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. 1990 Mar;10(3):898–909. doi: 10.1128/mcb.10.3.898

Vanadate-resistant mutants of Saccharomyces cerevisiae show alterations in protein phosphorylation and growth control.

C Kanik-Ennulat 1, N Neff 1
PMCID: PMC360929  PMID: 2137555

Abstract

This work describes two spontaneous vanadate-resistant mutants of Saccharomyces cerevisiae with constitutive alterations in protein phosphorylation, growth control, and sporulation. Vanadate has been shown by a number of studies to be an efficient competitor of phosphate in biochemical reactions, especially those that involve phosphoproteins as intermediates or substrates. Resistance to toxic concentrations of vanadate can arise in S. cerevisiae by both recessive and dominant spontaneous mutations in a large number of loci. Mutations in two of the recessive loci, van1-18 and van2-93, resulted in alterations in the phosphorylation of a number of proteins. The mutant van1-18 gene also showed an increase in plasma membrane ATPase activity in vitro and a lowered basal phosphatase activity under alkaline conditions. Cells containing the van2-93 mutant allele had normal levels of plasma membrane ATPase activity, but this activity was not inhibited by vanadate. Both of these mutants failed to enter stationary phase, were heat shock sensitive, showed lowered long-term viability, and sporulated on rich medium in the presence of 2% glucose. The wild-type VAN1 gene was isolated and sequenced. The open reading frame predicts a protein of 522 amino acids, with no significant homology to any genes that have been identified. Diploid cells that contained two mutant alleles of this gene demonstrated defects in spore viability. These data suggest that the VAN1 gene product is involved in regulation of the phosphorylation of a number of proteins, some of which appear to be important in cell growth control.

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Selected References

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  1. Barnes W. M., Bevan M., Son P. H. Kilo-sequencing: creation of an ordered nest of asymmetric deletions across a large target sequence carried on phage M13. Methods Enzymol. 1983;101:98–122. doi: 10.1016/0076-6879(83)01008-3. [DOI] [PubMed] [Google Scholar]
  2. Bisson L. F., Thorner J. Mutations in the pho80 gene confer permeability to 5'-mononucleotides in Saccharomyces cerevisiae. Genetics. 1982 Nov;102(3):341–359. doi: 10.1093/genetics/102.3.341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Borst-Pauwels G. W. Ion transport in yeast. Biochim Biophys Acta. 1981 Dec;650(2-3):88–127. doi: 10.1016/0304-4157(81)90002-2. [DOI] [PubMed] [Google Scholar]
  4. Bowman B. J., Slayman C. W. The effects of vanadate on the plasma membrane ATPase of Neurospora crassa. J Biol Chem. 1979 Apr 25;254(8):2928–2934. [PubMed] [Google Scholar]
  5. Bowman B. J. Vanadate uptake in Neurospora crassa occurs via phosphate transport system II. J Bacteriol. 1983 Jan;153(1):286–291. doi: 10.1128/jb.153.1.286-291.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown D. J., Gordon J. A. The stimulation of pp60v-src kinase activity by vanadate in intact cells accompanies a new phosphorylation state of the enzyme. J Biol Chem. 1984 Aug 10;259(15):9580–9586. [PubMed] [Google Scholar]
  7. Brugge J. S., Jarosik G., Andersen J., Queral-Lustig A., Fedor-Chaiken M., Broach J. R. Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells. Mol Cell Biol. 1987 Jun;7(6):2180–2187. doi: 10.1128/mcb.7.6.2180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cameron S., Levin L., Zoller M., Wigler M. cAMP-independent control of sporulation, glycogen metabolism, and heat shock resistance in S. cerevisiae. Cell. 1988 May 20;53(4):555–566. doi: 10.1016/0092-8674(88)90572-7. [DOI] [PubMed] [Google Scholar]
  9. Cannon J. F., Gibbs J. B., Tatchell K. Suppressors of the ras2 mutation of Saccharomyces cerevisiae. Genetics. 1986 Jun;113(2):247–264. doi: 10.1093/genetics/113.2.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cantley L. C., Jr, Resh M. D., Guidotti G. Vanadate inhibits the red cell (Na+, K+) ATPase from the cytoplasmic side. Nature. 1978 Apr 6;272(5653):552–554. doi: 10.1038/272552a0. [DOI] [PubMed] [Google Scholar]
  11. Carle G. F., Olson M. V. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. doi: 10.1073/pnas.82.11.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Castellanos R. M., Mazón M. J. Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane. J Biol Chem. 1985 Jul 15;260(14):8240–8242. [PubMed] [Google Scholar]
  13. Celenza J. L., Carlson M. Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Jan;4(1):49–53. doi: 10.1128/mcb.4.1.49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  15. Cooper J. A., Sefton B. M., Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. doi: 10.1016/0076-6879(83)99075-4. [DOI] [PubMed] [Google Scholar]
  16. Dubyak G. R., Kleinzeller A. The insulin-mimetic effects of vanadate in isolated rat adipocytes. Dissociation from effects of vanadate as a (Na+-K+)ATPase inhibitor. J Biol Chem. 1980 Jun 10;255(11):5306–5312. [PubMed] [Google Scholar]
  17. Dufour J. P., Boutry M., Goffeau A. Plasma membrane ATPase of yeast. Comparative inhibition studies of the purified and membrane-bound enzymes. J Biol Chem. 1980 Jun 25;255(12):5735–5741. [PubMed] [Google Scholar]
  18. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  19. Ek B., Heldin C. H. Use of an antiserum against phosphotyrosine for the identification of phosphorylated components in human fibroblasts stimulated by platelet-derived growth factor. J Biol Chem. 1984 Sep 10;259(17):11145–11152. [PubMed] [Google Scholar]
  20. English L. H., Macara I. G., Cantley L. C. Vanadium stimulates the (Na+,K+) pump in friend erythroleukemia cells and blocks erythropoiesis. J Cell Biol. 1983 Oct;97(4):1299–1302. doi: 10.1083/jcb.97.4.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Foulkes J. G., Erikson E., Erikson R. L. Separation of multiple phosphotyrosyl-and phosphoseryl-protein phosphatases from chicken brain. J Biol Chem. 1983 Jan 10;258(1):431–438. [PubMed] [Google Scholar]
  22. Frackelton A. R., Jr, Ross A. H., Eisen H. N. Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells. Mol Cell Biol. 1983 Aug;3(8):1343–1352. doi: 10.1128/mcb.3.8.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gallwitz D. Construction of a yeast actin gene intron deletion mutant that is defective in splicing and leads to the accumulation of precursor RNA in transformed yeast cells. Proc Natl Acad Sci U S A. 1982 Jun;79(11):3493–3497. doi: 10.1073/pnas.79.11.3493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gould K. L., Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. doi: 10.1038/342039a0. [DOI] [PubMed] [Google Scholar]
  25. Haguenauer-Tsapis R., Hinnen A. A deletion that includes the signal peptidase cleavage site impairs processing, glycosylation, and secretion of cell surface yeast acid phosphatase. Mol Cell Biol. 1984 Dec;4(12):2668–2675. doi: 10.1128/mcb.4.12.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  27. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  28. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Jove R., Kornbluth S., Hanafusa H. Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region. Cell. 1987 Sep 11;50(6):937–943. doi: 10.1016/0092-8674(87)90520-4. [DOI] [PubMed] [Google Scholar]
  30. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  31. Klarlund J. K. Transformation of cells by an inhibitor of phosphatases acting on phosphotyrosine in proteins. Cell. 1985 Jul;41(3):707–717. doi: 10.1016/s0092-8674(85)80051-9. [DOI] [PubMed] [Google Scholar]
  32. Kornbluth S., Jove R., Hanafusa H. Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4455–4459. doi: 10.1073/pnas.84.13.4455. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  34. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  35. 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]
  36. Lindquist R. N., Lynn J. L., Jr, Lienhard G. E. Possible transition-state analogs for ribonuclease. The complexes of uridine with oxovanadium(IV) ion and vanadium(V) ion. J Am Chem Soc. 1973 Dec 26;95(26):8762–8768. doi: 10.1021/ja00807a043. [DOI] [PubMed] [Google Scholar]
  37. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  38. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Matsumoto K., Uno I., Ishikawa T. Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase. Cell. 1983 Feb;32(2):417–423. doi: 10.1016/0092-8674(83)90461-0. [DOI] [PubMed] [Google Scholar]
  40. Matsumoto K., Uno I., Kato K., Ishikawa T. Isolation and characterization of a phosphoprotein phosphatase-deficient mutant in yeast. Yeast. 1985 Sep;1(1):25–38. doi: 10.1002/yea.320010104. [DOI] [PubMed] [Google Scholar]
  41. McCusker J. H., Perlin D. S., Haber J. E. Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Nov;7(11):4082–4088. doi: 10.1128/mcb.7.11.4082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nechay B. R. Mechanisms of action of vanadium. Annu Rev Pharmacol Toxicol. 1984;24:501–524. doi: 10.1146/annurev.pa.24.040184.002441. [DOI] [PubMed] [Google Scholar]
  44. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Yeast transformation: a model system for the study of recombination. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6354–6358. doi: 10.1073/pnas.78.10.6354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roon R. J., Larimore F. S., Meyer G. M., Kreisle R. A. Characterization of a Dio-9-resistant strain of Saccharomyces cerevisiae. Arch Biochem Biophys. 1978 Jan 15;185(1):142–150. doi: 10.1016/0003-9861(78)90153-4. [DOI] [PubMed] [Google Scholar]
  46. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schieven G., Thorner J., Martin G. S. Protein-tyrosine kinase activity in Saccharomyces cerevisiae. Science. 1986 Jan 24;231(4736):390–393. doi: 10.1126/science.2417318. [DOI] [PubMed] [Google Scholar]
  49. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  50. Serrano R., Kielland-Brandt M. C., Fink G. R. Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature. 1986 Feb 20;319(6055):689–693. doi: 10.1038/319689a0. [DOI] [PubMed] [Google Scholar]
  51. Shih C. K., Wagner R., Feinstein S., Kanik-Ennulat C., Neff N. A dominant trifluoperazine resistance gene from Saccharomyces cerevisiae has homology with F0F1 ATP synthase and confers calcium-sensitive growth. Mol Cell Biol. 1988 Aug;8(8):3094–3103. doi: 10.1128/mcb.8.8.3094. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Smith J. B. Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6162–6166. doi: 10.1073/pnas.80.20.6162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Struhl K. The yeast his3 promoter contains at least two distinct elements. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7385–7389. doi: 10.1073/pnas.79.23.7385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sunada H., MacLeod C., Mendelsohn J. A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor. Anal Biochem. 1985 Sep;149(2):438–447. doi: 10.1016/0003-2697(85)90595-0. [DOI] [PubMed] [Google Scholar]
  55. Swarup G., Cohen S., Garbers D. L. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Res Commun. 1982 Aug;107(3):1104–1109. doi: 10.1016/0006-291x(82)90635-0. [DOI] [PubMed] [Google Scholar]
  56. Swarup G., Cohen S., Garbers D. L. Selective dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J Biol Chem. 1981 Aug 10;256(15):8197–8201. [PubMed] [Google Scholar]
  57. Tanaka K., Waxman L., Goldberg A. L. Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation. J Biol Chem. 1984 Mar 10;259(5):2803–2809. [PubMed] [Google Scholar]
  58. Tatchell K., Robinson L. C., Breitenbach M. RAS2 of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3785–3789. doi: 10.1073/pnas.82.11.3785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Toda T., Cameron S., Sass P., Zoller M., Scott J. D., McMullen B., Hurwitz M., Krebs E. G., Wigler M. Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Apr;7(4):1371–1377. doi: 10.1128/mcb.7.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. 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]
  61. Ulaszewski S., Balzi E., Goffeau A. Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae. Mol Gen Genet. 1987 Apr;207(1):38–46. doi: 10.1007/BF00331488. [DOI] [PubMed] [Google Scholar]
  62. Ulaszewski S., Grenson M., Goffeau A. Modified plasma-membrane ATPase in mutants of Saccharomyces cerevisiae. Eur J Biochem. 1983 Feb 1;130(2):235–239. doi: 10.1111/j.1432-1033.1983.tb07141.x. [DOI] [PubMed] [Google Scholar]
  63. Willsky G. R., Leung J. O., Offermann P. V., Jr, Plotnick E. K., Dosch S. F. Isolation and characterization of vanadate-resistant mutants of Saccharomyces cerevisiae. J Bacteriol. 1985 Nov;164(2):611–617. doi: 10.1128/jb.164.2.611-617.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Willsky G. R., White D. A., McCabe B. C. Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae. J Biol Chem. 1984 Nov 10;259(21):13273–13281. [PubMed] [Google Scholar]

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