Molecular cell biology of GlcNAc-Pl de-N-acetylase (PIG-L), the second enzyme of the GPI biosynthetic pathway

The second step of glycosylphosphatidylinositol (GPI) biosynthesis, de- N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) to glucosaminylphosphatidylinositol (GlcN-PI), is catalyzed by GlcNAc-PI de-N-acetylase (PIG-L). This thesis presents a detailed analysis of human PIG-L with respect to its membrane topology, subcellular localization and molecular interactions with other components of the pathway.; Chapter 2 describes experiments that establish the membrane topology, subcellular localization and ER targeting signals in PIG-L. We used a combination of immunofluorescence analyses and glycosidase treatments to confirm that PIG-L is a single transmembrane domain containing protein with a long cytoplasmic tail. Subcellular fractionation studies provided evidence that PIG-L is uniformly distributed in the ER membrane. Analyses of a series of PIG-L truncations fused to Tac antigen, a cell surface protein, identified two independent ER localization signals in PIG-L, one in the cytoplasmic domain and another weak signal in the transmembrane and luminal domains.; In chapter 3 we further explored the molecular properties of PIG-L that might cause its ER retention. We provide evidence indicating that PIG-L is mobile in the ER membrane with a diffusion coefficient of 1.5 x 10 -9 cm2s-1, comparable to that of other laterally mobile ER membrane proteins. Co-immunoprecipitation studies ruled out homotypic interactions between PIG-L molecules but indicated that ER-retained PIG-L forms a 1:1 stoichiometric complex with PIG-H, a subunit of GPI-N-acetylglucosaminyltransferase (GPI-GnT), the multi-subunit enzyme complex catalyzing the first step of GPI biosynthesis. The PIG-L:PIG-H complex did not interact with PIG-A, the catalytic subunit of GPI-GnT. Our results suggest the existence of two independent pools of PIG-H, one complexed with PIG-L and another within the GPI-GnT complex.; The results presented in this thesis represent the first step towards characterizing the molecular cell biology of PIG-L protein.