title: Disulfide Bond Formation and N-Glycosylation Modulate Protein-Protein Interactions in GPI-Transamidase (GPIT).
abstract: Glycosylphosphatidylinositol (GPI) transamidase (GPIT), the enzyme that attaches GPI anchors to proteins as they enter the lumen of the endoplasmic reticulum, is a membrane-bound hetero-pentameric complex consisting of Gpi8, Gpi16, Gaa1, Gpi17 and Gab1. Here, we expressed and purified the luminal domain of Saccharomyces cerevisiae (S. cerevisiae) Gpi8 using different expression systems, and examined its interaction with insect cell expressed luminal domain of S. cerevisiae Gpi16. We found that the N-terminal caspase-like domain of Gpi8 forms a disulfide-linked dimer, which is strengthened by N-glycosylation. The non-core domain of Gpi8 following the caspase-like domain inhibits this dimerization. In contrast to the previously reported disulfide linkage between Gpi8 and Gpi16 in human and trypanosome GPIT, our data show that the luminal domains of S. cerevisiae Gpi8 and S. cerevisiae Gpi16 do not interact directly, nor do they form a disulfide bond in the intact S. cerevisiae GPIT. Our data suggest that subunit interactions within the GPIT complex from different species may vary, a feature that should be taken into account in future structural and functional studies.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.|Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
authors.forename: Lina
authors.initials: L
authors.lastname: Yi
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
authors.forename: Gunes
authors.initials: G
authors.lastname: Bozkurt
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.
authors.forename: Qiubai
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, NY, 10065, USA.
authors.forename: Stanley
authors.initials: S
authors.lastname: Lo
authors.affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, NY, 10065, USA.
authors.forename: Anant K
authors.initials: AK
authors.lastname: Menon
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.|Program in Cellular and Molecular Medicine, Boston Children's Hospital, Boston, MA 02115, USA.|Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Wu
mesh_terms: D000217:Acyltransferases / Q000235:genetics*; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics; D000818:Animals; D015815:Cell Adhesion Molecules / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007313:Insecta / Q000235:genetics; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D000072417:Protein Domains / Q000235:genetics; D060066:Protein Interaction Maps / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D014345:Trypanosoma / Q000235:genetics / Q000472:pathogenicity
chemical_list: D015815:Cell Adhesion Molecules; D004220:Disulfides; D017261:Glycosylphosphatidylinositols; C444433:Gpi16 protein, S cerevisiae; D008562:Membrane Glycoproteins; C491190:PIGK protein, human; D029701:Saccharomyces cerevisiae Proteins; D000217:Acyltransferases; D019881:Aminoacyltransferases; C081007:COOH-terminal signal transamidase; C103133:GPI8 protein, S cerevisiae