Gaa1p and Gpi8p Are Components of a Glycosylphosphatidylinositol (GPI) Transamidase That Mediates Attachment of GPI to Proteins

Abstract

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (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 reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.

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

“…To us, it seemed important to show that the complex is present under physiological conditions. A previous study demonstrated by coimmunoprecipitation that hGpi8p and hGaa1p interact stably when they are strongly overexpressed (Ohishi et al, 2000). In the situation of overexpression, the ER folding machinery may get overwhelmed, and incompletely folded proteins may accumulate and aggregate in a nonspecific way through hydrophobic interactions.…”

“…Although a stable interaction of Gpi8p and Gaa1p has been demonstrated by coimmunoprecipitation (Ohishi et al, 2000), it appeared possible that proteins in the GPI transamidase would not remain firmly associated throughout the catalytic cycle but that the normal workings of this complex required that some subunits dissociate at a certain stage. In fact, if overexpression is used to demonstrate the interaction, a large proportion of overexpressed proteins may never be engaged in GPI anchoring, and this could lead to the artificial perpetuation of a normally transient interaction.…”

“…Gpi8p has 25-28% homology to a family of cysteine proteinases, one of which is able to act as a transamidase (Benghezal et al, 1996). The Cys and His residues predicted to be active sites by sequence comparison with caspases indeed are essential, and their mutation to Ala yields nonfunctional GPI8 alleles (Chen et al, 1998;Meyer et al, 2000;Ohishi et al, 2000). Recent evidence shows that Gpi8p may be part of a larger protein complex.…”

“…Recent evidence shows that Gpi8p may be part of a larger protein complex. First, the human Gpi8p is efficiently coimmunoprecipitated with human Gaa1p when tagged forms are coexpressed in CHO cells, and even truncated versions of Gpi8p lacking the C-terminal TMD still can be coprecipitated with Gaa1p (Ohishi et al, 2000). Second, the overexpression in wild-type (wt) cells of any GPI8 allele that is mutated in one of the active site residues leads to cell growth arrest and causes the accumulation of unprocessed GPI lipids and protein precursors (Meyer et al, 2000).…”

Profiling the expression pattern of GPI transamidase complex subunits in human cancer

“…1 Gaa1p and Gpi8p were the first to be identified as proteins essential for GPI anchor attachment onto proteins, 2,3 and were subsequently shown to form a complex. 4 Other subunits were identified as they co-immunoprecipitated with the GPI8/Gpi8p-GAA1/Gaa1p complex. 5,6 Trypanosomatids such as Trypanosoma brucei, share three subunits with mammalian/yeast GPIT (homologues of GPI8, GAA1 and PIG-T termed TbGPI8, TbGAA1 and bGPI16/PIGT, respectively) but have two novel subunits (TTA1 and TTA2) in lieu of PIG-S and PIG-U.…”