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. 1998 Apr;148(4):1715–1729. doi: 10.1093/genetics/148.4.1715

Identification and characterization of an essential family of inositol polyphosphate 5-phosphatases (INP51, INP52 and INP53 gene products) in the yeast Saccharomyces cerevisiae.

L E Stolz 1, C V Huynh 1, J Thorner 1, J D York 1
PMCID: PMC1460112  PMID: 9560389

Abstract

We recently demonstrated that the S. cerevisiae INP51 locus (YIL002c) encodes an inositol polyphosphate 5-phosphatase. Here we describe two related yeast loci, INP52 (YNL106c) and INP53 (YOR109w). Like Inp51p, the primary structures of Inp52p and Inp53p resemble the mammalian synaptic vesicle-associated protein, synaptojanin, and contain a carboxy-terminal catalytic domain and an amino-terminal SAC1-like segment. Inp51p (108 kD), Inp52p (136 kD) and Inp53p (124 kD) are membrane-associated. Single null mutants (inp51, inp52, or inp53) are viable. Both inp51 inp52 and inp52 inp53 double mutants display compromised cell growth, whereas an inp51 inp53 double mutant does not. An inp51 inp52 inp53 triple mutant is inviable on standard medium, but can grow weakly on media supplemented with an osmotic stabilizer (1 M sorbitol). An inp51 mutation, and to a lesser degree an inp52 mutation, confers cold-resistant growth in a strain background that cannot grow at temperatures below 15 degrees. Analysis of inositol metabolites in vivo showed measurable accumulation of phosphatidylinositol 4,5-bisphosphate in the inp51 mutant. Electron microscopy revealed plasma membrane invaginations and cell wall thickening in double mutants and the triple mutant grown in sorbitol-containing medium. A fluorescent dye that detects endocytic and vacuolar membranes suggests that the vacuole is highly fragmented in inp51 inp52 double mutants. Our observations indicate that Inp51p, Inp52p, and Inp53p have distinct functions and that substrates and/or products of inositol polyphosphate 5-phosphatases may have roles in vesicle trafficking, membrane structure, and/or cell wall formation.

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Selected References

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  1. Attree O., Olivos I. M., Okabe I., Bailey L. C., Nelson D. L., Lewis R. A., McInnes R. R., Nussbaum R. L. The Lowe's oculocerebrorenal syndrome gene encodes a protein highly homologous to inositol polyphosphate-5-phosphatase. Nature. 1992 Jul 16;358(6383):239–242. doi: 10.1038/358239a0. [DOI] [PubMed] [Google Scholar]
  2. Bansal V. S., Majerus P. W. Phosphatidylinositol-derived precursors and signals. Annu Rev Cell Biol. 1990;6:41–67. doi: 10.1146/annurev.cb.06.110190.000353. [DOI] [PubMed] [Google Scholar]
  3. Chowdhury S., Smith K. W., Gustin M. C. Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J Cell Biol. 1992 Aug;118(3):561–571. doi: 10.1083/jcb.118.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Communi D., Lecocq R., Erneux C. Arginine 343 and 350 are two active residues involved in substrate binding by human Type I D-myo-inositol 1,4,5,-trisphosphate 5-phosphatase. J Biol Chem. 1996 May 17;271(20):11676–11683. doi: 10.1074/jbc.271.20.11676. [DOI] [PubMed] [Google Scholar]
  5. Damen J. E., Liu L., Rosten P., Humphries R. K., Jefferson A. B., Majerus P. W., Krystal G. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1689–1693. doi: 10.1073/pnas.93.4.1689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Smedt F., Boom A., Pesesse X., Schiffmann S. N., Erneux C. Post-translational modification of human brain type I inositol-1,4,5-trisphosphate 5-phosphatase by farnesylation. J Biol Chem. 1996 Apr 26;271(17):10419–10424. doi: 10.1074/jbc.271.17.10419. [DOI] [PubMed] [Google Scholar]
  7. De Smedt F., Verjans B., Mailleux P., Erneux C. Cloning and expression of human brain type I inositol 1,4,5-trisphosphate 5-phosphatase. High levels of mRNA in cerebellar Purkinje cells. FEBS Lett. 1994 Jun 20;347(1):69–72. doi: 10.1016/0014-5793(94)00509-5. [DOI] [PubMed] [Google Scholar]
  8. Dove S. K., Cooke F. T., Douglas M. R., Sayers L. G., Parker P. J., Michell R. H. Osmotic stress activates phosphatidylinositol-3,5-bisphosphate synthesis. Nature. 1997 Nov 13;390(6656):187–192. doi: 10.1038/36613. [DOI] [PubMed] [Google Scholar]
  9. Drayer A. L., Pesesse X., De Smedt F., Communi D., Moreau C., Erneux C. The family of inositol and phosphatidylinositol polyphosphate 5-phosphatases. Biochem Soc Trans. 1996 Nov;24(4):1001–1005. doi: 10.1042/bst0241001. [DOI] [PubMed] [Google Scholar]
  10. Flanagan C. A., Schnieders E. A., Emerick A. W., Kunisawa R., Admon A., Thorner J. Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability. Science. 1993 Nov 26;262(5138):1444–1448. doi: 10.1126/science.8248783. [DOI] [PubMed] [Google Scholar]
  11. Flick J. S., Thorner J. Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Sep;13(9):5861–5876. doi: 10.1128/mcb.13.9.5861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harlan J. E., Hajduk P. J., Yoon H. S., Fesik S. W. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature. 1994 Sep 8;371(6493):168–170. doi: 10.1038/371168a0. [DOI] [PubMed] [Google Scholar]
  13. Hartwig J. H., Bokoch G. M., Carpenter C. L., Janmey P. A., Taylor L. A., Toker A., Stossel T. P. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell. 1995 Aug 25;82(4):643–653. doi: 10.1016/0092-8674(95)90036-5. [DOI] [PubMed] [Google Scholar]
  14. Hawkins P. T., Jackson T. R., Stephens L. R. Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature. 1992 Jul 9;358(6382):157–159. doi: 10.1038/358157a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hokin L. E. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem. 1985;54:205–235. doi: 10.1146/annurev.bi.54.070185.001225. [DOI] [PubMed] [Google Scholar]
  17. Irvine R. F., Moor R. M., Pollock W. K., Smith P. M., Wreggett K. A. Inositol phosphates: proliferation, metabolism and function. Philos Trans R Soc Lond B Biol Sci. 1988 Jul 26;320(1199):281–298. doi: 10.1098/rstb.1988.0077. [DOI] [PubMed] [Google Scholar]
  18. Irvine R. Second messengers and Lowe syndrome. Nat Genet. 1992 Aug;1(5):315–316. doi: 10.1038/ng0892-315. [DOI] [PubMed] [Google Scholar]
  19. Jackson S. P., Schoenwaelder S. M., Matzaris M., Brown S., Mitchell C. A. Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase. EMBO J. 1995 Sep 15;14(18):4490–4500. doi: 10.1002/j.1460-2075.1995.tb00128.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Janmey P. A., Lamb J., Allen P. G., Matsudaira P. T. Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J Biol Chem. 1992 Jun 15;267(17):11818–11823. [PubMed] [Google Scholar]
  21. Jefferson A. B., Auethavekiat V., Pot D. A., Williams L. T., Majerus P. W. Signaling inositol polyphosphate-5-phosphatase. Characterization of activity and effect of GRB2 association. J Biol Chem. 1997 Feb 28;272(9):5983–5988. doi: 10.1074/jbc.272.9.5983. [DOI] [PubMed] [Google Scholar]
  22. Jefferson A. B., Majerus P. W. Properties of type II inositol polyphosphate 5-phosphatase. J Biol Chem. 1995 Apr 21;270(16):9370–9377. doi: 10.1074/jbc.270.16.9370. [DOI] [PubMed] [Google Scholar]
  23. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  24. Kearns B. G., McGee T. P., Mayinger P., Gedvilaite A., Phillips S. E., Kagiwada S., Bankaitis V. A. Essential role for diacylglycerol in protein transport from the yeast Golgi complex. Nature. 1997 May 1;387(6628):101–105. doi: 10.1038/387101a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunz J., Henriquez R., Schneider U., Deuter-Reinhard M., Movva N. R., Hall M. N. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. doi: 10.1016/0092-8674(93)90144-f. [DOI] [PubMed] [Google Scholar]
  26. Laxminarayan K. M., Chan B. K., Tetaz T., Bird P. I., Mitchell C. A. Characterization of a cDNA encoding the 43-kDa membrane-associated inositol-polyphosphate 5-phosphatase. J Biol Chem. 1994 Jun 24;269(25):17305–17310. [PubMed] [Google Scholar]
  27. Laxminarayan K. M., Matzaris M., Speed C. J., Mitchell C. A. Purification and characterization of a 43-kDa membrane-associated inositol polyphosphate 5-phosphatase from human placenta. J Biol Chem. 1993 Mar 5;268(7):4968–4974. [PubMed] [Google Scholar]
  28. Levin D. E., Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene display a cell cycle-specific osmotic stability defect. J Cell Biol. 1992 Mar;116(5):1221–1229. doi: 10.1083/jcb.116.5.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lioubin M. N., Algate P. A., Tsai S., Carlberg K., Aebersold A., Rohrschneider L. R. p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity. Genes Dev. 1996 May 1;10(9):1084–1095. doi: 10.1101/gad.10.9.1084. [DOI] [PubMed] [Google Scholar]
  30. Luo W. j., Chang A. Novel genes involved in endosomal traffic in yeast revealed by suppression of a targeting-defective plasma membrane ATPase mutant. J Cell Biol. 1997 Aug 25;138(4):731–746. doi: 10.1083/jcb.138.4.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Majerus P. W. Inositol phosphate biochemistry. Annu Rev Biochem. 1992;61:225–250. doi: 10.1146/annurev.bi.61.070192.001301. [DOI] [PubMed] [Google Scholar]
  32. Majerus P. W., Ross T. S., Cunningham T. W., Caldwell K. K., Jefferson A. B., Bansal V. S. Recent insights in phosphatidylinositol signaling. Cell. 1990 Nov 2;63(3):459–465. doi: 10.1016/0092-8674(90)90442-h. [DOI] [PubMed] [Google Scholar]
  33. Mayinger P., Bankaitis V. A., Meyer D. I. Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation. J Cell Biol. 1995 Dec;131(6 Pt 1):1377–1386. doi: 10.1083/jcb.131.6.1377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McLaughlin M. M., Kumar S., McDonnell P. C., Van Horn S., Lee J. C., Livi G. P., Young P. R. Identification of mitogen-activated protein (MAP) kinase-activated protein kinase-3, a novel substrate of CSBP p38 MAP kinase. J Biol Chem. 1996 Apr 5;271(14):8488–8492. doi: 10.1074/jbc.271.14.8488. [DOI] [PubMed] [Google Scholar]
  35. McPherson P. S., Czernik A. J., Chilcote T. J., Onofri F., Benfenati F., Greengard P., Schlessinger J., De Camilli P. Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I. Proc Natl Acad Sci U S A. 1994 Jul 5;91(14):6486–6490. doi: 10.1073/pnas.91.14.6486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. McPherson P. S., Garcia E. P., Slepnev V. I., David C., Zhang X., Grabs D., Sossin W. S., Bauerfeind R., Nemoto Y., De Camilli P. A presynaptic inositol-5-phosphatase. Nature. 1996 Jan 25;379(6563):353–357. doi: 10.1038/379353a0. [DOI] [PubMed] [Google Scholar]
  37. McPherson P. S., Takei K., Schmid S. L., De Camilli P. p145, a major Grb2-binding protein in brain, is co-localized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation. J Biol Chem. 1994 Dec 2;269(48):30132–30139. [PubMed] [Google Scholar]
  38. Mitchell C. A., Brown S., Campbell J. K., Munday A. D., Speed C. J. Regulation of second messengers by the inositol polyphosphate 5-phosphatases. Biochem Soc Trans. 1996 Nov;24(4):994–1000. doi: 10.1042/bst0240994. [DOI] [PubMed] [Google Scholar]
  39. Mitchell C. A., Connolly T. M., Majerus P. W. Identification and isolation of a 75-kDa inositol polyphosphate-5-phosphatase from human platelets. J Biol Chem. 1989 May 25;264(15):8873–8877. [PubMed] [Google Scholar]
  40. Novick P., Osmond B. C., Botstein D. Suppressors of yeast actin mutations. Genetics. 1989 Apr;121(4):659–674. doi: 10.1093/genetics/121.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Olivos-Glander I. M., Jänne P. A., Nussbaum R. L. The oculocerebrorenal syndrome gene product is a 105-kD protein localized to the Golgi complex. Am J Hum Genet. 1995 Oct;57(4):817–823. [PMC free article] [PubMed] [Google Scholar]
  42. Posas F., Casamayor A., Ariño J. The PPZ protein phosphatases are involved in the maintenance of osmotic stability of yeast cells. FEBS Lett. 1993 Mar 8;318(3):282–286. doi: 10.1016/0014-5793(93)80529-4. [DOI] [PubMed] [Google Scholar]
  43. Rameh L. E., Chen C. S., Cantley L. C. Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell. 1995 Dec 1;83(5):821–830. doi: 10.1016/0092-8674(95)90195-7. [DOI] [PubMed] [Google Scholar]
  44. Reed R. H., Chudek J. A., Foster R., Gadd G. M. Osmotic significance of glycerol accumulation in exponentially growing yeasts. Appl Environ Microbiol. 1987 Sep;53(9):2119–2123. doi: 10.1128/aem.53.9.2119-2123.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Singh A., Manney T. R. Genetic analysis of mutations affecting growth of Saccharomyces cerevisiae at low temperature. Genetics. 1974 Aug;77(4):651–659. doi: 10.1093/genetics/77.4.651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Srinivasan S., Seaman M., Nemoto Y., Daniell L., Suchy S. F., Emr S., De Camilli P., Nussbaum R. Disruption of three phosphatidylinositol-polyphosphate 5-phosphatase genes from Saccharomyces cerevisiae results in pleiotropic abnormalities of vacuole morphology, cell shape, and osmohomeostasis. Eur J Cell Biol. 1997 Dec;74(4):350–360. [PubMed] [Google Scholar]
  48. Stack J. H., DeWald D. B., Takegawa K., Emr S. D. Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast. J Cell Biol. 1995 Apr;129(2):321–334. doi: 10.1083/jcb.129.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Verjans B., De Smedt F., Lecocq R., Vanweyenberg V., Moreau C., Erneux C. Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase. Biochem J. 1994 May 15;300(Pt 1):85–90. doi: 10.1042/bj3000085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. Whitters E. A., Cleves A. E., McGee T. P., Skinner H. B., Bankaitis V. A. SAC1p is an integral membrane protein that influences the cellular requirement for phospholipid transfer protein function and inositol in yeast. J Cell Biol. 1993 Jul;122(1):79–94. doi: 10.1083/jcb.122.1.79. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Woscholski R., Waterfield M. D., Parker P. J. Purification and biochemical characterization of a mammalian phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase. J Biol Chem. 1995 Dec 29;270(52):31001–31007. doi: 10.1074/jbc.270.52.31001. [DOI] [PubMed] [Google Scholar]
  53. York J. D., Majerus P. W. Nuclear phosphatidylinositols decrease during S-phase of the cell cycle in HeLa cells. J Biol Chem. 1994 Mar 18;269(11):7847–7850. [PubMed] [Google Scholar]
  54. Yoshida S., Ohya Y., Goebl M., Nakano A., Anraku Y. A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae. J Biol Chem. 1994 Jan 14;269(2):1166–1172. [PubMed] [Google Scholar]
  55. Zhang F. L., Casey P. J. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem. 1996;65:241–269. doi: 10.1146/annurev.bi.65.070196.001325. [DOI] [PubMed] [Google Scholar]
  56. Zhou M. M., Ravichandran K. S., Olejniczak E. F., Petros A. M., Meadows R. P., Sattler M., Harlan J. E., Wade W. S., Burakoff S. J., Fesik S. W. Structure and ligand recognition of the phosphotyrosine binding domain of Shc. Nature. 1995 Dec 7;378(6557):584–592. doi: 10.1038/378584a0. [DOI] [PubMed] [Google Scholar]

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