Biosynthesis pathway of GPI anchored proteins.

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Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a prolacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GOI. It was known that the GPI transamidase is a complex containing GAAI and GPI8 To further characterize GPI transamidase, we affinity-purified the enzyme complex and found two new components PIG-S and PIG-T. To determinc roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins. We established Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the responsible gene, termed PIG-U, that encodes a 435-amino-acid hydrophobic protein. GPI transamidase complex affnity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U as well as four other known components. Cells lacking PIG-U normally formed complexes of four other components but had no ability to cleave the GPI attachment signal peptide. Therefore, these results demonstrated that GPI transamidase consists of five proteins. We also demonstrated that two subunits, GPI8 and GPI-T, from a functionally important disulfide bond through conserved cysteine residues.