PIG-A and PIG-H, Which Participate in Glycosylphosphatidylinositol Anchor Biosynthesis, Form a Protein Complex in the Endoplasmic Reticulum

Abstract

Many eukaryotic cell surface proteins are bound to the membrane via a glycosylphosphatidylinositol (GPI) anchor. Assembly of the GPI anchor precursor is a sequential addition of components to phosphatidylinositol (PI) in the endoplasmic reticulum (ER). The first step is the transfer of N-acetylglucosamine (GlcNAc) to PI from UDP-GlcNAc to generate GlcNAc-PI. This simple step, however, is regulated by at least three genes because in both mammals and yeasts, there are three mutants of different complementation classes. To clarify this complexity, we analyzed the products of two cloned human genes, PIG-A and PIG-H. Here we demonstrate 1) that PIG-A is an ER transmembrane protein with a large cytoplasmic domain that has homology to a bacterial GlcNAc transferase and a small lumenal domain; 2) that PIG-H is a cytoplasmically oriented, ER-associated protein; and 3) that they form a protein complex. We also show that part of the small lumenal domain of PIG-A plays an essential functional role in targeting itself to the rough ER. Taken together with the cytoplasmic orientation of GlcNAc-PI, these results indicated that PIG-A and PIG-H are subunits of the GPI GlcNAc transferase that transfers GlcNAc to PI on the cytoplasmic side of the ER.

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

“…This is consistent with the membrane orientation of GlcN-PI [36]. Taken together with previous reports that both the first-step enzyme [18] and GlcNAc-PI [36] are cytoplasmically oriented, it is now clear that the first two reactions proceed on the cytoplasmic side of the ER.…”

Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

“…The acyl chains of the PI species that receive are the same length as those in other membrane phospholipids (Sipos et al 1997). Evidence that GlcNAc transfer occurs at the cytoplasmic face of the ER membrane is that i) the catalytic domain of Gpi3's human orthologue faces the cytoplasm (Watanabe et al 1996;Tiede et al 2000), and ii) GlcNAc--PI can be labeled with membrane topological probes on the cytoplasmic side of the mammalian ER membrane (Vidugiriene and Menon, 1993).…”

“…A complex of at least six proteins (GPI-GnT) is involved, of which Gpi3 is catalytic because it can be labeled with a photoactivatable UDP-GlcNAc analog (Kostova et al 2000). GlcNAc transfer occurs at the cytoplasmic face of the ER membrane (Vidugiriene and Menon 1993;Watanabe et al 1996;Tiede et al 2000). Essential Gpi2, Gpi15, and Gpi19 (Leidich et al 1995;Yan et al 2001;Newman et al 2005), and nonessential Gpi1 and Eri1 (Leidich and Orlean 1996;Sobering et al 2004), are also required for GlcNAc-PI synthesis.…”

Murine embryonic stem cells without pig-a gene activity are competent for hematopoiesis with the PNH phenotype but not for clonal expansion.

“…Specifically, this protein is required for the transfer of N -acetyl-glucosamine to phosphatidylinositol to form N -acetyl glucosaminyl phosphatidylinositol (6). Stretches of sequence homology to a bacterial N -acetyl glucosaminyl transferase suggest that PIG-A is part of the ␣ -1,6, N -acetyl glucosaminyl phosphatidylinositol transferase enzyme complex (7,8). In all patients analyzed, the mutation leads to a partial or total deficiency of the PIG-A protein with consequent impaired synthesis of GPI anchors (5,9,10).…”