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. 2000 May 1;347(Pt 3):837–843.

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae sec14p are found in the same cell.

P Swigart 1, R Insall 1, A Wilkins 1, S Cockcroft 1
PMCID: PMC1221023  PMID: 10769190

Abstract

Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (alpha and beta) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPalpha and PITPbeta. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

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Selected References

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  1. Aikawa Y., Hara H., Watanabe T. Molecular cloning and characterization of mammalian homologues of the Drosophila retinal degeneration B gene. Biochem Biophys Res Commun. 1997 Jul 30;236(3):559–564. doi: 10.1006/bbrc.1997.7009. [DOI] [PubMed] [Google Scholar]
  2. Allen V., Swigart P., Cheung R., Cockcroft S., Katan M. Regulation of inositol lipid-specific phospholipase cdelta by changes in Ca2+ ion concentrations. Biochem J. 1997 Oct 15;327(Pt 2):545–552. doi: 10.1042/bj3270545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bankaitis V. A., Aitken J. R., Cleves A. E., Dowhan W. An essential role for a phospholipid transfer protein in yeast Golgi function. Nature. 1990 Oct 11;347(6293):561–562. doi: 10.1038/347561a0. [DOI] [PubMed] [Google Scholar]
  4. Bankaitis V. A., Malehorn D. E., Emr S. D., Greene R. The Saccharomyces cerevisiae SEC14 gene encodes a cytosolic factor that is required for transport of secretory proteins from the yeast Golgi complex. J Cell Biol. 1989 Apr;108(4):1271–1281. doi: 10.1083/jcb.108.4.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berks M., Kay R. R. Combinatorial control of cell differentiation by cAMP and DIF-1 during development of Dictyostelium discoideum. Development. 1990 Nov;110(3):977–984. doi: 10.1242/dev.110.3.977. [DOI] [PubMed] [Google Scholar]
  6. Buczynski G., Grove B., Nomura A., Kleve M., Bush J., Firtel R. A., Cardelli J. Inactivation of two Dictyostelium discoideum genes, DdPIK1 and DdPIK2, encoding proteins related to mammalian phosphatidylinositide 3-kinases, results in defects in endocytosis, lysosome to postlysosome transport, and actin cytoskeleton organization. J Cell Biol. 1997 Mar 24;136(6):1271–1286. doi: 10.1083/jcb.136.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cockcroft S. Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic. Bioessays. 1998 May;20(5):423–432. doi: 10.1002/(SICI)1521-1878(199805)20:5<423::AID-BIES9>3.0.CO;2-O. [DOI] [PubMed] [Google Scholar]
  8. Cunningham E., Tan S. K., Swigart P., Hsuan J., Bankaitis V., Cockcroft S. The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6589–6593. doi: 10.1073/pnas.93.13.6589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cunningham E., Thomas G. M., Ball A., Hiles I., Cockcroft S. Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2. Curr Biol. 1995 Jul 1;5(7):775–783. doi: 10.1016/s0960-9822(95)00154-0. [DOI] [PubMed] [Google Scholar]
  10. De Camilli P., Emr S. D., McPherson P. S., Novick P. Phosphoinositides as regulators in membrane traffic. Science. 1996 Mar 15;271(5255):1533–1539. doi: 10.1126/science.271.5255.1533. [DOI] [PubMed] [Google Scholar]
  11. De Vries K. J., Westerman J., Bastiaens P. I., Jovin T. M., Wirtz K. W., Snoek G. T. Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites. Exp Cell Res. 1996 Aug 25;227(1):33–39. doi: 10.1006/excr.1996.0246. [DOI] [PubMed] [Google Scholar]
  12. Fensome A., Cunningham E., Prosser S., Tan S. K., Swigart P., Thomas G., Hsuan J., Cockcroft S. ARF and PITP restore GTP gamma S-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis. Curr Biol. 1996 Jun 1;6(6):730–738. doi: 10.1016/s0960-9822(09)00454-0. [DOI] [PubMed] [Google Scholar]
  13. Guo J., Yu F. X. Cloning and characterization of human homologue of Drosophila retinal degeneration B: a candidate gene for degenerative retinal diseases. Dev Genet. 1997;20(3):235–245. doi: 10.1002/(SICI)1520-6408(1997)20:3<235::AID-DVG6>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  14. Hamilton B. A., Smith D. J., Mueller K. L., Kerrebrock A. W., Bronson R. T., van Berkel V., Daly M. J., Kruglyak L., Reeve M. P., Nemhauser J. L. The vibrator mutation causes neurodegeneration via reduced expression of PITP alpha: positional complementation cloning and extragenic suppression. Neuron. 1997 May;18(5):711–722. doi: 10.1016/s0896-6273(00)80312-8. [DOI] [PubMed] [Google Scholar]
  15. Hara S., Swigart P., Jones D., Cockcroft S. The first 5 amino acids of the carboxyl terminus of phosphatidylinositol transfer protein (PITP) alpha play a critical role in inositol lipid signaling. Transfer activity of PITP is essential but not sufficient for restoration of phospholipase C signaling. J Biol Chem. 1997 Jun 6;272(23):14908–14913. doi: 10.1074/jbc.272.23.14908. [DOI] [PubMed] [Google Scholar]
  16. Hay J. C., Martin T. F. Phosphatidylinositol transfer protein required for ATP-dependent priming of Ca(2+)-activated secretion. Nature. 1993 Dec 9;366(6455):572–575. doi: 10.1038/366572a0. [DOI] [PubMed] [Google Scholar]
  17. Jones S. M., Alb J. G., Jr, Phillips S. E., Bankaitis V. A., Howell K. E. A phosphatidylinositol 3-kinase and phosphatidylinositol transfer protein act synergistically in formation of constitutive transport vesicles from the trans-Golgi network. J Biol Chem. 1998 Apr 24;273(17):10349–10354. doi: 10.1074/jbc.273.17.10349. [DOI] [PubMed] [Google Scholar]
  18. Kauffmann-Zeh A., Thomas G. M., Ball A., Prosser S., Cunningham E., Cockcroft S., Hsuan J. J. Requirement for phosphatidylinositol transfer protein in epidermal growth factor signaling. Science. 1995 May 26;268(5214):1188–1190. doi: 10.1126/science.7761838. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kular G., Loubtchenkov M., Swigart P., Whatmore J., Ball A., Cockcroft S., Wetzker R. Co-operation of phosphatidylinositol transfer protein with phosphoinositide 3-kinase gamma in the formylmethionyl-leucylphenylalanine-dependent production of phosphatidylinositol 3,4,5-trisphosphate in human neutrophils. Biochem J. 1997 Jul 15;325(Pt 2):299–301. doi: 10.1042/bj3250299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Leevers S. J., Vanhaesebroeck B., Waterfield M. D. Signalling through phosphoinositide 3-kinases: the lipids take centre stage. Curr Opin Cell Biol. 1999 Apr;11(2):219–225. doi: 10.1016/s0955-0674(99)80029-5. [DOI] [PubMed] [Google Scholar]
  22. Lev S., Hernandez J., Martinez R., Chen A., Plowman G., Schlessinger J. Identification of a novel family of targets of PYK2 related to Drosophila retinal degeneration B (rdgB) protein. Mol Cell Biol. 1999 Mar;19(3):2278–2288. doi: 10.1128/mcb.19.3.2278. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lopez M. C., Nicaud J. M., Skinner H. B., Vergnolle C., Kader J. C., Bankaitis V. A., Gaillardin C. A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form. J Cell Biol. 1994 Apr;125(1):113–127. doi: 10.1083/jcb.125.1.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Martin T. F. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Rev Cell Dev Biol. 1998;14:231–264. doi: 10.1146/annurev.cellbio.14.1.231. [DOI] [PubMed] [Google Scholar]
  25. Milligan S. C., Alb J. G., Jr, Elagina R. B., Bankaitis V. A., Hyde D. R. The phosphatidylinositol transfer protein domain of Drosophila retinal degeneration B protein is essential for photoreceptor cell survival and recovery from light stimulation. J Cell Biol. 1997 Oct 20;139(2):351–363. doi: 10.1083/jcb.139.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Parent C. A., Devreotes P. N. A cell's sense of direction. Science. 1999 Apr 30;284(5415):765–770. doi: 10.1126/science.284.5415.765. [DOI] [PubMed] [Google Scholar]
  28. Parent C. A., Devreotes P. N. Molecular genetics of signal transduction in Dictyostelium. Annu Rev Biochem. 1996;65:411–440. doi: 10.1146/annurev.bi.65.070196.002211. [DOI] [PubMed] [Google Scholar]
  29. Phillips S. E., Sha B., Topalof L., Xie Z., Alb J. G., Klenchin V. A., Swigart P., Cockcroft S., Martin T. F., Luo M. Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo. Mol Cell. 1999 Aug;4(2):187–197. doi: 10.1016/s1097-2765(00)80366-4. [DOI] [PubMed] [Google Scholar]
  30. Prosser S., Sarra R., Swigart P., Ball A., Cockcroft S. Deletion of 24 amino acids from the C-terminus of phosphatidylinositol transfer protein causes loss of phospholipase C-mediated inositol lipid signalling. Biochem J. 1997 May 15;324(Pt 1):19–23. doi: 10.1042/bj3240019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stock S. D., Hama H., DeWald D. B., Takemoto J. Y. SEC14-dependent secretion in Saccharomyces cerevisiae. Nondependence on sphingolipid synthesis-coupled diacylglycerol production. J Biol Chem. 1999 May 7;274(19):12979–12983. doi: 10.1074/jbc.274.19.12979. [DOI] [PubMed] [Google Scholar]
  32. Tanaka S., Hosaka K. Cloning of a cDNA encoding a second phosphatidylinositol transfer protein of rat brain by complementation of the yeast sec14 mutation. J Biochem. 1994 May;115(5):981–984. doi: 10.1093/oxfordjournals.jbchem.a124448. [DOI] [PubMed] [Google Scholar]
  33. Thomas G. M., Cunningham E., Fensome A., Ball A., Totty N. F., Truong O., Hsuan J. J., Cockcroft S. An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling. Cell. 1993 Sep 10;74(5):919–928. doi: 10.1016/0092-8674(93)90471-2. [DOI] [PubMed] [Google Scholar]
  34. Wirtz K. W. Phospholipid transfer proteins revisited. Biochem J. 1997 Jun 1;324(Pt 2):353–360. doi: 10.1042/bj3240353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Vries K. J., Heinrichs A. A., Cunningham E., Brunink F., Westerman J., Somerharju P. J., Cockcroft S., Wirtz K. W., Snoek G. T. An isoform of the phosphatidylinositol-transfer protein transfers sphingomyelin and is associated with the Golgi system. Biochem J. 1995 Sep 1;310(Pt 2):643–649. doi: 10.1042/bj3100643. [DOI] [PMC free article] [PubMed] [Google Scholar]

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