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

Reika Watanabe,Taroh Kinoshita,Ryuichi Masaki,Akitsugu Yamamoto,Junji Takeda,Norimitsu Inoue

doi:10.1074/jbc.271.43.26868

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.

Highlights

Many eukaryotic proteins are bound to the cell surface membrane by a glycosylphosphatidylinositol (GPI)1 anchor [1, 2]
A region spanning amino acids 304 –395 has homology to the bacterial GlcNAc transferase, RfaK, which is involved in the synthesis of lipopolysaccharides, suggesting that PIG-A protein has a catalytic site for the transfer of GlcNAc [14]
In this study we showed that the products of two genes, PIG-A and PIG-H, which participate in the first step of GPI anchor biosynthesis, are endoplasmic reticulum (ER) membrane proteins that together form a protein complex

Summary

Introduction

Many eukaryotic proteins are bound to the cell surface membrane by a glycosylphosphatidylinositol (GPI) anchor [1, 2]. The anchor is linked to the carboxyl terminus via an amide bond with ethanolamine This linkage is formed by the transfer of a synthesized GPI anchor precursor to protein in the endoplasmic reticulum (ER) following the cleavage of a carboxylterminal GPI signal sequence. Many mutant mammalian cell lines [5, 6] and yeasts [7,8,9] that are deficient in biosynthesis or attachment of the GPI anchor have been established They have been characterized biochemically and grouped into complementation classes. This orientation is in agreement with the notion that the first step of GPI anchor synthesis occurs on the cytoplasmic side of the ER membrane

Methods

Results

Conclusion