Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N‐acetylglucosamine (GlcNAc) from UDP‐GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG‐A and PIG‐C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG‐H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG‐L, which is involved in the second step in GPI synthesis, GlcNAc‐PI de‐N‐acetylation, did not associate with the isolated complex. The protein complex had GPI–GlcNAc transferase (GPI–GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized ∼100‐fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI–GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI–GnT may be relevant to selective usage of PI and/or regulation.

Glycosylphosphatidylinositol (GPI) represents an important anchoring molecule for cell surface proteins. The first step in its synthesis is the transfer of N-acetylglucosamine (GlcNAc) from UDP to phosphatidylinositol (PI). The products of three mammalian genes, PIG-A, PIG-C and PIG-H, have previously been shown to be involved in the putative enzymic complex. Here we report the cloning of human and mouse cDNAs encoding a fourth participant in the GlcNAc transfer reaction which are homologues of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Gpi1 proteins. To provide evidence for their function, these proteins were expressed in GPI1-disrupted yeast strains. In Sacch. cerevisiae, where GPI1 disruption results in a temperature-sensitive phenotype and abolishes in vitro GlcNAc-PI synthesis, restoration of growth could be demonstrated in a temperature-dependent manner. In addition, in vitro GlcNAc-PI synthetic activity was again detectable. In Schiz. pombe, gpi1+ disruption is lethal. Using random spore analysis, we were able to show that the mammalian GPI1 homologues can rescue haploids harbouring the lethal gpi1+::his7+ allele. Our data demonstrate that the genes identified are indeed involved in the first step of GPI biosynthesis, and allow conclusions about a specific function for Gpi1p in stabilizing the enzymic complex. The finding that, despite a low degree of identity, the mammalian Gpi1 proteins are able to participate in the yeast GlcNAc-PI synthetic machinery as heterologous components further demonstrates that GPI biosynthesis has been highly conserved throughout evolution.