title: Two subunits of glycosylphosphatidylinositol transamidase, GPI8 and PIG-T, form a functionally important intermolecular disulfide bridge.
abstract: Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.
authors.affiliation: Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Ohishi
authors.forename: Kisaburo
authors.initials: K
authors.lastname: Nagamune
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Maeda
authors.forename: Taroh
authors.initials: T
authors.lastname: Kinoshita
mesh_terms: D000217:Acyltransferases / Q000737:chemistry*; D000818:Animals; D018957:CD59 Antigens / Q000096:biosynthesis; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005434:Flow Cytometry; D006801:Humans; D020014:K562 Cells; D009154:Mutation; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000378:metabolism
chemical_list: D018957:CD59 Antigens; D015815:Cell Adhesion Molecules; D004220:Disulfides; C491190:PIGK protein, human; D000217:Acyltransferases; C081007:COOH-terminal signal transamidase; D003545:Cysteine