Mammalian PIG-L and its yeast homologue Gpi12p are N-acetylglucosaminylphosphatidylinositol de-N-acetylases essential in glycosylphosphatidylinositolbiosynthesis

Glycosylphosphatidylinositol (GPI) is used as a membrane anchor by many euk aryotic cell-surface proteins. The second step of GPI biosynthesis is de-N- acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI). We hav e previously cloned the rat PIG-L gene by expression cloning that complemen ted a mutant Chinese hamster ovary cell line defective in this step. Here w e show that recombinant rat PIG-L protein purified from Escherichia coli as a complex with GroEL has GlcNAc-PI de-N-acetylase activity in vitro. The a ctivity was not enhanced by GTP, which is known to enhance the de-N-acetyla se activity of mammalian cell microsomes. As with other de-N-acetylases tha t act on the GlcNAc moiety, metal ions, in particular Mn2+ and Ni2+, enhanc ed the enzyme activity of PIG-L. The Saccharomyces cerevisiae YMR281W open reading frame encodes a protein (termed Gpi12p) with 24%, amino acid identi ty with rat PIG-L. On transfection into mammalian PIG-L-deficient cells, th is gene, GPI12, restored the cell-surface expression of GPI-anchored protei ns and GlcNAc-PI de-N-acetylase activity. The disruption of the gene caused lethality in S. cerevisiae. These results indicate that GlcNAc-PI de-N-ace tylase is conserved between mammals and yeasts and that the de-N-acetylatio n step is also indispensable in yeasts.