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. 2001 Aug;158(4):1431–1444. doi: 10.1093/genetics/158.4.1431

SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae.

S A Rudge 1, T R Pettitt 1, C Zhou 1, M J Wakelam 1, J A Engebrecht 1
PMCID: PMC1461740  PMID: 11514437

Abstract

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

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Selected References

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  1. Athenstaedt K., Daum G. Phosphatidic acid, a key intermediate in lipid metabolism. Eur J Biochem. 1999 Nov;266(1):1–16. doi: 10.1046/j.1432-1327.1999.00822.x. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  4. Cleves A. E., McGee T. P., Whitters E. A., Champion K. M., Aitken J. R., Dowhan W., Goebl M., Bankaitis V. A. Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein. Cell. 1991 Feb 22;64(4):789–800. doi: 10.1016/0092-8674(91)90508-v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cluett E. B., Machamer C. E. The envelope of vaccinia virus reveals an unusual phospholipid in Golgi complex membranes. J Cell Sci. 1996 Aug;109(Pt 8):2121–2131. doi: 10.1242/jcs.109.8.2121. [DOI] [PubMed] [Google Scholar]
  6. Elledge S. J., Davis R. W. A family of versatile centromeric vectors designed for use in the sectoring-shuffle mutagenesis assay in Saccharomyces cerevisiae. Gene. 1988 Oct 30;70(2):303–312. doi: 10.1016/0378-1119(88)90202-8. [DOI] [PubMed] [Google Scholar]
  7. Exton J. H. Phospholipase D. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):105–115. doi: 10.1016/s0005-2760(98)00124-6. [DOI] [PubMed] [Google Scholar]
  8. Grant A. M., Hanson P. K., Malone L., Nichols J. W. NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane. Traffic. 2001 Jan;2(1):37–50. doi: 10.1034/j.1600-0854.2001.020106.x. [DOI] [PubMed] [Google Scholar]
  9. Henneberry A. L., Lagace T. A., Ridgway N. D., McMaster C. R. Phosphatidylcholine synthesis influences the diacylglycerol homeostasis required for SEC14p-dependent Golgi function and cell growth. Mol Biol Cell. 2001 Mar;12(3):511–520. doi: 10.1091/mbc.12.3.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Henry S. A., Halvorson H. O. Lipid synthesis during sporulation of Saccharomyces cerevisiae. J Bacteriol. 1973 Jun;114(3):1158–1163. doi: 10.1128/jb.114.3.1158-1163.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  12. Hollingsworth N. M., Ponte L., Halsey C. MSH5, a novel MutS homolog, facilitates meiotic reciprocal recombination between homologs in Saccharomyces cerevisiae but not mismatch repair. Genes Dev. 1995 Jul 15;9(14):1728–1739. doi: 10.1101/gad.9.14.1728. [DOI] [PubMed] [Google Scholar]
  13. Honda A., Nogami M., Yokozeki T., Yamazaki M., Nakamura H., Watanabe H., Kawamoto K., Nakayama K., Morris A. J., Frohman M. A. Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell. 1999 Nov 24;99(5):521–532. doi: 10.1016/s0092-8674(00)81540-8. [DOI] [PubMed] [Google Scholar]
  14. Honigberg S. M., Conicella C., Espositio R. E. Commitment to meiosis in Saccharomyces cerevisiae: involvement of the SPO14 gene. Genetics. 1992 Apr;130(4):703–716. doi: 10.1093/genetics/130.4.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Jones D., Morgan C., Cockcroft S. Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding. Biochim Biophys Acta. 1999 Jul 30;1439(2):229–244. doi: 10.1016/s1388-1981(99)00097-9. [DOI] [PubMed] [Google Scholar]
  17. Kean L. S., Fuller R. S., Nichols J. W. Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane. J Cell Biol. 1993 Dec;123(6 Pt 1):1403–1419. doi: 10.1083/jcb.123.6.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Krisak L., Strich R., Winters R. S., Hall J. P., Mallory M. J., Kreitzer D., Tuan R. S., Winter E. SMK1, a developmentally regulated MAP kinase, is required for spore wall assembly in Saccharomyces cerevisiae. Genes Dev. 1994 Sep 15;8(18):2151–2161. doi: 10.1101/gad.8.18.2151. [DOI] [PubMed] [Google Scholar]
  19. Li X., Routt S. M., Xie Z., Cui X., Fang M., Kearns M. A., Bard M., Kirsch D. R., Bankaitis V. A. Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth. Mol Biol Cell. 2000 Jun;11(6):1989–2005. doi: 10.1091/mbc.11.6.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Liscovitch M., Cantley L. C. Signal transduction and membrane traffic: the PITP/phosphoinositide connection. Cell. 1995 Jun 2;81(5):659–662. doi: 10.1016/0092-8674(95)90525-1. [DOI] [PubMed] [Google Scholar]
  21. Matsuoka K., Orci L., Amherdt M., Bednarek S. Y., Hamamoto S., Schekman R., Yeung T. COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes. Cell. 1998 Apr 17;93(2):263–275. doi: 10.1016/s0092-8674(00)81577-9. [DOI] [PubMed] [Google Scholar]
  22. Mayr J. A., Kohlwein S. D., Paltauf F. Identification of a novel, Ca(2+)-dependent phospholipase D with preference for phosphatidylserine and phosphatidylethanolamine in Saccharomyces cerevisiae. FEBS Lett. 1996 Sep 16;393(2-3):236–240. doi: 10.1016/0014-5793(96)00893-9. [DOI] [PubMed] [Google Scholar]
  23. Morris A. J., Engebrecht J., Frohman M. A. Structure and regulation of phospholipase D. Trends Pharmacol Sci. 1996 May;17(5):182–185. doi: 10.1016/0165-6147(96)10016-x. [DOI] [PubMed] [Google Scholar]
  24. Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
  25. Neiman A. M. Prospore membrane formation defines a developmentally regulated branch of the secretory pathway in yeast. J Cell Biol. 1998 Jan 12;140(1):29–37. doi: 10.1083/jcb.140.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  27. Patton-Vogt J. L., Griac P., Sreenivas A., Bruno V., Dowd S., Swede M. J., Henry S. A. Role of the yeast phosphatidylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation. J Biol Chem. 1997 Aug 15;272(33):20873–20883. doi: 10.1074/jbc.272.33.20873. [DOI] [PubMed] [Google Scholar]
  28. Pettitt T. R., Martin A., Horton T., Liossis C., Lord J. M., Wakelam M. J. Diacylglycerol and phosphatidate generated by phospholipases C and D, respectively, have distinct fatty acid compositions and functions. Phospholipase D-derived diacylglycerol does not activate protein kinase C in porcine aortic endothelial cells. J Biol Chem. 1997 Jul 11;272(28):17354–17359. doi: 10.1074/jbc.272.28.17354. [DOI] [PubMed] [Google Scholar]
  29. Ponting C. P. Novel domains in NADPH oxidase subunits, sorting nexins, and PtdIns 3-kinases: binding partners of SH3 domains? Protein Sci. 1996 Nov;5(11):2353–2357. doi: 10.1002/pro.5560051122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rivas M. P., Kearns B. G., Xie Z., Guo S., Sekar M. C., Hosaka K., Kagiwada S., York J. D., Bankaitis V. A. Pleiotropic alterations in lipid metabolism in yeast sac1 mutants: relationship to "bypass Sec14p" and inositol auxotrophy. Mol Biol Cell. 1999 Jul;10(7):2235–2250. doi: 10.1091/mbc.10.7.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rose K., Rudge S. A., Frohman M. A., Morris A. J., Engebrecht J. Phospholipase D signaling is essential for meiosis. Proc Natl Acad Sci U S A. 1995 Dec 19;92(26):12151–12155. doi: 10.1073/pnas.92.26.12151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  33. Rudge S. A., Cavenagh M. M., Kamath R., Sciorra V. A., Morris A. J., Kahn R. A., Engebrecht J. ADP-Ribosylation factors do not activate yeast phospholipase Ds but are required for sporulation. Mol Biol Cell. 1998 Aug;9(8):2025–2036. doi: 10.1091/mbc.9.8.2025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rudge S. A., Engebrecht J. Regulation and function of PLDs in yeast. Biochim Biophys Acta. 1999 Jul 30;1439(2):167–174. doi: 10.1016/s1388-1981(99)00092-x. [DOI] [PubMed] [Google Scholar]
  35. Schmidt A., Wolde M., Thiele C., Fest W., Kratzin H., Podtelejnikov A. V., Witke W., Huttner W. B., Söling H. D. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature. 1999 Sep 9;401(6749):133–141. doi: 10.1038/43613. [DOI] [PubMed] [Google Scholar]
  36. Sciorra V. A., Morris A. J. Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells. Mol Biol Cell. 1999 Nov;10(11):3863–3876. doi: 10.1091/mbc.10.11.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Siddhanta A., Backer J. M., Shields D. Inhibition of phosphatidic acid synthesis alters the structure of the Golgi apparatus and inhibits secretion in endocrine cells. J Biol Chem. 2000 Apr 21;275(16):12023–12031. doi: 10.1074/jbc.275.16.12023. [DOI] [PubMed] [Google Scholar]
  38. Sreenivas A., Patton-Vogt J. L., Bruno V., Griac P., Henry S. A. A role for phospholipase D (Pld1p) in growth, secretion, and regulation of membrane lipid synthesis in yeast. J Biol Chem. 1998 Jul 3;273(27):16635–16638. doi: 10.1074/jbc.273.27.16635. [DOI] [PubMed] [Google Scholar]
  39. Steed P. M., Clark K. L., Boyar W. C., Lasala D. J. Characterization of human PLD2 and the analysis of PLD isoform splice variants. FASEB J. 1998 Oct;12(13):1309–1317. doi: 10.1096/fasebj.12.13.1309. [DOI] [PubMed] [Google Scholar]
  40. Sung T. C., Roper R. L., Zhang Y., Rudge S. A., Temel R., Hammond S. M., Morris A. J., Moss B., Engebrecht J., Frohman M. A. Mutagenesis of phospholipase D defines a superfamily including a trans-Golgi viral protein required for poxvirus pathogenicity. EMBO J. 1997 Aug 1;16(15):4519–4530. doi: 10.1093/emboj/16.15.4519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Waksman M., Tang X., Eli Y., Gerst J. E., Liscovitch M. Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1. J Biol Chem. 1997 Jan 3;272(1):36–39. doi: 10.1074/jbc.272.1.36. [DOI] [PubMed] [Google Scholar]
  42. Waksman M., Tang X., Eli Y., Gerst J. E., Liscovitch M. Identification of a novel Ca2+-dependent, phosphatidylethanolamine-hydrolyzing phospholipase D in yeast bearing a disruption in PLD1. J Biol Chem. 1997 Jan 3;272(1):36–39. doi: 10.1074/jbc.272.1.36. [DOI] [PubMed] [Google Scholar]
  43. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  44. Xie Z., Fang M., Rivas M. P., Faulkner A. J., Sternweis P. C., Engebrecht J. A., Bankaitis V. A. Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects. Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12346–12351. doi: 10.1073/pnas.95.21.12346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. van Blitterswijk W. J., Hilkmann H. Rapid attenuation of receptor-induced diacylglycerol and phosphatidic acid by phospholipase D-mediated transphosphatidylation: formation of bisphosphatidic acid. EMBO J. 1993 Jul;12(7):2655–2662. doi: 10.1002/j.1460-2075.1993.tb05926.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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