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. 1997 Dec;17(12):6847–6858. doi: 10.1128/mcb.17.12.6847

Vac7p, a novel vacuolar protein, is required for normal vacuole inheritance and morphology.

C J Bonangelino 1, N L Catlett 1, L S Weisman 1
PMCID: PMC232541  PMID: 9372916

Abstract

During cell division, the vacuole of Saccharomyces cerevisiae partitions between mother and daughter cells. A portion of the parental vacuole membrane moves into the bud, and ultimately membrane scission divides the vacuole into two separate structures. Here we characterize two yeast mutations causing defects in vacuole membrane scission, vac7-1 and vac14-1. A third mutant, afab1-2 strain, isolated in a nonrelated screen (A. Yamamoto et al., Mol. Biol. Cell 6:525-539, 1995) shares the vacuolar phenotypes of the vac7-1 and vac14-1 strains. Unlike the wild type, mutant vacuoles are not multilobed structures; in many cases, a single vacuole spans both the mother and bud, with a distinct gap in the mother-bud neck. Thus, even where the membranes are closely opposed, vacuole fission is arrested. Simply enlarging the vacuole does not produce this mutant phenotype. An additional common phenotype of these mutants is a defect in vacuole acidification; however, vacuole scission in most other vacuole acidification mutants is normal. An alteration in vacuole membrane lipids could account for both the vacuole membrane scission and acidification defects. Because a directed screen has not identified additional class III complementation groups, it is likely that all three genes are involved in a similar process. Interestingly, FAB1, was previously shown to encode a putative phosphatidylinositol-4-phosphate 5-kinase. Moreover, overexpression of FAB1 suppresses the vac14-1 mutation, which suggests that VAC14 and FAB1 act at a common step. VAC7 encodes a novel 128-kDa protein that is localized at the vacuole membrane. This location of Vac7p is consistent with its involvement in vacuole morphology and inheritance.

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Selected References

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  1. Acharya U., Jacobs R., Peters J. M., Watson N., Farquhar M. G., Malhotra V. The formation of Golgi stacks from vesiculated Golgi membranes requires two distinct fusion events. Cell. 1995 Sep 22;82(6):895–904. doi: 10.1016/0092-8674(95)90269-4. [DOI] [PubMed] [Google Scholar]
  2. Banta L. M., Robinson J. S., Klionsky D. J., Emr S. D. Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol. 1988 Oct;107(4):1369–1383. doi: 10.1083/jcb.107.4.1369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baudin A., Ozier-Kalogeropoulos O., Denouel A., Lacroute F., Cullin C. A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 1993 Jul 11;21(14):3329–3330. doi: 10.1093/nar/21.14.3329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belde P. J., Vossen J. H., Borst-Pauwels G. W., Theuvenet A. P. Inositol 1,4,5-trisphosphate releases Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. FEBS Lett. 1993 May 24;323(1-2):113–118. doi: 10.1016/0014-5793(93)81460-h. [DOI] [PubMed] [Google Scholar]
  5. Bergez P., Doignon F., Crouzet M. The sequence of a 44 420 bp fragment located on the left arm of chromosome XIV from Saccharomyces cerevisiae. Yeast. 1995 Aug;11(10):967–974. doi: 10.1002/yea.320111008. [DOI] [PubMed] [Google Scholar]
  6. Berkower C., Loayza D., Michaelis S. Metabolic instability and constitutive endocytosis of STE6, the a-factor transporter of Saccharomyces cerevisiae. Mol Biol Cell. 1994 Nov;5(11):1185–1198. doi: 10.1091/mbc.5.11.1185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cardenas M. E., Heitman J. FKBP12-rapamycin target TOR2 is a vacuolar protein with an associated phosphatidylinositol-4 kinase activity. EMBO J. 1995 Dec 1;14(23):5892–5907. doi: 10.1002/j.1460-2075.1995.tb00277.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conibear E., Stevens T. H. Vacuolar biogenesis in yeast: sorting out the sorting proteins. Cell. 1995 Nov 17;83(4):513–516. doi: 10.1016/0092-8674(95)90088-8. [DOI] [PubMed] [Google Scholar]
  9. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  10. De Camilli P. The eighth Datta Lecture. Molecular mechanisms in synaptic vesicle recycling. FEBS Lett. 1995 Aug 1;369(1):3–12. doi: 10.1016/0014-5793(95)00739-v. [DOI] [PubMed] [Google Scholar]
  11. Düzgüneş N., Wilschut J., Fraley R., Papahadjopoulos D. Studies on the mechanism of membrane fusion. Role of head-group composition in calcium- and magnesium-induced fusion of mixed phospholipid vesicles. Biochim Biophys Acta. 1981 Mar 20;642(1):182–195. doi: 10.1016/0005-2736(81)90148-6. [DOI] [PubMed] [Google Scholar]
  12. Gammie A. E., Kurihara L. J., Vallee R. B., Rose M. D. DNM1, a dynamin-related gene, participates in endosomal trafficking in yeast. J Cell Biol. 1995 Aug;130(3):553–566. doi: 10.1083/jcb.130.3.553. [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. Gomes de Mesquita D. S., van den Hazel H. B., Bouwman J., Woldringh C. L. Characterization of new vacuolar segregation mutants, isolated by screening for loss of proteinase B self-activation. Eur J Cell Biol. 1996 Nov;71(3):237–247. [PubMed] [Google Scholar]
  16. Guan K., Farh L., Marshall T. K., Deschenes R. J. Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene. Curr Genet. 1993 Jul-Aug;24(1-2):141–148. doi: 10.1007/BF00324678. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hay J. C., Fisette P. L., Jenkins G. H., Fukami K., Takenawa T., Anderson R. A., Martin T. F. ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion. Nature. 1995 Mar 9;374(6518):173–177. doi: 10.1038/374173a0. [DOI] [PubMed] [Google Scholar]
  19. Heitman J., Movva N. R., Hall M. N. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science. 1991 Aug 23;253(5022):905–909. doi: 10.1126/science.1715094. [DOI] [PubMed] [Google Scholar]
  20. Hill K. L., Catlett N. L., Weisman L. S. Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae. J Cell Biol. 1996 Dec;135(6 Pt 1):1535–1549. doi: 10.1083/jcb.135.6.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones B. A., Fangman W. L. Mitochondrial DNA maintenance in yeast requires a protein containing a region related to the GTP-binding domain of dynamin. Genes Dev. 1992 Mar;6(3):380–389. doi: 10.1101/gad.6.3.380. [DOI] [PubMed] [Google Scholar]
  22. Kane P. M., Kuehn M. C., Howald-Stevenson I., Stevens T. H. Assembly and targeting of peripheral and integral membrane subunits of the yeast vacuolar H(+)-ATPase. J Biol Chem. 1992 Jan 5;267(1):447–454. [PubMed] [Google Scholar]
  23. Klionsky D. J., Nelson H., Nelson N. Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae. J Biol Chem. 1992 Feb 15;267(5):3416–3422. [PubMed] [Google Scholar]
  24. Koenig J. H., Ikeda K. Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci. 1989 Nov;9(11):3844–3860. doi: 10.1523/JNEUROSCI.09-11-03844.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Latterich M., Fröhlich K. U., Schekman R. Membrane fusion and the cell cycle: Cdc48p participates in the fusion of ER membranes. Cell. 1995 Sep 22;82(6):885–893. doi: 10.1016/0092-8674(95)90268-6. [DOI] [PubMed] [Google Scholar]
  27. Liscovitch M., Chalifa V., Pertile P., Chen C. S., Cantley L. C. Novel function of phosphatidylinositol 4,5-bisphosphate as a cofactor for brain membrane phospholipase D. J Biol Chem. 1994 Aug 26;269(34):21403–21406. [PubMed] [Google Scholar]
  28. Ohashi M., Jan de Vries K., Frank R., Snoek G., Bankaitis V., Wirtz K., Huttner W. B. A role for phosphatidylinositol transfer protein in secretory vesicle formation. Nature. 1995 Oct 12;377(6549):544–547. doi: 10.1038/377544a0. [DOI] [PubMed] [Google Scholar]
  29. Rabouille C., Levine T. P., Peters J. M., Warren G. An NSF-like ATPase, p97, and NSF mediate cisternal regrowth from mitotic Golgi fragments. Cell. 1995 Sep 22;82(6):905–914. doi: 10.1016/0092-8674(95)90270-8. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Rothman J. E., Wieland F. T. Protein sorting by transport vesicles. Science. 1996 Apr 12;272(5259):227–234. doi: 10.1126/science.272.5259.227. [DOI] [PubMed] [Google Scholar]
  33. Rothman J. H., Raymond C. K., Gilbert T., O'Hara P. J., Stevens T. H. A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting. Cell. 1990 Jun 15;61(6):1063–1074. doi: 10.1016/0092-8674(90)90070-u. [DOI] [PubMed] [Google Scholar]
  34. Schneider B. L., Seufert W., Steiner B., Yang Q. H., Futcher A. B. Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast. 1995 Oct;11(13):1265–1274. doi: 10.1002/yea.320111306. [DOI] [PubMed] [Google Scholar]
  35. Sears L. E., Moran L. S., Kissinger C., Creasey T., Perry-O'Keefe H., Roskey M., Sutherland E., Slatko B. E. CircumVent thermal cycle sequencing and alternative manual and automated DNA sequencing protocols using the highly thermostable VentR (exo-) DNA polymerase. Biotechniques. 1992 Oct;13(4):626–633. [PubMed] [Google Scholar]
  36. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Simon J. P., Ivanov I. E., Adesnik M., Sabatini D. D. The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity. J Cell Biol. 1996 Oct;135(2):355–370. doi: 10.1083/jcb.135.2.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Skinner H. B., McGee T. P., McMaster C. R., Fry M. R., Bell R. M., Bankaitis V. A. The Saccharomyces cerevisiae phosphatidylinositol-transfer protein effects a ligand-dependent inhibition of choline-phosphate cytidylyltransferase activity. Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):112–116. doi: 10.1073/pnas.92.1.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stack J. H., Horazdovsky B., Emr S. D. Receptor-mediated protein sorting to the vacuole in yeast: roles for a protein kinase, a lipid kinase and GTP-binding proteins. Annu Rev Cell Dev Biol. 1995;11:1–33. doi: 10.1146/annurev.cb.11.110195.000245. [DOI] [PubMed] [Google Scholar]
  40. Sullivan K. M., Busa W. B., Wilson K. L. Calcium mobilization is required for nuclear vesicle fusion in vitro: implications for membrane traffic and IP3 receptor function. Cell. 1993 Jul 2;73(7):1411–1422. doi: 10.1016/0092-8674(93)90366-x. [DOI] [PubMed] [Google Scholar]
  41. Sundler R., Düzgüneş N., Papahadjopoulos D. Control of membrane fusion by phospholipid head groups. II. The role of phosphatidylethanolamine in mixtures with phosphatidate and phosphatidylinositol. Biochim Biophys Acta. 1981 Dec 21;649(3):751–758. doi: 10.1016/0005-2736(81)90180-2. [DOI] [PubMed] [Google Scholar]
  42. Sundler R., Papahadjopoulos D. Control of membrane fusion by phospholipid head groups. I. Phosphatidate/phosphatidylinositol specificity. Biochim Biophys Acta. 1981 Dec 21;649(3):743–750. doi: 10.1016/0005-2736(81)90179-6. [DOI] [PubMed] [Google Scholar]
  43. Takei K., McPherson P. S., Schmid S. L., De Camilli P. Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma S in nerve terminals. Nature. 1995 Mar 9;374(6518):186–190. doi: 10.1038/374186a0. [DOI] [PubMed] [Google Scholar]
  44. Takei K., Mundigl O., Daniell L., De Camilli P. The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin. J Cell Biol. 1996 Jun;133(6):1237–1250. doi: 10.1083/jcb.133.6.1237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Uchida E., Ohsumi Y., Anraku Y. Purification of yeast vacuolar membrane H+-ATPase and enzymological discrimination of three ATP-driven proton pumps in Saccharomyces cerevisiae. Methods Enzymol. 1988;157:544–562. doi: 10.1016/0076-6879(88)57103-3. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. 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]
  49. 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]
  50. 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]
  51. 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]
  52. 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]
  53. 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]
  54. Xu Z., Mayer A., Muller E., Wickner W. A heterodimer of thioredoxin and I(B)2 cooperates with Sec18p (NSF) to promote yeast vacuole inheritance. J Cell Biol. 1997 Jan 27;136(2):299–306. doi: 10.1083/jcb.136.2.299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yamamoto A., DeWald D. B., Boronenkov I. V., Anderson R. A., Emr S. D., Koshland D. Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast. Mol Biol Cell. 1995 May;6(5):525–539. doi: 10.1091/mbc.6.5.525. [DOI] [PMC free article] [PubMed] [Google Scholar]

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