Functional analysis of the yeast "Saccharomyces cerevisiae" Gpi8 protein and characterization of the purified GPI-transamidase complex

Many glycoproteins of lower and higher eucaryotes are attached to the plasma membrane by means of a glycosylphosphatidylinositol (GPI). GPI anchoring of proteins is essential for the growth of Saccharomyces cerevisiae . S.cerevisiae contains about 70 open reading frames predicting GPI proteins and many of these have been found to be cell wall glycoproteins. The precursors of GPI anchored proteins have a classical signal sequence for import into the endoplasmic reticulum at their N-terminus and a GPI anchoring signal at their C-terminus which is necessary and sufficient to direct GPI addition. The C-terminal GPI anchoring signal (composed of a C-terminal hydrophobic domain separated by a short hydrophilic spacer from the cleavage/attachment ω site) is recognized and removed by a GPI transamidase, which replaces it by a preformed GPI. The GPI transferase is believed to act as a transamidase, i.e. to jointly remove the GPI anchoring signal and transfer the preformed GPI. Transamidasedeficient cells are expected to accumulate complete GPI’s as well as GPI precursor proteins retaining the GPI anchoring signal. This phenotype is exhibited by two yeast mutants, gaa1 and gpi8. Gpi8p and Gaa1p are essential components of the GPI transamidase which adds GPIs to newly synthesized proteins. GPI8 is essential and encodes a 50 kDa type I ER membrane protein with a single membrane spanning domain (MSD), which is entirely lacking in C. elegans and some protozoan Gpi8p. Gpi8p has 25-28% homology to a family of cysteine proteinase (C13 family), one of which is able to act as a transamidase. The Cys and His residues, predicted to be active sites by sequence comparison with caspases indeed are essential and their mutation to Ala yields non-functional GPI8 alleles. Yeast Gpi8p is fully functional without its C-terminal membrane spanning domain. This domain however helps to retain Gpi8p in the ER. Purification of the water soluble part of Gpi8p to homogeneity yields pure Gpi8p that has no detectable transamidase activity towards suitable peptides in vitro. GAA1 is essential and encodes a 68 kDa ER protein with a large, hydrophilic, lumenal domain, followed by several MSDs and a cytosolic ER retrieval signal on its extreme Cterminus. After solubilization in 1.5 % digitonin and separation by blue native polyacrylamide gel electrophoresis, Gpi8p is found in 430 to 650 kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, it has a single C-terminal transmembrane domain and a small Cterminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430 to 650 kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact. Recently, the identification of Gpi17p, another new component of the GPI transamidase complex was reported (Ohishi et al., 2001). Gpi17p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and two transmembrane domains are predicted near the Nand C-termini. Depletion of Gpi17p results in the accumulation of the complete GPI lipid CP2. The interaction of the hydrophilic, lumenal parts of each component of the transamidase complex, Gpi8p, Gaa1p, Gpi16p, and Gpi17p, with each other, was studied in a GAL4-based two hybrid system. From thus, a model of the macromolecular organization of the transamidase complex is proposed.