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.
Highlights
Many eukaryotic cell surface proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI).1 The GPI moiety is synthesized in the endoplasmic reticulum (ER) by the sequential addition of sugars and ethanolamine phosphate to phosphatidylinositol [1, 2]
We demonstrate that an inactive human GPI transamidase complex that consists of nonfunctional 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
We demonstrate that GPI8 and PIG-T form a disulfide bridge that is required for normal transamidase activity via conserved cysteine residues in mammalian cells and in T. brucei and provide direct evidence that a transamidase complex consisting of five components associates with translocated substrate proteins
Summary
Tated with GAA1 [18]. We have proposed that PIG-T/Gpi16p has a central role in the formation of the transamidase complex because stable expression of GPI8/Gpi8p exclusively depends on PIG-T/Gpi16p [12, 14]. We demonstrate that GPI8 and PIG-T form a disulfide bridge that is required for normal transamidase activity via conserved cysteine residues in mammalian cells and in T. brucei and provide direct evidence that a transamidase complex consisting of five components associates with translocated substrate proteins
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