulator in yeast. Science. 1994 Nov 25;266(5189):1391–1395. doi: 10.1126/science.7973731. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Nash R., Tokiwa G., Anand S., Erickson K., Futcher A. B. The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 1988 Dec 20;7(13):4335–4346. doi: 10.1002/j.1460-2075.1988.tb03332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ogawa N., Oshima Y. Functional domains of a positive regulatory protein, PHO4, for transcriptional control of the phosphatase regulon in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2224–2236. doi: 10.1128/mcb.10.5.2224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ohsumi M., Uchiyama K., Ohsumi Y. Density fluctuation during the cell cycle in the defective vacuolar morphology mutants of Saccharomyces cerevisiae. J Bacteriol. 1993 Sep;175(17):5714–5716. doi: 10.1128/jb.175.17.5714-5716.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Okada H., Toh-e A. A novel mutation occurring in the PHO80 gene suppresses the PHO4c mutations of Saccharomyces cerevisiae. Curr Genet. 1992 Feb;21(2):95–99. doi: 10.1007/BF00318466. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Payne G. S., Hasson T. B., Hasson M. S., Schekman R. Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae. Mol Cell Biol. 1987 Nov;7(11):3888–3898. doi: 10.1128/mcb.7.11.3888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Preston R. A., Murphy R. F., Jones E. W. Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran. J Cell Biol. 1987 Nov;105(5):1981–1987. doi: 10.1083/jcb.105.5.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Raymond C. K., Roberts C. J., Moore K. E., Howald I., Stevens T. H. Biogenesis of the vacuole in Saccharomyces cerevisiae. Int Rev Cytol. 1992;139:59–120. doi: 10.1016/s0074-7696(08)61410-2. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Sakurai H., Hiraoka Y., Fukasawa T. Yeast GAL11 protein is a distinctive type transcription factor that enhances basal transcription in vitro. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8382–8386. doi: 10.1073/pnas.90.18.8382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Salama S. R., Hendricks K. B., Thorner J. G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover. Mol Cell Biol. 1994 Dec;14(12):7953–7966. doi: 10.1128/mcb.14.12.7953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sanger F. Determination of nucleotide sequences in DNA. Science. 1981 Dec 11;214(4526):1205–1210. doi: 10.1126/science.7302589. [DOI] [PubMed] [Google Scholar]
  51. Schneider K. R., Smith R. L., O'Shea E. K. Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81. Science. 1994 Oct 7;266(5182):122–126. doi: 10.1126/science.7939631. [DOI] [PubMed] [Google Scholar]
  52. Seeger M., Payne G. S. Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae. J Cell Biol. 1992 Aug;118(3):531–540. doi: 10.1083/jcb.118.3.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Sogo L. F., Yaffe M. P. Regulation of mitochondrial morphology and inheritance by Mdm10p, a protein of the mitochondrial outer membrane. J Cell Biol. 1994 Sep;126(6):1361–1373. doi: 10.1083/jcb.126.6.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. Toh-e A., Shimauchi T. Cloning and sequencing of the PHO80 gene and CEN15 of Saccharomyces cerevisiae. Yeast. 1986 Jun;2(2):129–139. doi: 10.1002/yea.320020209. [DOI] [PubMed] [Google Scholar]
  57. Toh-e A., Tanaka K., Uesono Y., Wickner R. B. PHO85, a negative regulator of the PHO system, is a homolog of the protein kinase gene, CDC28, of Saccharomyces cerevisiae. Mol Gen Genet. 1988 Sep;214(1):162–164. doi: 10.1007/BF00340196. [DOI] [PubMed] [Google Scholar]
  58. Tyers M., Tokiwa G., Nash R., Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. doi: 10.1002/j.1460-2075.1992.tb05229.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ueda Y., To-E A., Oshima Y. Isolation and characterization of recessive, constitutive mutations for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. J Bacteriol. 1975 Jun;122(3):911–922. doi: 10.1128/jb.122.3.911-922.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Uesono Y., Tanaka K., Toh-e A. Negative regulators of the PHO system in Saccharomyces cerevisiae: isolation and structural characterization of PHO85. Nucleic Acids Res. 1987 Dec 23;15(24):10299–10309. doi: 10.1093/nar/15.24.10299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Warren G. Membrane partitioning during cell division. Annu Rev Biochem. 1993;62:323–348. doi: 10.1146/annurev.bi.62.070193.001543. [DOI] [PubMed] [Google Scholar]
  62. 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]
  63. 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]
  64. 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]
  65. 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]
  66. Yoshida K., Ogawa N., Oshima Y. Function of the PHO regulatory genes for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. Mol Gen Genet. 1989 May;217(1):40–46. doi: 10.1007/BF00330940. [DOI] [PubMed] [Google Scholar]
  67. Zeligs J. D., Wollman S. H. Mitosis in rat thyroid epithelial cells in vivo. I. Ultrastructural changes in cytoplasmic organelles during the mitotic cycle. J Ultrastruct Res. 1979 Jan;66(1):53–77. doi: 10.1016/s0022-5320(79)80065-9. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES