Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1994 Oct 2;127(2):333–341. doi: 10.1083/jcb.127.2.333

The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum

PMCID: PMC2120202  PMID: 7929579

Abstract

Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-specific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Abeijon C., Hirschberg C. B. Topography of glycosylation reactions in the endoplasmic reticulum. Trends Biochem Sci. 1992 Jan;17(1):32–36. doi: 10.1016/0968-0004(92)90424-8. [DOI] [PubMed] [Google Scholar]
  2. Amthauer R., Kodukula K., Brink L., Udenfriend S. Phosphatidylinositol-glycan (PI-G)-anchored membrane proteins: requirement of ATP and GTP for translation-independent COOH-terminal processing. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6124–6128. doi: 10.1073/pnas.89.13.6124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Bangs J. D., Uyetake L., Brickman M. J., Balber A. E., Boothroyd J. C. Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J Cell Sci. 1993 Aug;105(Pt 4):1101–1113. doi: 10.1242/jcs.105.4.1101. [DOI] [PubMed] [Google Scholar]
  6. Bell R. M., Ballas L. M., Coleman R. A. Lipid topogenesis. J Lipid Res. 1981 Mar;22(3):391–403. [PubMed] [Google Scholar]
  7. Booth C., Koch G. L. Perturbation of cellular calcium induces secretion of luminal ER proteins. Cell. 1989 Nov 17;59(4):729–737. doi: 10.1016/0092-8674(89)90019-6. [DOI] [PubMed] [Google Scholar]
  8. Brown D. A. Interactions between GPI-anchored proteins and membrane lipids. Trends Cell Biol. 1992 Nov;2(11):338–343. [PubMed] [Google Scholar]
  9. Brown D. The tyrosine kinase connection: how GPI-anchored proteins activate T cells. Curr Opin Immunol. 1993 Jun;5(3):349–354. doi: 10.1016/0952-7915(93)90052-t. [DOI] [PubMed] [Google Scholar]
  10. Bölow R., Griffiths G., Webster P., Stierhof Y. D., Opperdoes F. R., Overath P. Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei. J Cell Sci. 1989 Jun;93(Pt 2):233–240. doi: 10.1242/jcs.93.2.233. [DOI] [PubMed] [Google Scholar]
  11. Cross G. A. Glycolipid anchoring of plasma membrane proteins. Annu Rev Cell Biol. 1990;6:1–39. doi: 10.1146/annurev.cb.06.110190.000245. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gilmore R. Protein translocation across the endoplasmic reticulum: a tunnel with toll booths at entry and exit. Cell. 1993 Nov 19;75(4):589–592. doi: 10.1016/0092-8674(93)90476-7. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Haselbeck A., Tanner W. Dolichyl phosphate-mediated mannosyl transfer through liposomal membranes. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1520–1524. doi: 10.1073/pnas.79.5.1520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Higgins J. A., Hitchin B. W., Low M. G. Phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis as a probe for the distribution of phosphatidylinositol in hepatocyte membranes. Biochem J. 1989 May 1;259(3):913–916. doi: 10.1042/bj2590913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hubbard S. C., Ivatt R. J. Synthesis and processing of asparagine-linked oligosaccharides. Annu Rev Biochem. 1981;50:555–583. doi: 10.1146/annurev.bi.50.070181.003011. [DOI] [PubMed] [Google Scholar]
  19. Hutson J. L., Higgins J. A. Asymmetric synthesis followed by transmembrane movement of phosphatidylethanolamine in rat liver endoplasmic reticulum. Biochim Biophys Acta. 1982 May 7;687(2):247–256. doi: 10.1016/0005-2736(82)90553-3. [DOI] [PubMed] [Google Scholar]
  20. Jackson R. C., Blobel G. Post-translational cleavage of presecretory proteins with an extract of rough microsomes from dog pancreas containing signal peptidase activity. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5598–5602. doi: 10.1073/pnas.74.12.5598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kinoshita T., Takeda J. GPI-anchor synthesis. Parasitol Today. 1994 Apr;10(4):139–143. doi: 10.1016/0169-4758(94)90261-5. [DOI] [PubMed] [Google Scholar]
  22. Krakow J. L., Doering T. L., Masterson W. J., Hart G. W., Englund P. T. A glycolipid from Trypanosoma brucei related to the variant surface glycoprotein membrane anchor. Mol Biochem Parasitol. 1989 Oct;36(3):263–270. doi: 10.1016/0166-6851(89)90174-6. [DOI] [PubMed] [Google Scholar]
  23. Lambert N., Freedman R. B. Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure. Biochem J. 1983 Jul 1;213(1):225–234. doi: 10.1042/bj2130225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Low M. G., Kincade P. W. Phosphatidylinositol is the membrane-anchoring domain of the Thy-1 glycoprotein. Nature. 1985 Nov 7;318(6041):62–64. doi: 10.1038/318062a0. [DOI] [PubMed] [Google Scholar]
  25. Masterson W. J., Doering T. L., Hart G. W., Englund P. T. A novel pathway for glycan assembly: biosynthesis of the glycosyl-phosphatidylinositol anchor of the trypanosome variant surface glycoprotein. Cell. 1989 Mar 10;56(5):793–800. doi: 10.1016/0092-8674(89)90684-3. [DOI] [PubMed] [Google Scholar]
  26. Masterson W. J., Ferguson M. A. Phenylmethanesulphonyl fluoride inhibits GPI anchor biosynthesis in the African trypanosome. EMBO J. 1991 Aug;10(8):2041–2045. doi: 10.1002/j.1460-2075.1991.tb07734.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mayor S., Menon A. K., Cross G. A. Glycolipid precursors for the membrane anchor of Trypanosoma brucei variant surface glycoproteins. II. Lipid structures of phosphatidylinositol-specific phospholipase C sensitive and resistant glycolipids. J Biol Chem. 1990 Apr 15;265(11):6174–6181. [PubMed] [Google Scholar]
  28. Mayor S., Menon A. K., Cross G. A. Transfer of glycosyl-phosphatidylinositol membrane anchors to polypeptide acceptors in a cell-free system. J Cell Biol. 1991 Jul;114(1):61–71. doi: 10.1083/jcb.114.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Menon A. K., Eppinger M., Mayor S., Schwarz R. T. Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols. EMBO J. 1993 May;12(5):1907–1914. doi: 10.1002/j.1460-2075.1993.tb05839.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Menon A. K., Mayor S., Schwarz R. T. Biosynthesis of glycosyl-phosphatidylinositol lipids in Trypanosoma brucei: involvement of mannosyl-phosphoryldolichol as the mannose donor. EMBO J. 1990 Dec;9(13):4249–4258. doi: 10.1002/j.1460-2075.1990.tb07873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Menon A. K., Schwarz R. T., Mayor S., Cross G. A. Cell-free synthesis of glycosyl-phosphatidylinositol precursors for the glycolipid membrane anchor of Trypanosoma brucei variant surface glycoproteins. Structural characterization of putative biosynthetic intermediates. J Biol Chem. 1990 Jun 5;265(16):9033–9042. [PubMed] [Google Scholar]
  33. Mensa-Wilmot K., LeBowitz J. H., Chang K. P., al-Qahtani A., McGwire B. S., Tucker S., Morris J. C. A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways. J Cell Biol. 1994 Mar;124(6):935–947. doi: 10.1083/jcb.124.6.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nagel S. D., Boothroyd J. C. The major surface antigen, P30, of Toxoplasma gondii is anchored by a glycolipid. J Biol Chem. 1989 Apr 5;264(10):5569–5574. [PubMed] [Google Scholar]
  35. Ogata S., Muramatsu T., Kobata A. Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose. J Biochem. 1975 Oct;78(4):687–696. doi: 10.1093/oxfordjournals.jbchem.a130956. [DOI] [PubMed] [Google Scholar]
  36. Paver J. L., Hawkins H. C., Freedman R. B. Preparation and characterization of dog pancreas microsomal membranes specifically depleted of protein disulphide-isomerase. Biochem J. 1989 Feb 1;257(3):657–663. doi: 10.1042/bj2570657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Roberts W. L., Myher J. J., Kuksis A., Low M. G., Rosenberry T. L. Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C. J Biol Chem. 1988 Dec 15;263(35):18766–18775. [PubMed] [Google Scholar]
  38. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  39. Snider M. D., Robbins P. W. Transmembrane organization of protein glycosylation. Mature oligosaccharide-lipid is located on the luminal side of microsomes from Chinese hamster ovary cells. J Biol Chem. 1982 Jun 25;257(12):6796–6801. [PubMed] [Google Scholar]
  40. Snider M. D., Rogers O. C. Transmembrane movement of oligosaccharide-lipids during glycoprotein synthesis. Cell. 1984 Mar;36(3):753–761. doi: 10.1016/0092-8674(84)90355-6. [DOI] [PubMed] [Google Scholar]
  41. Stevens V. L., Raetz C. R. Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants. J Biol Chem. 1991 Jun 5;266(16):10039–10042. [PubMed] [Google Scholar]
  42. Tanner W., Lehle L. Protein glycosylation in yeast. Biochim Biophys Acta. 1987 Apr 27;906(1):81–99. doi: 10.1016/0304-4157(87)90006-2. [DOI] [PubMed] [Google Scholar]
  43. Vidugiriene J., Menon A. K. Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. J Cell Biol. 1993 Jun;121(5):987–996. doi: 10.1083/jcb.121.5.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zachowski A. Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement. Biochem J. 1993 Aug 15;294(Pt 1):1–14. doi: 10.1042/bj2940001. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES