Glycosylation is an essential modification that functions in a variety of biological roles ranging from the stabilization of protein structure to the regulation of cell surface properties. Despite its importance, glycosylation remains one of the most poorly understood of all the metabolic processes. The overall goal of this research is to understand how glycosylation is regulated and how carbohydrate modifications mediate their biological roles. The proposed experiments will focus on two key families of proteins that are required for terminal carbohydrate additions in the Golgi. These proteins are the nucleotide sugar transporters (NSTs), which provide the lumenal sugar substrates, and the glycosyltransferases, which catalyze sugar additions on proteins and lipids. Using the yeast S. cerevisiae as a model system, a combined genetic and biochemical approach will be used to study these enzymes. The first specific aim will be to study the mechanism by which NSTs transport nucleotide sugars into the Golgi. Using the VRG4-encoded GDP-mannose transporter as a model, novel vrg4 mutants will be isolated and assayed for defects in the binding or transport of GDP-mannose and in dimer formation. Each mutant allele will be characterized to determine the molecular basis for the defective phenotype. The in vivo function of other VRG4-homologs will also be investigated. The proposed experiments will provide important new information relating the structure of NSTs to their biological function and will contribute to our understanding of these proteins in other species. An understanding of the Vrg4 protein also has important implications for human disease. VRG4 is indispenable for the synthesis of cell wall mannoproteins which are key determinants in fungal pathogenicity. Although VRG4 is essential for yeast viability, it does not have a mammalian counterpart. These features make VRG4 an attractive target for anti-fungal therapies. As a first step in our long term goal of developing VRG4 as a drug target, the second specific aim is to investigate the biological role of the VRG4 gene in Candida albicans, the most frequently isolated fungal pathogen in humans. Another major aspect of this application addresses basic questions about the mannosyltransferases that function in the cis-Golgi. A subset of these proteins exists together in the membrane as a multiprotein complex. The third specific aim is to determine the requirements for complex assembly and to examine whether complex assembly and localization in the Golgi are related processes. These studies could reveal important principles for glycan synthesis in other systems.
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