Glycosylation in the Yeast Golgi

DESCRIPTION (provided by applicant): Carbohydrate modifications in the Golgi function in a variety of biological roles ranging from the stabilization of protein structure to the regulation of cell surface properties. The overall goal of this research is to understand how glycosylation is regulated and how carbohydrate modifications mediate their biological roles at the cell surface. Glycosyl transferases catalyze a set of ubiquitous reactions that contribute to and are affected by the ionic equilibrium of the Golgi lumen. As a byproduct of glycosylation, vast quantities of inorganic phosphate are generated. This phosphate must be removed to prevent an overly acidic lumen that would otherwise inhibit the activity of the resident enzymes. Our first aim is to determine how this phosphate is removed from the Golgi, using S. cerevisiae as an experimental model system. Our past studies identified ERD1 as an important, highly conserved regulator of phosphate efflux from the Golgi. To better understand how ERD1 regulates the lumenal environment, experiments are proposed to generate new mutants and analyze mutants already identified that interact with ERD1 or that are similarly defective in regulating the lumenal environment of the Golgi. In a complementary approach we will apply biochemical assays to identify and characterize the Gotgi phosphate transporter. Our second aim is to determine how the byproducts of sugar consumption contribute to other metabolic pathways in yeast, specifically phosphate homeostasis. To achieve this aim, we will apply sensitive biochemical assays to directly measure phosphate production from glycosylation. The proposed experiments will reveal important information about the establishment and maintenance of an optimal Golgi environment and how the reactions that occur in the Golgi intersect with other pathways. Our third aim is to examine the role of the Golgi and the glycans themselves in contributing to the cell surface properties of Candida albicans, the most frequently isolated human fungat pathogen. Our past studies demonstrated that C. albicans has evolved an alternate strategy for establishing polarity during hyphal growth, in which the entire Golgi complex redistributes sub-apicality during filamentation. Specifically, we will use cell biological and molecular techniques to study the regulation and cytoskeletal requirements of Golgi movement to the hyphal tip.