The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After s olubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p i s found in 430-650-kDa protein complexes. These complexes can be affinity p urified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi 16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk res ides on the lumenal side of the ER, and it has a single C-terminal transmem brane domain and a small C-terminal, cytosolic extension with an ER retriev al motif. Depletion of Gpi16p results in the accumulation of the complete G PI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p ar e unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The trans amidase complex is not associated with the Sec61p complex and oligosacchary ltransferase complex required for ER insertion and N-glycosylation of GPI p roteins, respectively. When GPI precursor proteins or GPI lipids are deplet ed, the transamidase complex remains intact.