Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Oct 2;115(2):289–295. doi: 10.1083/jcb.115.2.289

Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport

PMCID: PMC2289159  PMID: 1833410

Abstract

In this study we show that the plasma membrane [H+]ATPase of Saccharomyces cerevisiae is phosphorylated on multiple Ser and Thr residues in vivo. Phosphorylation occurs during the movement of newly synthesized ATPase from the ER to the cell surface, as revealed by the analysis of temperature-sensitive sec mutants blocked at successive steps of the secretory pathway. Two-dimensional phosphopeptide analysis of the ATPase indicates that, although most sites are phosphorylated at or before arrival in secretory vesicles, some phosphopeptides are unique to the plasma membrane. Phosphorylation of plasma membrane- specific site(s) is associated with increased ATPase activity during growth on glucose. Upon glucose starvation, dephosphorylation occurs concomitantly with a decrease in enzymatic activity, and both are rapidly reversed (within 2 min) upon readdition of glucose. We suggest that reversible, site-specific phosphorylation serves to adjust ATPase activity in response to nutritional signals.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  2. Bidwai A. P., Takemoto J. Y. Bacterial phytotoxin, syringomycin, induces a protein kinase-mediated phosphorylation of red beet plasma membrane polypeptides. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6755–6759. doi: 10.1073/pnas.84.19.6755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brada D., Schekman R. Coincident localization of secretory and plasma membrane proteins in organelles of the yeast secretory pathway. J Bacteriol. 1988 Jun;170(6):2775–2783. doi: 10.1128/jb.170.6.2775-2783.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cannon J. F., Gitan R., Tatchell K. Yeast cAMP-dependent protein kinase regulatory subunit mutations display a variety of phenotypes. J Biol Chem. 1990 Jul 15;265(20):11897–11904. [PubMed] [Google Scholar]
  5. Cid A., Serrano R. Mutations of the yeast plasma membrane H+-ATPase which cause thermosensitivity and altered regulation of the enzyme. J Biol Chem. 1988 Oct 5;263(28):14134–14139. [PubMed] [Google Scholar]
  6. Eraso P., Gancedo C. Activation of yeast plasma membrane ATPase by acid pH during growth. FEBS Lett. 1987 Nov 16;224(1):187–192. doi: 10.1016/0014-5793(87)80445-3. [DOI] [PubMed] [Google Scholar]
  7. Fujiyama A., Tamanoi F. Processing and fatty acid acylation of RAS1 and RAS2 proteins in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1266–1270. doi: 10.1073/pnas.83.5.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gillies R. J., Ugurbil K., den Hollander J. A., Shulman R. G. 31P NMR studies of intracellular pH and phosphate metabolism during cell division cycle of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2125–2129. doi: 10.1073/pnas.78.4.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hager K. M., Mandala S. M., Davenport J. W., Speicher D. W., Benz E. J., Jr, Slayman C. W. Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences. Proc Natl Acad Sci U S A. 1986 Oct;83(20):7693–7697. doi: 10.1073/pnas.83.20.7693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Herman P. K., Stack J. H., DeModena J. A., Emr S. D. A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell. 1991 Jan 25;64(2):425–437. doi: 10.1016/0092-8674(91)90650-n. [DOI] [PubMed] [Google Scholar]
  11. Holcomb C. L., Hansen W. J., Etcheverry T., Schekman R. Secretory vesicles externalize the major plasma membrane ATPase in yeast. J Cell Biol. 1988 Mar;106(3):641–648. doi: 10.1083/jcb.106.3.641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. James P. H., Pruschy M., Vorherr T. E., Penniston J. T., Carafoli E. Primary structure of the cAMP-dependent phosphorylation site of the plasma membrane calcium pump. Biochemistry. 1989 May 16;28(10):4253–4258. doi: 10.1021/bi00436a020. [DOI] [PubMed] [Google Scholar]
  13. Kemp B. E., Pearson R. B. Protein kinase recognition sequence motifs. Trends Biochem Sci. 1990 Sep;15(9):342–346. doi: 10.1016/0968-0004(90)90073-k. [DOI] [PubMed] [Google Scholar]
  14. Kolarov J., Kulpa J., Baijot M., Goffeau A. Characterization of a protein serine kinase from yeast plasma membrane. J Biol Chem. 1988 Aug 5;263(22):10613–10619. [PubMed] [Google Scholar]
  15. 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]
  16. Lai Y., Nairn A. C., Gorelick F., Greengard P. Ca2+/calmodulin-dependent protein kinase II: identification of autophosphorylation sites responsible for generation of Ca2+/calmodulin-independence. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5710–5714. doi: 10.1073/pnas.84.16.5710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levin D. E., Fields F. O., Kunisawa R., Bishop J. M., Thorner J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell. 1990 Jul 27;62(2):213–224. doi: 10.1016/0092-8674(90)90360-q. [DOI] [PubMed] [Google Scholar]
  18. Lowndes J. M., Hokin-Neaverson M., Bertics P. J. Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C. Biochim Biophys Acta. 1990 Apr 9;1052(1):143–151. doi: 10.1016/0167-4889(90)90069-p. [DOI] [PubMed] [Google Scholar]
  19. Margolis R. N., Schell M. J., Taylor S. I., Hubbard A. L. Hepatocyte plasma membrane ECTO-ATPase (pp120/HA4) is a substrate for tyrosine kinase activity of the insulin receptor. Biochem Biophys Res Commun. 1990 Jan 30;166(2):562–566. doi: 10.1016/0006-291x(90)90845-e. [DOI] [PubMed] [Google Scholar]
  20. McDonough J. P., Mahler H. P. Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes. J Biol Chem. 1982 Dec 25;257(24):14579–14581. [PubMed] [Google Scholar]
  21. Miyakawa T., Oka Y., Tsuchiya E., Fukui S. Saccharomyces cerevisiae protein kinase dependent on Ca2+ and calmodulin. J Bacteriol. 1989 Mar;171(3):1417–1422. doi: 10.1128/jb.171.3.1417-1422.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nairn A. C., Greengard P. Purification and characterization of Ca2+/calmodulin-dependent protein kinase I from bovine brain. J Biol Chem. 1987 May 25;262(15):7273–7281. [PubMed] [Google Scholar]
  23. Nakamoto R. K., Rao R., Slayman C. W. Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis. J Biol Chem. 1991 Apr 25;266(12):7940–7949. [PubMed] [Google Scholar]
  24. Novick P., Schekman R. Export of major cell surface proteins is blocked in yeast secretory mutants. J Cell Biol. 1983 Feb;96(2):541–547. doi: 10.1083/jcb.96.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perona R., Serrano R. Increased pH and tumorigenicity of fibroblasts expressing a yeast proton pump. Nature. 1988 Aug 4;334(6181):438–440. doi: 10.1038/334438a0. [DOI] [PubMed] [Google Scholar]
  26. Portillo F., Mazón M. J. Activation of yeast plasma membrane ATPase by phorbol ester. FEBS Lett. 1985 Nov 11;192(1):95–98. doi: 10.1016/0014-5793(85)80050-8. [DOI] [PubMed] [Google Scholar]
  27. Portillo F., de Larrinoa I. F., Serrano R. Deletion analysis of yeast plasma membrane H+-ATPase and identification of a regulatory domain at the carboxyl-terminus. FEBS Lett. 1989 Apr 24;247(2):381–385. doi: 10.1016/0014-5793(89)81375-4. [DOI] [PubMed] [Google Scholar]
  28. Serrano R. H+-ATPase from plasma membranes of Saccharomyces cerevisiae and Avena sativa roots: purification and reconstitution. Methods Enzymol. 1988;157:533–544. doi: 10.1016/0076-6879(88)57102-1. [DOI] [PubMed] [Google Scholar]
  29. Serrano R. In vivo glucose activation of the yeast plasma membrane ATPase. FEBS Lett. 1983 May 30;156(1):11–14. doi: 10.1016/0014-5793(83)80237-3. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Smith K. E., Hammes G. G. Studies of the phosphoenzyme intermediate of the yeast plasma membrane proton-translocating ATPase. J Biol Chem. 1988 Sep 25;263(27):13774–13778. [PubMed] [Google Scholar]
  32. Tschopp J., Esmon P. C., Schekman R. Defective plasma membrane assembly in yeast secretory mutants. J Bacteriol. 1984 Dec;160(3):966–970. doi: 10.1128/jb.160.3.966-970.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Walworth N. C., Novick P. J. Purification and characterization of constitutive secretory vesicles from yeast. J Cell Biol. 1987 Jul;105(1):163–174. doi: 10.1083/jcb.105.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Yanagita Y., Abdel-Ghany M., Raden D., Nelson N., Racker E. Polypeptide-dependent protein kinase from bakers' yeast. Proc Natl Acad Sci U S A. 1987 Feb;84(4):925–929. doi: 10.1073/pnas.84.4.925. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES