Gaa1p and Gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins

Many eukaryotic cell surface proteins are anchored to the membrane via glyc osylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticu lum by a transamidation reaction through the formation of a carbonyl interm ediate. CPI transamidase is a key enzyme of this posttranslational modifica tion. Here we report that Gaa1p and Cpi8p are components of a GPI transamid ase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F 9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and tran samidase as determined by an in vitro GPI-anchoring assay. We also show tha t cysteine and histidine residues of Cpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl i ntermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are as sociated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI trans amidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.