The mechanisms of glucose-induced catabolite degradation in the yeast Saccharomyces cerevisiae

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The sugar glucose is essential for energy metabolism and the generation of building blocks in cells. Besides this, glucose is a central signalling molecule. It has a strong impact on the entire cell metabolism up to the cell cycle. The consequences of a disturbed glucose metabolism are impressively demonstrated by the widespread human disease diabetes. Besides the regulation of transcription and translation glucose induces the activation and inactivation of enzymes and especially their degradation. In the project applied, the regulation of the two central antagonistic pathways, glycolysis and gluconeogenesis, are analyzed in the eukaryotic model organism yeast. Central to this project is the elucidation of the glucose induced degradation of three key enzymes of gluconeogenesis, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and cytoplasmic malate dehydrogenase, by two different degradation pathways: the ubiquitin proteasome system and the vacuole (lysosome). Glucose addition to cells growing for up to 24 hours on a nonfermentable carbon source induces a signalling pathway which leads to degradation of the enzymes via the ubiquitin proteasome pathway. Specific for this degradation pathway is a novel RING E3 ligase complex, the Gid complex, which catalyzes ubiquitination of the enzymes and thus triggers their degradation by the proteasome. The glucose signal induces the rapid synthesis of one of the subunits of the Gid ligase complex, Gid4, which binds to the core complex and thereby stimulates ubiquitination of the gluconeogenic enzymes and their subsequent proteasomal degradation. The aim of this grant proposal is to elucidate the entire signal cascade, which leads to degradation of the enzymes as well as to examine the regulation of Gid4 by proteolysis. In addition, the mechanistic events occurring at the Gid ubiquitin ligase complex during polyubiquitination of the enzymes will be investigated. Glucose addition to cells which have been grown for about 48 to 72 hours on a non-fermentable carbon source until they reach stationary phase induces degradation of the gluconeogenic enzymes in the vacuole (lysosome). Obviously, during this growth phase a switch occurs which channels degradation of the enzymes from proteasomal proteolysis to vacuolar (lysosomal) proteolysis. Preliminary results indicate that the Gid ubiquitin ligase complex is also required for the vacuolar (lysosomal) proteolysis of the enzymes and that this process is related to selective autophagy. Our aim is to understand in which manner the trigger molecule glucose can induce these two different degradation pathways.The studies are not only meant to better understand the regulation of gluconeogenesis but are additionally supposed to shed light on the glucose induced proteolysis of gluconeogenic enzymes by two completely different proteolytic systems, the proteasome and the vacuole.