PIG-W Is Critical for Inositol Acylation but Not for Flipping of Glycosylphosphatidylinositol-Anchor

Abstract

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.

Inositol Deacylation of Glycosylphosphatidylinositol-anchored Proteins Is Mediated by Mammalian PGAP1 and Yeast Bst1p

“…cerevisiae, an acyl chain is added to the 2-position of inositol early in the GPI biosynthetic pathway (24). The acylation is mediated by PIG-W in mammalian cells (5) and by Bwt1p, the PIG-W orthologue, in S. cerevisiae (25). The acceptor substrate for these acyltransferases is glucosaminyl-phosphatidylinositol (26,27).…”

“…In contrast, precursors of the GPI-anchor present in the endoplasmic reticulum (ER) are resistant to PI-PLC due to an acyl chain linked to the 2-position of inositol (4). The inositol ring of GPI is acylated at an early step in GPI biosynthesis by the action of PIG-W protein, an acyltransferase that adds a palmitoyl chain to the inositol of glucosaminyl-phosphatidylinositol, the second intermediate in the pathway (5). The inositol acylation is critical for the attachment of GPI to proteins, because mutant cells defective in PIG-W express only very low levels of GPI-APs (5).…”

“…The inositol ring of GPI is acylated at an early step in GPI biosynthesis by the action of PIG-W protein, an acyltransferase that adds a palmitoyl chain to the inositol of glucosaminyl-phosphatidylinositol, the second intermediate in the pathway (5). The inositol acylation is critical for the attachment of GPI to proteins, because mutant cells defective in PIG-W express only very low levels of GPI-APs (5). It is very likely that the acyl group is required for a later step in the pathway when "bridging" ethanolamine phosphate, which links GPI to the protein, is added to the third mannose to generate mature GPI.…”

Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis

“…Wild-type 3B2A cells (22) were cultured in Ham's F12 medium (Sigma). All mutant cell lines [M2S2 (accumulating N-acetyl glucosamine-PI; GlcNAc-PI) (22), CHOPA10.14 (accumulating GlcN-PI) (23), and Lec15 (accumulating GlcN-acyl-PI) (24)] were cultured in Ham's F12 medium supplemented with 10% fetal calf serum and 600 mg/ml G418.…”

Potential Roles of GPI-Anchor Remodeling in Protein Trafficking and Raft Association in Mammalian Cells

“…This reaction is performed by PIG-W (its yeast homolog is named Gwt1p, and the names of the functional homologous proteins in budding yeast are henceforth shown like (yeast Gwt1p)) (30,31). Membrane topological analysis of yeast Gwt1p has shown that acylation of inositol is performed on the luminal side of the ER membrane (32).…”