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. 1997 Jun 15;16(12):3506–3518. doi: 10.1093/emboj/16.12.3506

Lipid remodeling leads to the introduction and exchange of defined ceramides on GPI proteins in the ER and Golgi of Saccharomyces cerevisiae.

F Reggiori 1, E Canivenc-Gansel 1, A Conzelmann 1
PMCID: PMC1169976  PMID: 9218793

Abstract

Previous experiments with Saccharomyces cerevisiae had suggested that diacylglycerol-containing glycosylphosphatidylinositols (GPIs) are added to newly synthesized proteins in the endoplasmic reticulum (ER) and that ceramides subsequently are incorporated into GPI proteins by lipid remodeling. Here we prove this hypothesis by labeling yeast cells with [3H]dihydrosphingosine ([3H]DHS) and showing that this tracer is incorporated into many GPI proteins even when protein synthesis and, hence, anchor addition, is blocked by cycloheximide. [3H]DHS incorporation is greatly enhanced if endogenous synthesis of DHS is inhibited by myriocin. Labeled GPI anchors contain three types of ceramides which, based on previous and present results, are identified as DHS-C26:0, phytosphingosine-C26:0 and phytosphingosine-C26:0-OH, the latter being found only on proteins which have reached the Golgi. Lipid remodeling can occur both in the ER and in a later secretory compartment. In addition, ceramide is incorporated into GPI proteins a long time after their initial synthesis by a process in which one ceramide gets replaced by another ceramide. Remodeling outside the ER requires vesicular flow from the ER to the Golgi, possibly to supply the remodeling enzymes with ceramides.

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Selected References

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  1. Benghezal M., Benachour A., Rusconi S., Aebi M., Conzelmann A. Yeast Gpi8p is essential for GPI anchor attachment onto proteins. EMBO J. 1996 Dec 2;15(23):6575–6583. [PMC free article] [PubMed] [Google Scholar]
  2. Brennwald P., Kearns B., Champion K., Keränen S., Bankaitis V., Novick P. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell. 1994 Oct 21;79(2):245–258. doi: 10.1016/0092-8674(94)90194-5. [DOI] [PubMed] [Google Scholar]
  3. Buede R., Rinker-Schaffer C., Pinto W. J., Lester R. L., Dickson R. C. Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids. J Bacteriol. 1991 Jul;173(14):4325–4332. doi: 10.1128/jb.173.14.4325-4332.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buxbaum L. U., Milne K. G., Werbovetz K. A., Englund P. T. Myristate exchange on the Trypanosoma brucei variant surface glycoprotein. Proc Natl Acad Sci U S A. 1996 Feb 6;93(3):1178–1183. doi: 10.1073/pnas.93.3.1178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Collins R. N., Warren G. Sphingolipid transport in mitotic HeLa cells. J Biol Chem. 1992 Dec 5;267(34):24906–24911. [PubMed] [Google Scholar]
  6. Conzelmann A., Fankhauser C., Desponds C. Myoinositol gets incorporated into numerous membrane glycoproteins of Saccharomyces cerevisiae; incorporation is dependent on phosphomannomutase (sec53). EMBO J. 1990 Mar;9(3):653–661. doi: 10.1002/j.1460-2075.1990.tb08157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davitz M. A., Hom J., Schenkman S. Purification of a glycosyl-phosphatidylinositol-specific phospholipase D from human plasma. J Biol Chem. 1989 Aug 15;264(23):13760–13764. [PubMed] [Google Scholar]
  8. Graham T. R., Emr S. D. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol. 1991 Jul;114(2):207–218. doi: 10.1083/jcb.114.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guide to yeast genetics and molecular biology. Methods Enzymol. 1991;194:1–863. [PubMed] [Google Scholar]
  10. Güther M. L., Masterson W. J., Ferguson M. A. The effects of phenylmethylsulfonyl fluoride on inositol-acylation and fatty acid remodeling in African trypanosomes. J Biol Chem. 1994 Jul 15;269(28):18694–18701. [PubMed] [Google Scholar]
  11. Güther M. L., de Almeida M. L., Yoshida N., Ferguson M. A. Structural studies on the glycosylphosphatidylinositol membrane anchor of Trypanosoma cruzi 1G7-antigen. The structure of the glycan core. J Biol Chem. 1992 Apr 5;267(10):6820–6828. [PubMed] [Google Scholar]
  12. Hamburger D., Egerton M., Riezman H. Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins. J Cell Biol. 1995 May;129(3):629–639. doi: 10.1083/jcb.129.3.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hanson B. A., Lester R. L. The extraction of inositol-containing phospholipids and phosphatidylcholine from Saccharomyces cerevisiae and Neurospora crassa. J Lipid Res. 1980 Mar;21(3):309–315. [PubMed] [Google Scholar]
  14. Harris S. L., Waters M. G. Localization of a yeast early Golgi mannosyltransferase, Och1p, involves retrograde transport. J Cell Biol. 1996 Mar;132(6):985–998. doi: 10.1083/jcb.132.6.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heise N., Raper J., Buxbaum L. U., Peranovich T. M., de Almeida M. L. Identification of complete precursors for the glycosylphosphatidylinositol protein anchors of Trypanosoma cruzi. J Biol Chem. 1996 Jul 12;271(28):16877–16887. doi: 10.1074/jbc.271.28.16877. [DOI] [PubMed] [Google Scholar]
  16. Horvath A., Sütterlin C., Manning-Krieg U., Movva N. R., Riezman H. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. EMBO J. 1994 Aug 15;13(16):3687–3695. doi: 10.1002/j.1460-2075.1994.tb06678.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hunter K., Rose A. H. Lipid composition of Saccharomyces cerevisiae as influenced by growth temperature. Biochim Biophys Acta. 1972 Apr 18;260(4):639–653. doi: 10.1016/0005-2760(72)90013-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lu C. F., Kurjan J., Lipke P. N. A pathway for cell wall anchorage of Saccharomyces cerevisiae alpha-agglutinin. Mol Cell Biol. 1994 Jul;14(7):4825–4833. doi: 10.1128/mcb.14.7.4825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lu C. F., Montijn R. C., Brown J. L., Klis F., Kurjan J., Bussey H., Lipke P. N. Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol. 1995 Feb;128(3):333–340. doi: 10.1083/jcb.128.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Maxwell S. E., Ramalingam S., Gerber L. D., Brink L., Udenfriend S. An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase. J Biol Chem. 1995 Aug 18;270(33):19576–19582. doi: 10.1074/jbc.270.33.19576. [DOI] [PubMed] [Google Scholar]
  22. McConville M. J., Ferguson M. A. The structure, biosynthesis and function of glycosylated phosphatidylinositols in the parasitic protozoa and higher eukaryotes. Biochem J. 1993 Sep 1;294(Pt 2):305–324. doi: 10.1042/bj2940305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miyake Y., Kozutsumi Y., Nakamura S., Fujita T., Kawasaki T. Serine palmitoyltransferase is the primary target of a sphingosine-like immunosuppressant, ISP-1/myriocin. Biochem Biophys Res Commun. 1995 Jun 15;211(2):396–403. doi: 10.1006/bbrc.1995.1827. [DOI] [PubMed] [Google Scholar]
  24. Nickels J. T., Broach J. R. A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae. Genes Dev. 1996 Feb 15;10(4):382–394. doi: 10.1101/gad.10.4.382. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Nuoffer C., Horvath A., Riezman H. Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein. J Biol Chem. 1993 May 15;268(14):10558–10563. [PubMed] [Google Scholar]
  27. 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]
  28. Patton J. L., Lester R. L. The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane. J Bacteriol. 1991 May;173(10):3101–3108. doi: 10.1128/jb.173.10.3101-3108.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pinto W. J., Wells G. W., Lester R. L. Characterization of enzymatic synthesis of sphingolipid long-chain bases in Saccharomyces cerevisiae: mutant strains exhibiting long-chain-base auxotrophy are deficient in serine palmitoyltransferase activity. J Bacteriol. 1992 Apr;174(8):2575–2581. doi: 10.1128/jb.174.8.2575-2581.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Puoti A., Conzelmann A. Characterization of abnormal free glycophosphatidylinositols accumulating in mutant lymphoma cells of classes B, E, F, and H. J Biol Chem. 1993 Apr 5;268(10):7215–7224. [PubMed] [Google Scholar]
  31. Puoti A., Desponds C., Conzelmann A. Biosynthesis of mannosylinositolphosphoceramide in Saccharomyces cerevisiae is dependent on genes controlling the flow of secretory vesicles from the endoplasmic reticulum to the Golgi. J Cell Biol. 1991 May;113(3):515–525. doi: 10.1083/jcb.113.3.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sefton B. M., Trowbridge I. S., Cooper J. A., Scolnick E. M. The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell. 1982 Dec;31(2 Pt 1):465–474. doi: 10.1016/0092-8674(82)90139-8. [DOI] [PubMed] [Google Scholar]
  33. Serrano A. A., Schenkman S., Yoshida N., Mehlert A., Richardson J. M., Ferguson M. A. The lipid structure of the glycosylphosphatidylinositol-anchored mucin-like sialic acid acceptors of Trypanosoma cruzi changes during parasite differentiation from epimastigotes to infective metacyclic trypomastigote forms. J Biol Chem. 1995 Nov 10;270(45):27244–27253. doi: 10.1074/jbc.270.45.27244. [DOI] [PubMed] [Google Scholar]
  34. Sipos G., Puoti A., Conzelmann A. Biosynthesis of the side chain of yeast glycosylphosphatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic reticulum and the Golgi apparatus. J Biol Chem. 1995 Aug 25;270(34):19709–19715. doi: 10.1074/jbc.270.34.19709. [DOI] [PubMed] [Google Scholar]
  35. Sipos G., Puoti A., Conzelmann A. Glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae: absence of ceramides from complete precursor glycolipids. EMBO J. 1994 Jun 15;13(12):2789–2796. doi: 10.1002/j.1460-2075.1994.tb06572.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sipos G., Reggiori F., Vionnet C., Conzelmann A. Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae. EMBO J. 1997 Jun 16;16(12):3494–3505. doi: 10.1093/emboj/16.12.3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smith S. W., Lester R. L. Inositol phosphorylceramide, a novel substance and the chief member of a major group of yeast sphingolipids containing a single inositol phosphate. J Biol Chem. 1974 Jun 10;249(11):3395–3405. [PubMed] [Google Scholar]
  38. Stadler J., Keenan T. W., Bauer G., Gerisch G. The contact site A glycoprotein of Dictyostelium discoideum carries a phospholipid anchor of a novel type. EMBO J. 1989 Feb;8(2):371–377. doi: 10.1002/j.1460-2075.1989.tb03387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stratford M. Another brick in the wall? Recent developments concerning the yeast cell envelope. Yeast. 1994 Dec;10(13):1741–1752. doi: 10.1002/yea.320101307. [DOI] [PubMed] [Google Scholar]
  40. Thomas J. R., Dwek R. A., Rademacher T. W. Structure, biosynthesis, and function of glycosylphosphatidylinositols. Biochemistry. 1990 Jun 12;29(23):5413–5422. doi: 10.1021/bi00475a001. [DOI] [PubMed] [Google Scholar]
  41. Wells G. B., Dickson R. C., Lester R. L. Isolation and composition of inositolphosphorylceramide-type sphingolipids of hyphal forms of Candida albicans. J Bacteriol. 1996 Nov;178(21):6223–6226. doi: 10.1128/jb.178.21.6223-6226.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wells G. B., Lester R. L. The isolation and characterization of a mutant strain of Saccharomyces cerevisiae that requires a long chain base for growth and for synthesis of phosphosphingolipids. J Biol Chem. 1983 Sep 10;258(17):10200–10203. [PubMed] [Google Scholar]
  43. van Meer G., Burger K. N. Sphingolipid trafficking--sorted out? Trends Cell Biol. 1992 Nov;2(11):332–337. [PubMed] [Google Scholar]

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