The goal of this research is to understand better the molecular mechanisms of cellular signal transduction in the yeast Saccharomyces cerevisiae by analysis of the nutritional controls that govern glycogen storage. Yeast provides an excellent experimental model system with which to combine biochemical, genetic and cellular approaches, and is now amenable to genome-wide analysis.
Aim (1) Glucose control of glycogen synthesis. Glucose controls a variety of cellular functions, including glycogen synthesis, by several signaling pathways. A means of controlling glycogen synthase may be through regulation of glycogen synthase kinase (Gsy2p) activity.
The aim explores the mechanisms of this control. It incorporates genome wide analyses based on the availability in the near future of a complete gene deletion set of about5000 yeast strains systematically disrupted for individual genes. These genome-wide screens will be aimed at identifying genes involved in the control of glycogen synthase kinase and glycogen accumulation. (ii) Substrate specificity of Pho85p cyclin dependent protein kinases. Pho85p is a cyclin-dependent protein kinase that interacts with some 10 different cyclins or Pcls. These are thought to confer specificity in vivo. For example, Pc11Op targets Pho85p to phosphorylate Gsy2p. We plan to examine the substrate specificity of Pho85p, and the molecular basis by which different Pcls specify substrates and hence function.
The aim i ncludes a proteomic approach to identifying physiological substrates as well as study of some novel Pho85p/ Pcl kinases. (iii) Autophagy and glycogen storage. Autophagy, the engulfment of cytosolic materials for transport to the vacuole for recycling, is an important component of the response of yeast and mammalian cells to nutritional status. Two autophagy genes, APG1 and APG13, one of which encodes a protein kinase, have been implicated in controlling glycogen metabolism.
The aim seeks to explore this connection through characterization of these and other potentially related genes, as well as by examining vacuolar glycogen deposits. (iv) Relative roles of covalent and non-covalent controls of glycogen synthase. Glycogen synthase (Gsy2p), an important regulated enzyme in the glycogen biosynthetic pathway, is inactivated by phosphorylation and activated by glucose-6-P. Site-directed mutagenesis will be used to examine the relative roles of the two mechanisms in vivo. Control of glycogen metabolism (aims 1 and iv) is of interest in its own right, and impaired glucose metabolism is associated with metabolic diseases including diabetes. The study of protein kinase specificity (aim ii) is of more general importance to understanding signal transduction. Yeast has proven to be a useful experimental system to study vesicular trafficking and such may be true for the study of autophagy and its relationship to glycogen metabolism (aim iii).
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