Characterisation of the enzymatic complex for the first step in glycosylphosphatidylinositol biosynthesis

The mammalian N-acetylglucosaminyl transferase for the first step in glycos ylphosphalidylinositol biosynthesis has been shown to consist of at least f our components: PIG-A, PIG-C, PIG-H and GPII. Here, the enzymatic complex i s further characterised. PIG-A protein. which is thought to represent the c atalytic subunit of the complex, was expressed in an epitope-tagged form in the PIG-A deficient JY5 lymphoblastoid cell line. Subcellular localisation of this protein was studied using immunofluorescence and immunoelectron mi croscopy. The protein was localised to both perinuclear and mitochondria-as sociated lamellae of the endoplasmic reticulum. Using affinity chromatograp hy, epitope-tagged PIG-A protein was partially purified. To identify region s that might be involved in the catalytic process, computer-aided compariso n was performed between PIG-A and 26 distantly related glycosyl transferase s. A number of residues in the membrane-proximal region of the cytoplasmic domain (230-340) were found highly conserved. Finally, a topological model of the four partners participating in the enzymatic complex is introduced t o provide a working model for further structural and functional analysis. ( C) 2000 Elsevier Science Ltd. All rights reserved.