(Verbatim from the applicant's description) In yeast and other fungi, cell shape, growth, and cell division depend on the coordinated synthesis and degradation of cell wall components. A quantitatively minor but functionally critical component of the yeast cell wall is the polymer beta-1,6 glucan. This polysaccharide is covalently cross-linked to other cell wall components, such as chitin, 3'-1,3 glucan and mannoproteins. These cross-links stabilize and organize the cell wall. The long-term goal of this project is to investigate the mechanisms by which beta-1,6 glucan is synthesized and associated with other cell wall components. We hypothesize that key aspects of beta-1,6 synthesis differ from those of the more abundant 3'-1,3 glucan polymer. Specifically, the proposed work will examine the hypothesis that beta-1,6 glucan synthesis is initiated in the lumen of the endoplasmic reticulum. Chain elongation and branching occurs in the Golgi apparatus, where the assembled polymers are packaged into secretory vesicles for delivery to the periplasmic space and assembly into the cell wall. In previous work we demonstrated the transport of UDP-glucose, the sugar donor for polymer assembly, into the lumen of the endoplasmic reticulum in Saccharomyces cerevisiae. In order to test our hypothesis we will identify the UDP-glucose transporter gene in yeast and generate a null mutant. Our model predicts that '3-1,6 glucan synthesis, but not 3'-1,3 glucan synthesis, will be prevented in the null mutant. A second, independent, strategy is to determine directly the subcellular sites in which beta-1,6 glucan chains are initiated and extended. This will be accomplished by in vivo radiolabeling experiments in combination with cell fractionation. This work will be facilitated by the use of mutants defective in beta-1,6 glucan synthesis (kre) and secretion (sec). The final approach consists of identifying and characterizing the enzymes that are responsible for synthesis of '3-1,6 glucan. The information derived from these studies will provide important new knowledge on the molecular mechanism of fungal cell wall assembly.
