Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Feb 1;124(3):351–363. doi: 10.1083/jcb.124.3.351

Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca2+ ATPases

PMCID: PMC2119937  PMID: 7507493

Abstract

Ca2+ ATPases deplete the cytosol of Ca2+ ions and are crucial to cellular Ca2+ homeostasis. The PMC1 gene of Saccharomyces cerevisiae encodes a vacuole membrane protein that is 40% identical to the plasma membrane Ca2+ ATPases (PMCAs) of mammalian cells. Mutants lacking PMC1 grow well in standard media, but sequester Ca2+ into the vacuole at 20% of the wild-type levels. pmc1 null mutants fail to grow in media containing high levels of Ca2+, suggesting a role of PMC1 in Ca2+ tolerance. The growth inhibitory effect of added Ca2+ requires activation of calcineurin, a Ca2+ and calmodulin-dependent protein phosphatase. Mutations in calcineurin A or B subunits or the inhibitory compounds FK506 and cyclosporin A restore growth of pmc1 mutants in high Ca2+ media. Also, growth is restored by recessive mutations that inactivate the high-affinity Ca(2+)-binding sites in calmodulin. This mutant calmodulin has apparently lost the ability to activate calcineurin in vivo. These results suggest that activation of calcineurin by Ca2+ and calmodulin can negatively affect yeast growth. A second Ca2+ ATPase homolog encoded by the PMR1 gene acts together with PMC1 to prevent lethal activation of calcineurin even in standard (low Ca2+) conditions. We propose that these Ca2+ ATPase homologs are essential in yeast to deplete the cytosol of Ca2+ ions which, at elevated concentrations, inhibits yeast growth through inappropriate activation of calcineurin.

Full Text

The Full Text of this article is available as a PDF (3.0 MB).

Selected References

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

  1. Anraku Y., Ohya Y., Iida H. Cell cycle control by calcium and calmodulin in Saccharomyces cerevisiae. Biochim Biophys Acta. 1991 Jul 10;1093(2-3):169–177. doi: 10.1016/0167-4889(91)90119-i. [DOI] [PubMed] [Google Scholar]
  2. Antebi A., Fink G. R. The yeast Ca(2+)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol Biol Cell. 1992 Jun;3(6):633–654. doi: 10.1091/mbc.3.6.633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bierer B. E., Schreiber S. L., Burakoff S. J. The effect of the immunosuppressant FK-506 on alternate pathways of T cell activation. Eur J Immunol. 1991 Feb;21(2):439–445. doi: 10.1002/eji.1830210228. [DOI] [PubMed] [Google Scholar]
  4. Carafoli E. Intracellular calcium homeostasis. Annu Rev Biochem. 1987;56:395–433. doi: 10.1146/annurev.bi.56.070187.002143. [DOI] [PubMed] [Google Scholar]
  5. Carafoli E. The Ca2+ pump of the plasma membrane. J Biol Chem. 1992 Feb 5;267(4):2115–2118. [PubMed] [Google Scholar]
  6. Clipstone N. A., Crabtree G. R. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–697. doi: 10.1038/357695a0. [DOI] [PubMed] [Google Scholar]
  7. Cyert M. S., Kunisawa R., Kaim D., Thorner J. Yeast has homologs (CNA1 and CNA2 gene products) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7376–7380. doi: 10.1073/pnas.88.16.7376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cyert M. S., Thorner J. Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone. Mol Cell Biol. 1992 Aug;12(8):3460–3469. doi: 10.1128/mcb.12.8.3460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis T. N., Urdea M. S., Masiarz F. R., Thorner J. Isolation of the yeast calmodulin gene: calmodulin is an essential protein. Cell. 1986 Nov 7;47(3):423–431. doi: 10.1016/0092-8674(86)90599-4. [DOI] [PubMed] [Google Scholar]
  10. Eilam Y. Studies on calcium efflux in the yeast Saccharomyces cerevisiae. Microbios. 1982;35(140):99–110. [PubMed] [Google Scholar]
  11. Eilam Y. The effect of monovalent cations on calcium efflux in yeasts. Biochim Biophys Acta. 1982 Apr 23;687(1):8–16. doi: 10.1016/0005-2736(82)90164-x. [DOI] [PubMed] [Google Scholar]
  12. Foor F., Parent S. A., Morin N., Dahl A. M., Ramadan N., Chrebet G., Bostian K. A., Nielsen J. B. Calcineurin mediates inhibition by FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature. 1992 Dec 17;360(6405):682–684. doi: 10.1038/360682a0. [DOI] [PubMed] [Google Scholar]
  13. Gardner P. Calcium and T lymphocyte activation. Cell. 1989 Oct 6;59(1):15–20. doi: 10.1016/0092-8674(89)90865-9. [DOI] [PubMed] [Google Scholar]
  14. Geiser J. R., van Tuinen D., Brockerhoff S. E., Neff M. M., Davis T. N. Can calmodulin function without binding calcium? Cell. 1991 Jun 14;65(6):949–959. doi: 10.1016/0092-8674(91)90547-c. [DOI] [PubMed] [Google Scholar]
  15. Halachmi D., Eilam Y. Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1. FEBS Lett. 1989 Oct 9;256(1-2):55–61. doi: 10.1016/0014-5793(89)81717-x. [DOI] [PubMed] [Google Scholar]
  16. Haro R., Garciadeblas B., Rodríguez-Navarro A. A novel P-type ATPase from yeast involved in sodium transport. FEBS Lett. 1991 Oct 21;291(2):189–191. doi: 10.1016/0014-5793(91)81280-l. [DOI] [PubMed] [Google Scholar]
  17. Hashimoto Y., Perrino B. A., Soderling T. R. Identification of an autoinhibitory domain in calcineurin. J Biol Chem. 1990 Feb 5;265(4):1924–1927. [PubMed] [Google Scholar]
  18. Heitman J., Movva N. R., Hiestand P. C., Hall M. N. FK 506-binding protein proline rotamase is a target for the immunosuppressive agent FK 506 in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1948–1952. doi: 10.1073/pnas.88.5.1948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurwitz M. Y., Putkey J. A., Klee C. B., Means A. R. Domain II of calmodulin is involved in activation of calcineurin. FEBS Lett. 1988 Sep 26;238(1):82–86. doi: 10.1016/0014-5793(88)80230-8. [DOI] [PubMed] [Google Scholar]
  20. Iida H., Yagawa Y., Anraku Y. Essential role for induced Ca2+ influx followed by [Ca2+]i rise in maintaining viability of yeast cells late in the mating pheromone response pathway. A study of [Ca2+]i in single Saccharomyces cerevisiae cells with imaging of fura-2. J Biol Chem. 1990 Aug 5;265(22):13391–13399. [PubMed] [Google Scholar]
  21. 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]
  22. Kuno T., Tanaka H., Mukai H., Chang C. D., Hiraga K., Miyakawa T., Tanaka C. cDNA cloning of a calcineurin B homolog in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1991 Oct 31;180(2):1159–1163. doi: 10.1016/s0006-291x(05)81188-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. Liu J., Farmer J. D., Jr, Lane W. S., Friedman J., Weissman I., Schreiber S. L. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–815. doi: 10.1016/0092-8674(91)90124-h. [DOI] [PubMed] [Google Scholar]
  26. Liu Y., Ishii S., Tokai M., Tsutsumi H., Ohki O., Akada R., Tanaka K., Tsuchiya E., Fukui S., Miyakawa T. The Saccharomyces cerevisiae genes (CMP1 and CMP2) encoding calmodulin-binding proteins homologous to the catalytic subunit of mammalian protein phosphatase 2B. Mol Gen Genet. 1991 May;227(1):52–59. doi: 10.1007/BF00260706. [DOI] [PubMed] [Google Scholar]
  27. Manalan A. S., Klee C. B. Activation of calcineurin by limited proteolysis. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4291–4295. doi: 10.1073/pnas.80.14.4291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller A. J., Vogg G., Sanders D. Cytosolic calcium homeostasis in fungi: roles of plasma membrane transport and intracellular sequestration of calcium. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9348–9352. doi: 10.1073/pnas.87.23.9348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. O'Keefe S. J., Tamura J., Kincaid R. L., Tocci M. J., O'Neill E. A. FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature. 1992 Jun 25;357(6380):692–694. doi: 10.1038/357692a0. [DOI] [PubMed] [Google Scholar]
  30. Ohsumi Y., Anraku Y. Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J Biol Chem. 1983 May 10;258(9):5614–5617. [PubMed] [Google Scholar]
  31. Ohya Y., Ohsumi Y., Anraku Y. Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants. Mol Gen Genet. 1984;193(3):389–394. doi: 10.1007/BF00382073. [DOI] [PubMed] [Google Scholar]
  32. Ohya Y., Umemoto N., Tanida I., Ohta A., Iida H., Anraku Y. Calcium-sensitive cls mutants of Saccharomyces cerevisiae showing a Pet- phenotype are ascribable to defects of vacuolar membrane H(+)-ATPase activity. J Biol Chem. 1991 Jul 25;266(21):13971–13977. [PubMed] [Google Scholar]
  33. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  34. Reid G. A., Schatz G. Import of proteins into mitochondria. Yeast cells grown in the presence of carbonyl cyanide m-chlorophenylhydrazone accumulate massive amounts of some mitochondrial precursor polypeptides. J Biol Chem. 1982 Nov 10;257(21):13056–13061. [PubMed] [Google Scholar]
  35. Riles L., Dutchik J. E., Baktha A., McCauley B. K., Thayer E. C., Leckie M. P., Braden V. V., Depke J. E., Olson M. V. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. doi: 10.1093/genetics/134.1.81. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Rudolph H. K., Antebi A., Fink G. R., Buckley C. M., Dorman T. E., LeVitre J., Davidow L. S., Mao J. I., Moir D. T. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell. 1989 Jul 14;58(1):133–145. doi: 10.1016/0092-8674(89)90410-8. [DOI] [PubMed] [Google Scholar]
  37. Schlesser A., Ulaszewski S., Ghislain M., Goffeau A. A second transport ATPase gene in Saccharomyces cerevisiae. J Biol Chem. 1988 Dec 25;263(36):19480–19487. [PubMed] [Google Scholar]
  38. 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]
  39. Shull G. E., Greeb J. Molecular cloning of two isoforms of the plasma membrane Ca2+-transporting ATPase from rat brain. Structural and functional domains exhibit similarity to Na+,K+- and other cation transport ATPases. J Biol Chem. 1988 Jun 25;263(18):8646–8657. [PubMed] [Google Scholar]
  40. 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]
  41. Stewart A. A., Ingebritsen T. S., Manalan A., Klee C. B., Cohen P. Discovery of a Ca2+- and calmodulin-dependent protein phosphatase: probable identity with calcineurin (CaM-BP80). FEBS Lett. 1982 Jan 11;137(1):80–84. doi: 10.1016/0014-5793(82)80319-0. [DOI] [PubMed] [Google Scholar]
  42. Wallis J. W., Chrebet G., Brodsky G., Rolfe M., Rothstein R. A hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic topoisomerase. Cell. 1989 Jul 28;58(2):409–419. doi: 10.1016/0092-8674(89)90855-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES