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

葡萄糖对细胞的能量代谢和积木的产生是必不可少的。除此之外，葡萄糖是一种中心信号分子。它对整个细胞新陈代谢乃至细胞周期都有很大的影响。糖代谢紊乱的后果在广泛传播的人类疾病糖尿病中得到了令人印象深刻的证明。除了转录和翻译的调节外，葡萄糖还可以诱导酶的激活和失活，特别是它们的降解。在应用的项目中，在真核模式生物酵母中分析了糖酵解和糖异生这两条中心拮抗途径的调节。这个项目的核心是阐明葡萄糖通过两种不同的降解途径：泛素蛋白酶体系统和液泡(溶酶体)降解葡萄糖异生的三个关键酶--果糖-1，6-二磷酸酶、磷酸烯醇式丙酮酸羧激酶和细胞质苹果酸脱氢酶。葡萄糖添加到细胞中，在非发酵碳源上生长长达24小时，诱导一条信号通路，通过泛素蛋白酶体途径导致酶的降解。这种降解途径特有的是一种新的环E3连接酶复合体，GID复合体，它催化酶的泛素化，从而触发蛋白酶体对它们的降解。葡萄糖信号诱导GID连接酶复合体的一个亚基Gid4的快速合成，Gid4与核心复合体结合，从而刺激糖异生酶的泛素化及其随后的蛋白酶体降解。这项拨款提案的目的是阐明导致酶降解的整个信号级联反应，并研究蛋白质降解对Gid4的调节。此外，还将调查在酶的多泛素化过程中GID泛素连接酶复合体发生的机械性事件。细胞在不可发酵的碳源上生长约48至72小时，直到达到静止阶段，细胞中添加葡萄糖会导致液泡(溶酶体)中的糖异生酶降解。显然，在这个生长阶段，发生了一种转换，将酶的降解从蛋白酶体蛋白分解转换为空泡(溶酶体)蛋白分解。初步结果表明，GID泛素连接酶复合体也是酶的空泡(溶酶体)蛋白分解所必需的，这一过程与选择性自噬有关。我们的目的是了解触发分子葡萄糖以何种方式诱导这两种不同的降解途径。这些研究不仅是为了更好地了解糖异生的调节，而且还应该揭示葡萄糖通过两个完全不同的蛋白水解体--蛋白酶体和液泡--诱导的糖异生酶的蛋白分解。

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.