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. 1996 Jan 2;15(1):182–191.

GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

T L Doering 1, R Schekman 1
PMCID: PMC449930  PMID: 8598201

Abstract

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.

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Selected References

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

  1. Ambroziak J., Henry S. A. INO2 and INO4 gene products, positive regulators of phospholipid biosynthesis in Saccharomyces cerevisiae, form a complex that binds to the INO1 promoter. J Biol Chem. 1994 May 27;269(21):15344–15349. [PubMed] [Google Scholar]
  2. Amthauer R., Kodukula K., Gerber L., Udenfriend S. Evidence that the putative COOH-terminal signal transamidase involved in glycosylphosphatidylinositol protein synthesis is present in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3973–3977. doi: 10.1073/pnas.90.9.3973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Atkinson K. D., Kolat A. I., Henry S. A. Osmotic imbalance in inositol-starved spheroplasts of Saccharomyces cerevisiae. J Bacteriol. 1977 Dec;132(3):806–817. doi: 10.1128/jb.132.3.806-817.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker D., Hicke L., Rexach M., Schleyer M., Schekman R. Reconstitution of SEC gene product-dependent intercompartmental protein transport. Cell. 1988 Jul 29;54(3):335–344. doi: 10.1016/0092-8674(88)90196-1. [DOI] [PubMed] [Google Scholar]
  5. Baker D., Wuestehube L., Schekman R., Botstein D., Segev N. GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction. Proc Natl Acad Sci U S A. 1990 Jan;87(1):355–359. doi: 10.1073/pnas.87.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Balch W. E., Farquhar M. G. Beyond bulk flow. Trends Cell Biol. 1995 Jan;5(1):16–19. doi: 10.1016/s0962-8924(00)88928-x. [DOI] [PubMed] [Google Scholar]
  7. Bangs J. D., Doering T. L., Englund P. T., Hart G. W. Biosynthesis of a variant surface glycoprotein of Trypanosoma brucei. Processing of the glycolipid membrane anchor and N-linked oligosaccharides. J Biol Chem. 1988 Nov 25;263(33):17697–17705. [PubMed] [Google Scholar]
  8. Barlowe C., Orci L., Yeung T., Hosobuchi M., Hamamoto S., Salama N., Rexach M. F., Ravazzola M., Amherdt M., Schekman R. COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell. 1994 Jun 17;77(6):895–907. doi: 10.1016/0092-8674(94)90138-4. [DOI] [PubMed] [Google Scholar]
  9. Becker G. W., Lester R. L. Changes in phospholipids of Saccharomyces cerevisiae associated with inositol-less death. J Biol Chem. 1977 Dec 10;252(23):8684–8691. [PubMed] [Google Scholar]
  10. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  11. Brown D. GPI-anchored proteins and detergent-resistant membrane domains. Braz J Med Biol Res. 1994 Feb;27(2):309–315. [PubMed] [Google Scholar]
  12. Caras I. W. An internally positioned signal can direct attachment of a glycophospholipid membrane anchor. J Cell Biol. 1991 Apr;113(1):77–85. doi: 10.1083/jcb.113.1.77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Caras I. W., Weddell G. N. Signal peptide for protein secretion directing glycophospholipid membrane anchor attachment. Science. 1989 Mar 3;243(4895):1196–1198. doi: 10.1126/science.2466338. [DOI] [PubMed] [Google Scholar]
  14. Carman G. M., Henry S. A. Phospholipid biosynthesis in yeast. Annu Rev Biochem. 1989;58:635–669. doi: 10.1146/annurev.bi.58.070189.003223. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Conzelmann A., Puoti A., Lester R. L., Desponds C. Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae. EMBO J. 1992 Feb;11(2):457–466. doi: 10.1002/j.1460-2075.1992.tb05075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Conzelmann A., Riezman H., Desponds C., Bron C. A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 1988 Jul;7(7):2233–2240. doi: 10.1002/j.1460-2075.1988.tb03063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Costello L. C., Orlean P. Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A. J Biol Chem. 1992 Apr 25;267(12):8599–8603. [PubMed] [Google Scholar]
  19. Culbertson M. R., Henry S. A. Inositol-requiring mutants of Saccharomyces cerevisiae. Genetics. 1975 May;80(1):23–40. doi: 10.1093/genetics/80.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Doering T. L., Masterson W. J., Englund P. T., Hart G. W. Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine. J Biol Chem. 1989 Jul 5;264(19):11168–11173. [PubMed] [Google Scholar]
  21. Englund P. T. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu Rev Biochem. 1993;62:121–138. doi: 10.1146/annurev.bi.62.070193.001005. [DOI] [PubMed] [Google Scholar]
  22. Fankhauser C., Homans S. W., Thomas-Oates J. E., McConville M. J., Desponds C., Conzelmann A., Ferguson M. A. Structures of glycosylphosphatidylinositol membrane anchors from Saccharomyces cerevisiae. J Biol Chem. 1993 Dec 15;268(35):26365–26374. [PubMed] [Google Scholar]
  23. Fiedler K., Kobayashi T., Kurzchalia T. V., Simons K. Glycosphingolipid-enriched, detergent-insoluble complexes in protein sorting in epithelial cells. Biochemistry. 1993 Jun 29;32(25):6365–6373. doi: 10.1021/bi00076a009. [DOI] [PubMed] [Google Scholar]
  24. Field M. C., Moran P., Li W., Keller G. A., Caras I. W. Retention and degradation of proteins containing an uncleaved glycosylphosphatidylinositol signal. J Biol Chem. 1994 Apr 8;269(14):10830–10837. [PubMed] [Google Scholar]
  25. Gatti E., Popolo L., Vai M., Rota N., Alberghina L. O-linked oligosaccharides in yeast glycosyl phosphatidylinositol-anchored protein gp115 are clustered in a serine-rich region not essential for its function. J Biol Chem. 1994 Aug 5;269(31):19695–19700. [PubMed] [Google Scholar]
  26. Gerber L. D., Kodukula K., Udenfriend S. Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide. J Biol Chem. 1992 Jun 15;267(17):12168–12173. [PubMed] [Google Scholar]
  27. Griffiths G., Doms R. W., Mayhew T., Lucocq J. The bulk-flow hypothesis: not quite the end. Trends Cell Biol. 1995 Jan;5(1):9–13. doi: 10.1016/s0962-8924(00)88926-6. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Helenius A., Marquardt T., Braakman I. The endoplasmic reticulum as a protein-folding compartment. Trends Cell Biol. 1992 Aug;2(8):227–231. doi: 10.1016/0962-8924(92)90309-b. [DOI] [PubMed] [Google Scholar]
  30. Henry S. A., Atkinson K. D., Kolat A. I., Culbertson M. R. Growth and metabolism of inositol-starved Saccharomyces cerevisiae. J Bacteriol. 1977 Apr;130(1):472–484. doi: 10.1128/jb.130.1.472-484.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. Kurzchalia T. V., Hartmann E., Dupree P. Guilty by insolubility--does a protein's detergent insolubility reflect a caveolar location? Trends Cell Biol. 1995 May;5(5):187–189. doi: 10.1016/s0962-8924(00)88990-4. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. 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]
  37. McGee T. P., Skinner H. B., Whitters E. A., Henry S. A., Bankaitis V. A. A phosphatidylinositol transfer protein controls the phosphatidylcholine content of yeast Golgi membranes. J Cell Biol. 1994 Feb;124(3):273–287. doi: 10.1083/jcb.124.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Micanovic R., Gerber L. D., Berger J., Kodukula K., Udenfriend S. Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase. Proc Natl Acad Sci U S A. 1990 Jan;87(1):157–161. doi: 10.1073/pnas.87.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Nilsson T., Warren G. Retention and retrieval in the endoplasmic reticulum and the Golgi apparatus. Curr Opin Cell Biol. 1994 Aug;6(4):517–521. doi: 10.1016/0955-0674(94)90070-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Nuoffer C., Jenö P., Conzelmann A., Riezman H. Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane. Mol Cell Biol. 1991 Jan;11(1):27–37. doi: 10.1128/mcb.11.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pryer N. K., Wuestehube L. J., Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. doi: 10.1146/annurev.bi.61.070192.002351. [DOI] [PubMed] [Google Scholar]
  43. Rexach M. F., Latterich M., Schekman R. W. Characteristics of endoplasmic reticulum-derived transport vesicles. J Cell Biol. 1994 Sep;126(5):1133–1148. doi: 10.1083/jcb.126.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  45. Schekman R. Genetic and biochemical analysis of vesicular traffic in yeast. Curr Opin Cell Biol. 1992 Aug;4(4):587–592. doi: 10.1016/0955-0674(92)90076-o. [DOI] [PubMed] [Google Scholar]
  46. Schimmöller F., Singer-Krüger B., Schröder S., Krüger U., Barlowe C., Riezman H. The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi. EMBO J. 1995 Apr 3;14(7):1329–1339. doi: 10.1002/j.1460-2075.1995.tb07119.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schu P. V., Takegawa K., Fry M. J., Stack J. H., Waterfield M. D., Emr S. D. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. doi: 10.1126/science.8385367. [DOI] [PubMed] [Google Scholar]
  48. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  49. Simons K., van Meer G. Lipid sorting in epithelial cells. Biochemistry. 1988 Aug 23;27(17):6197–6202. doi: 10.1021/bi00417a001. [DOI] [PubMed] [Google Scholar]
  50. Singer S. J. It's important to concentrate. Trends Cell Biol. 1995 Jan;5(1):14–15. doi: 10.1016/s0962-8924(00)88927-8. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  53. Vidugiriene J., Menon A. K. Soluble constituents of the ER lumen are required for GPI anchoring of a model protein. EMBO J. 1995 Oct 2;14(19):4686–4694. doi: 10.1002/j.1460-2075.1995.tb00150.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. 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]
  55. Wuestehube L. J., Schekman R. W. Reconstitution of transport from endoplasmic reticulum to Golgi complex using endoplasmic reticulum-enriched membrane fraction from yeast. Methods Enzymol. 1992;219:124–136. doi: 10.1016/0076-6879(92)19015-x. [DOI] [PubMed] [Google Scholar]
  56. van Meer G., Simons K. Lipid polarity and sorting in epithelial cells. J Cell Biochem. 1988 Jan;36(1):51–58. doi: 10.1002/jcb.240360106. [DOI] [PubMed] [Google Scholar]

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