This application is for support of a genetic and biochemical analysis of three types of structure in cells that contain the sugar glucosamine. These structures are glycosyl phosphatidylinositol (GPI) membrane anchors, which have roles in cell surface growth, O-linked N-acetylglucosamine (O-GlcNAc) residues, which are found on nuclear proteins, and saccharides consisting of GlcNAc residues which can be structural polymers, or serve as signaling molecules in embryonic development. Little is known about how, where, and when in the cell these structures are made, how they are important for cell growth, or how synthesis of these structures is coordinated. These issues will be explored using yeasts as model eukaryotes. To identify genes involved in GPI synthesis, or which encode proteins dependent on GPI anchoring to fulfill a key function, genetic screens for synthetic lethality and for multicopy suppression will be used. These screens exploit the finding that GPIs are essential for the growth of yeasts. Biochemical studies, using cloned GPI genes, will be used to define the functions of- and interactions between proteins involved in GPI synthesis. GPIs are major surface components of pathogenic fungi and parasites. To evaluate the role of GPIs in the growth of a pathogenic fungus, the Candida albicans GPI1 gene will be disrupted. This gene is required for growth of bakers' yeast at high temperature, and, if required for C. albicans to grow in a human patient, the Gpi1 protein would be a potential target for an antimicrobial agent. Certain gpi mutations, which affect cell surface growth in yeast, also affect gene expression. This may be due to altered partitioning of the substrate UDPGlcNAc between GPI synthesis and O-GlcNAc attachment to transcription factors, which in turn alters the latter's gene-regulatory activity. Variation in levels of O-GlcNAc-bearing proteins will be examined in gpi mutants, as well as during cell cycle progression in yeast. Studies on the relationship between GPI synthesis and transcription in yeast may shed light on findings in humans that GPI anchoring mutations may exert effects on mechanisms involved in growth control. Schizosaccharomyces pombe, whose chitin synthase gene is essential, will be used to study the function, cellular localization, and product of the enzyme. The consequences of reduced or excessive expression of SpChs1 on growth of fission yeast should provide clues as to how chitin synthase-like proteins and their product function in vertebrate embryogenesis.
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