CYTOSOL/VESICLE/VACUOLAR PROTEIN DEGRADATION PATHWAY

细胞溶胶/囊泡/液泡蛋白降解途径

• 批准号: 6181488
• 负责人: HUI-LING CHIANG
• 金额: $ 23.47万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6181488
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; adenosinetriphosphatase; chromosome walking; cytoplasm; cytoskeletal proteins; density gradient ultracentrifugation; fluorescence microscopy; fructose biphosphatase; guanosine triphosphate; hydrolysis; immunofluorescence technique; molecular cloning; polymerase chain reaction; protein degradation; protein reconstitution; protein transport; vesicle /vacuole

Protein degradation is important for cell cycle control, signal transduction and cell growth. Abnormal protein degradation has been implicated in metabolic disorders, cancer development and muscular dystrophy. A novel pathway of protein degradation in the yeast vacuole has been established in our lab. They key gluconeogenic enzyme, fructose-1.6- biophosphatase (FBPase), is targeted from the cytosol to the yeast lysosome (vacuole) for degradation when Saccharomyces cerevisiae are replenished with glucose. Our long term goal is to understand the FBPase degradation pathway. We have reconstituted this glucose-regulated targeting pathway using semi-intact cells, purified FBPase, an ATP regenerating system and cytosol. FBPase is targeted to the vacuole in the reconstituted system. We have isolated 33 vid (vacuolar import and degradation) mutants defective in the glucose-induced degradation of FBPase. Mutant analysis led to the hypothesis that FBPase is targeted from the cytosol to the intermediate vesicle and then the vacuole for degradation. We have purified a novel FBPase-associated vesicle to near homogeneity. We cloned the VID24 gene involved in vesicle targeting to the vacuole. Vid24p is synthesized and localized to the vesicles. Our specific aims are: (1) Reconstitution of FBPase import into the vesicle using the vid24-1 mutant. We will examine whether the imported FTPase is indeed targeted to the intermediate vesicles. We will divide vid1-vid13 which accumulates FBPase in the cytosol into functional subgroups. (2) Cloning of the VID genes. We plan to clone the VID genes using the colony blotting procedure and study the expression and localization of the Vid proteins. As an alternative approach, we will clone the VID15 gene which is tightly linked to the URA3 gene by chromosomal walking. (3) Purification of cytosolic proteins required for FBPase import into the vesicles. We will use the vid1-vid13 mutants that contain defective cytosolic factor(s) and add fractionated wild type cytosol to identify the fractions that complement the mutant cytosol defect. If we identify such protein, we will make mutants and prepare cytosol from the mutants to test whether the cytosol is defective in FBPase import in vitro. We will examine whether FBPase import into the vesicles is regulated by ATP or GTP hydrolysis.

蛋白质降解对于细胞周期控制、信号传导非常重要 转导和细胞生长。蛋白质降解异常 与代谢紊乱、癌症发展和肌肉生长有关 营养不良。 酵母液泡中蛋白质降解的新途径 在我们的实验室建立。它们是关键的糖异生酶，果糖-1.6- 生物磷酸酶 (FBPase)，从细胞质到酵母 当酿酒酵母被溶酶体（液泡）降解时 补充葡萄糖。我们的长期目标是了解 FBPase 降解途径。我们重建了这种葡萄糖调节的 使用半完整细胞、纯化的 FBPase、ATP 的靶向途径 再生系统和细胞质。 FBPase 靶向于液泡 重构的系统。我们已经分离出 33 个视频（液泡导入和 降解）突变体在葡萄糖诱导的降解中有缺陷 FBP酶。突变分析得出这样的假设：FBPase 的目标是 细胞质到中间囊泡，然后到液泡 降解。我们已经纯化了一种新型 FBPase 相关囊泡，接近 同质性。我们克隆了参与囊泡靶向的 VID24 基因 液泡。 Vid24p 被合成并定位于囊泡。 我们的具体目标是： (1) 将 FBPase 导入重组 使用vid24-1突变体的囊泡。我们将检查是否进口 FTPase 确实针对中间囊泡。我们将分 vid1-vid13 将细胞质中的 FBPase 积累成功能性的 亚组。 (2)VID基因的克隆。我们计划克隆VID基因 使用菌落印迹程序并研究表达和 Vid 蛋白的定位。作为替代方法，我们将 克隆与 URA3 基因紧密连锁的 VID15 基因 染色体行走。 (3) 纯化所需的胞浆蛋白 FBPase 输入到囊泡中。我们将使用 vid1-vid13 突变体 含有缺陷的胞质因子并添加分级分离的野生型 胞质来鉴定补充突变体胞质的组分 缺点。如果我们鉴定出这样的蛋白质，我们将制造突变体并准备 从突变体中提取细胞质来测试细胞质是否有缺陷 FBPase 体外导入。我们将检查 FBPase 是否导入到 囊泡受 ATP 或 GTP 水解调节。