| A glycosylphosphatidylinositol (GPI) anchor is a lipid modification of protein that serves to attach a protein to the outer surface of the plasma membrane or to the lumenal face of a secretory vesicle. My research focused on developing S. cerevisiae as a model system to study GPI anchor biosynthesis. I wished to elucidate the steps involved in early GPI anchor assembly and to characterize the intermediates made in these steps.; I found that S.cerevisiae did have GPI biosynthetic activities and developed in vitro assays to detect the synthesis of the first three GPI biosynthetic intermediates. By chemical and enzymatic methods, I characterized these intermediates as GIcNAc-PI, GIcNH2-Pl, and GIcNH2(acyl-inositol)-PI. The origin of the acyl group esterified to the inositol, which renders the GPI molecule resistant to cleavage by phosphatidylinositol specific-phospholipase C (PI-PLC), was then explored in depth. I found that inositol acylation in yeast was acyl-CoA-dependent, a novel reaction in GPI anchor biosynthesis. Furthermore, I have developed a yeast crude lysate assay which detects the synthesis of putative mannosylated GPI anchor intermediates.; Next, it was desired to obtain structural information on in vivo GPI anchor intermediates. To address this issue, I exploited the fact that at non-permissive temperature, the yeast dpm1--6 mutant accumulates GlcNH2-(acyl-inositol)-Pl. This precursor molecule is the earliest biosynthetic intermediate that can be detected in in vivo radiolabeling experiments. I isolated this lipid and had it analyzed by liquid chromatography - mass spectroscopy (LC-MS) with an electrospray interface. The results show that this intermediate has very long chain fatty acids, most likely on its diacylglycerol moiety. This has significant implications for GPI anchor biosynthesis in yeast and suggests that GPI anchors are either assembled on a specialized PI or that the diacylglycerol moiety on early GPI precursor molecules are rapidly remodeled. |
