Logo of embojLink to Publisher's site
EMBO J. Feb 16, 1998; 17(4): 877–885.
PMCID: PMC1170437

The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1.

Abstract

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG-A and PIG-C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG-H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG-L, which is involved in the second step in GPI synthesis, GlcNAc-PI de-N-acetylation, did not associate with the isolated complex. The protein complex had GPI-GlcNAc transferase (GPI-GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized approximately 100-fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI-GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI-GnT may be relevant to selective usage of PI and/or regulation.

Full Text

The Full Text of this article is available as a PDF (660K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bessler M, Hillmen P, Longo L, Luzzatto L, Mason PJ. Genomic organization of the X-linked gene (PIG-A) that is mutated in paroxysmal nocturnal haemoglobinuria and of a related autosomal pseudogene mapped to 12q21. Hum Mol Genet. 1994 May;3(5):751–757. [PubMed]
  • BLIGH EG, DYER WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959 Aug;37(8):911–917. [PubMed]
  • Boguski MS, Lowe TM, Tolstoshev CM. dbEST--database for "expressed sequence tags". Nat Genet. 1993 Aug;4(4):332–333. [PubMed]
  • Colussi PA, Orlean P. The essential Schizosaccharomyces pombe gpil+ gene complements a bakers' yeast GPI anchoring mutant and is required for efficient cell separation. Yeast. 1997 Feb;13(2):139–150. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Conzelmann A, Puoti A, Lester RL, 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. [PMC free article] [PubMed]
  • Costello LC, 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]
  • Doering TL, Masterson WJ, Englund PT, Hart GW. 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]
  • Doering TL, Pessin MS, Hart GW, Raben DM, Englund PT. The fatty acids in unremodelled trypanosome glycosyl-phosphatidylinositols. Biochem J. 1994 May 1;299(Pt 3):741–746. [PMC free article] [PubMed]
  • Englund PT. The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu Rev Biochem. 1993;62:121–138. [PubMed]
  • Ferguson MA. What can GPI do for you? Parasitol Today. 1994 Feb;10(2):48–52. [PubMed]
  • Ferguson MA, Williams AF. Cell-surface anchoring of proteins via glycosyl-phosphatidylinositol structures. Annu Rev Biochem. 1988;57:285–320. [PubMed]
  • Heise N, Raper J, Buxbaum LU, Peranovich TM, de Almeida ML. Identification of complete precursors for the glycosylphosphatidylinositol protein anchors of Trypanosoma cruzi. J Biol Chem. 1996 Jul 12;271(28):16877–16887. [PubMed]
  • Hirose S, Ravi L, Hazra SV, Medof ME. Assembly and deacetylation of N-acetylglucosaminyl-plasmanylinositol in normal and affected paroxysmal nocturnal hemoglobinuria cells. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3762–3766. [PMC free article] [PubMed]
  • Inoue N, Watanabe R, Takeda J, Kinoshita T. PIG-C, one of the three human genes involved in the first step of glycosylphosphatidylinositol biosynthesis is a homologue of Saccharomyces cerevisiae GPI2. Biochem Biophys Res Commun. 1996 Sep 4;226(1):193–199. [PubMed]
  • Itoh N, Yonehara S, Ishii A, Yonehara M, Mizushima S, Sameshima M, Hase A, Seto Y, Nagata S. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991 Jul 26;66(2):233–243. [PubMed]
  • Jensen JW, Schutzbach JS. Activation of dolichyl-phospho-mannose synthase by phospholipids. Eur J Biochem. 1985 Nov 15;153(1):41–48. [PubMed]
  • Kamitani T, Chang HM, Rollins C, Waneck GL, Yeh ET. Correction of the class H defect in glycosylphosphatidylinositol anchor biosynthesis in Ltk- cells by a human cDNA clone. J Biol Chem. 1993 Oct 5;268(28):20733–20736. [PubMed]
  • Kawagoe K, Takeda J, Endo Y, Kinoshita T. Molecular cloning of murine pig-a, a gene for GPI-anchor biosynthesis, and demonstration of interspecies conservation of its structure, function, and genetic locus. Genomics. 1994 Oct;23(3):566–574. [PubMed]
  • Kawagoe K, Kitamura D, Okabe M, Taniuchi I, Ikawa M, Watanabe T, Kinoshita T, Takeda J. Glycosylphosphatidylinositol-anchor-deficient mice: implications for clonal dominance of mutant cells in paroxysmal nocturnal hemoglobinuria. Blood. 1996 May 1;87(9):3600–3606. [PubMed]
  • Kinoshita T, Inoue N, Takeda J. Defective glycosyl phosphatidylinositol anchor synthesis and paroxysmal nocturnal hemoglobinuria. Adv Immunol. 1995;60:57–103. [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Leidich SD, Orlean P. Gpi1, a Saccharomyces cerevisiae protein that participates in the first step in glycosylphosphatidylinositol anchor synthesis. J Biol Chem. 1996 Nov 1;271(44):27829–27837. [PubMed]
  • Leidich SD, Drapp DA, Orlean P. A conditionally lethal yeast mutant blocked at the first step in glycosyl phosphatidylinositol anchor synthesis. J Biol Chem. 1994 Apr 8;269(14):10193–10196. [PubMed]
  • Leidich SD, Kostova Z, Latek RR, Costello LC, Drapp DA, Gray W, Fassler JS, Orlean P. Temperature-sensitive yeast GPI anchoring mutants gpi2 and gpi3 are defective in the synthesis of N-acetylglucosaminyl phosphatidylinositol. Cloning of the GPI2 gene. J Biol Chem. 1995 Jun 2;270(22):13029–13035. [PubMed]
  • Lu CF, Montijn RC, Brown JL, Klis F, Kurjan J, Bussey H, Lipke PN. 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. [PMC free article] [PubMed]
  • Masterson WJ, Doering TL, Hart GW, Englund PT. 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. [PubMed]
  • Masterson WJ, Raper J, Doering TL, Hart GW, Englund PT. Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors. Cell. 1990 Jul 13;62(1):73–80. [PubMed]
  • McConville MJ, Ferguson MA. 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. [PMC free article] [PubMed]
  • Milne KG, Field RA, Masterson WJ, Cottaz S, Brimacombe JS, Ferguson MA. Partial purification and characterization of the N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase of glycosylphosphatidylinositol anchor biosynthesis in African trypanosomes. J Biol Chem. 1994 Jun 10;269(23):16403–16408. [PubMed]
  • Miyata T, Takeda J, Iida Y, Yamada N, Inoue N, Takahashi M, Maeda K, Kitani T, Kinoshita T. The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis. Science. 1993 Feb 26;259(5099):1318–1320. [PubMed]
  • Nakamura N, Inoue N, Watanabe R, Takahashi M, Takeda J, Stevens VL, Kinoshita T. Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase. J Biol Chem. 1997 Jun 20;272(25):15834–15840. [PubMed]
  • Schönbächler M, Horvath A, Fassler J, Riezman H. The yeast spt14 gene is homologous to the human PIG-A gene and is required for GPI anchor synthesis. EMBO J. 1995 Apr 18;14(8):1637–1645. [PMC free article] [PubMed]
  • Singh N, Zoeller RA, Tykocinski ML, Lazarow PB, Tartakoff AM. Addition of lipid substituents of mammalian protein glycosylphosphoinositol anchors. Mol Cell Biol. 1994 Jan;14(1):21–31. [PMC free article] [PubMed]
  • Stevens VL, Raetz CR. 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]
  • Sugiyama E, DeGasperi R, Urakaze M, Chang HM, Thomas LJ, Hyman R, Warren CD, Yeh ET. Identification of defects in glycosylphosphatidylinositol anchor biosynthesis in the Thy-1 expression mutants. J Biol Chem. 1991 Jul 5;266(19):12119–12122. [PubMed]
  • Takeda J, Miyata T, Kawagoe K, Iida Y, Endo Y, Fujita T, Takahashi M, Kitani T, Kinoshita T. Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria. Cell. 1993 May 21;73(4):703–711. [PubMed]
  • Thomas JR, Dwek RA, Rademacher TW. Structure, biosynthesis, and function of glycosylphosphatidylinositols. Biochemistry. 1990 Jun 12;29(23):5413–5422. [PubMed]
  • Vidugiriene J, Menon AK. 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. [PMC free article] [PubMed]
  • Vossen JH, Ram AF, Klis FM. Identification of SPT14/CWH6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors. Biochim Biophys Acta. 1995 Apr 13;1243(3):549–551. [PubMed]
  • Watanabe R, Kinoshita T, Masaki R, Yamamoto A, Takeda J, Inoue N. PIG-A and PIG-H, which participate in glycosylphosphatidylinositol anchor biosynthesis, form a protein complex in the endoplasmic reticulum. J Biol Chem. 1996 Oct 25;271(43):26868–26875. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...