The GPI Transamidase Complex ofSaccharomyces cerevisiaeContains Gaa1p, Gpi8p, and Gpi16p

Abstract

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430 -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, and it has a single C-terminal transmembrane domain and a small C-terminal, 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 -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 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.

Human PIG-U and Yeast Cdc91p Are the Fifth Subunit of GPI Transamidase That Attaches GPI-Anchors to Proteins

“…The present study and previous reports (Hamburger et al, 1995;Benghezal et al, 1996;Yu et al, 1997;Hiroi et al, 1998;Ohishi et al, 2000Ohishi et al, , 2001Fraering et al, 2001) indicate that human and S. cerevisiae GPI transamidases are very similar, both consisting of five proteins. Human GAA1, GPI8, PIG-S, PIG-T, and PIG-U are orthologous with S. cerevisiae Gaa1p,Gpi8p,Gpi17p,Gpi16p,and Cdc91p,respectively,having 25,44,23,30, and 28% amino acid identity, respectively.…”

“…The GPI transamidase complex extracted from HeLa cells with digitonin sedimented at ϳ17S corresponding to ϳ460 kDa (Vainauskas et al, 2002). The size of the digitonin-extracted GPI transamidase of S. cerevisiae was assessed to be 430 -650 kDa by blue native gel electrophoresis (Fraering et al, 2001). As discussed previously (Vainauskas et al, 2002), the large size of the digitonin-extracted GPI transamidase may be attributed to a number of possible reasons: a nonglobular shape of the complex, bound detergent, the presence of multiple copies of one or more subunits and/or the presence of unidentified subunits that may have been lost during the affinity-purification.…”

“…PIG-S and Gpi17p, and PIG-T and Gpi16p are orthologous to each other, respectively (Fraering et al, 2001;Ohishi et al, 2001). All the proteins are essential for GPI transamidase as shown by their mutant cells (Yu et al, 1997;Ohishi et al, 2000Ohishi et al, , 2001.…”

“…The GPI transamidases of humans and Saccharomyces cerevisiae are complexes of at least four proteins, GAA1, GPI8, PIG-S, and PIG-T in humans and Gaa1p, Gpi8p, Gpi17p, and Gpi16p in yeast (Fraering et al, 2001;Ohishi et al, 2001). PIG-S and Gpi17p, and PIG-T and Gpi16p are orthologous to each other, respectively (Fraering et al, 2001;Ohishi et al, 2001).…”

“…The recombinant GPI8 protein of protozoa, Trypanosoma brucei cleaved a synthetic peptide acetyl-S-V-L-N-aminomethyl-coumarine . PIG-T/Gpi16p are critical for the formation of the enzyme complex because the stable expressions of GPI8/Gpi8p are dependent upon PIG-T/Gpi16p (Fraering et al, 2001;Ohishi et al, 2001) and the expression of GAA1 is dependent on PIG-T (Ohishi et al, 2001). The roles of GAA1 and PIG-S/ Gpi17p have yet to be clarified.…”