This application is for support for a genetic and biochemical analysis of glycosyl phosphatidylinositol (GPI) membrane anchoring and O-mannosylation of protein in yeast. These are modifications of cell surface and secretory proteins that are carried out in the endoplasmic reticulum. Of these, GPI anchoring appears to be conserved among eukaryotic cells, whereas O-mannosylation has so far been demonstrated in fungi and in brain proteoglycan; neither process, however, is well understood at the biochemical and molecular level. The goals of the proposed work are to establish the importance of these modifications to the cell, to chart these biochemical pathways, and to analyze the structure and function of the enzymes in these pathways. Saccharomyces cerevisiae will be used as a model eukaryotic organism with which to study the biochemistry of these processes, and to isolate and characterize mutants blocked at steps in these pathways. Two approaches will be used to isolate genes for enzymes specific to the GPI anchoring pathway. In the first, colonies of mutagenized yeast cells will be screened for those blocked in the incorporation of [2-3H]- myoinositol into protein. The GPI anchoring mutants isolated will be used to order steps in the GPI anchoring pathway. Their enzymic defects will be characterized, and the structures of the accumulated GPI anchor precursor lipids determined. The wild type counterparts of the defective genes will be cloned by complementation. The second strategy will exploit a colony screen for the in vitro activity that synthesizes phosphatidylinositol-N-acetylglucosamine, the first enzyme in the GPI anchor assembly pathway. Yeast colonies transformed with a genomic DNA library will be screened for those that overproduce this enzyme activity. It will be shown whether plasmids conferring enzyme overproduction contain the structural gene for this enzyme. The gene will be disrupted to show whether GPI anchoring is essential for viability in yeast, and the phenotypes of null- or conditional alleles will be explored. A screen for overproduction of the O-mannosylating enzyme, dolichol phosphate mannose Ser/ Thr: Omannosyltransferase, will be used to clone this yeast enzyme. This O-mannosyltransferase will provide the first opportunity to determine the structure and membrane topography of a protein that transfers sugar to protein. The phenotypes of yeast O-mannosylation mutants will be explored in order to establish the biological significance this modification. It will also be determined whether a mammalian counterpart of Omannosyltransferase is present in membranes from brain tissue. These studies will allow evaluation of O- mannosylation as a potential target for the selective action of an antifungal agent.
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