CYTOSOL/VESICLE/VACUOLAR PROTEIN DEGRADATION PATHWAY

细胞溶胶/囊泡/液泡蛋白降解途径

• 批准号: 6386503
• 负责人: HUI-LING CHIANG
• 金额: $ 24.18万
• 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/6386503
• 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.

蛋白质降解对细胞周期控制、信号传递很重要 转导和细胞生长。蛋白质的异常降解 与代谢紊乱、癌症发展和肌肉发达有关 营养不良。 酵母液泡中蛋白质降解的一种新途径是 在我们的实验室建立。它们是关键的葡萄糖异生酶，果糖-1.6- 生物磷酸酶(FBPase)，是从胞浆到酵母的靶标 用于酿酒酵母降解的溶酶体(液泡) 补充葡萄糖的。我们的长期目标是了解FBPase 降解途径。我们已经重建了这个由葡萄糖调节的 利用半完整细胞、纯化的三磷酸腺苷FBPase的靶向途径 再生系统和胞浆。FBPase定位于植物体内的液泡。 重组后的系统。我们已经分离出33个VID(空泡导入和 降解)突变株在葡萄糖诱导的降解中存在缺陷 FBPase。突变分析导致假设FBPase的靶点来自 从胞浆到中间小泡，然后是液泡 退化。我们已经纯化了一个新的与FBPase相关的囊泡 同质性。我们克隆了与囊泡靶向有关的VID24基因 液泡。合成了Vid24p，并将其定位于小泡。 我们的具体目标是：(1)将FBPase导入到 使用vid24-1突变体的囊泡。我们将审查进口的 FTPase确实是以中间小泡为靶点的。我们将分裂 VID1-VID13，它将胞浆中的FBPase积累成功能性的 子组。(2)VID基因的克隆。我们计划克隆病毒基因 利用菌落印迹法研究其表达和表达 病毒蛋白的定位。作为一种替代方法，我们将 通过以下方法克隆与URA3基因紧密连锁的VID15基因 染色体行走。(3)提纯所需的胞浆蛋白 FBPase导入囊泡。我们将使用vid1-vid13突变体 含有缺陷胞浆因子(S)和添加分离的野生型 胞浆以鉴定补充突变胞浆的组分 叛逃。如果我们鉴定出这种蛋白质，我们将制造突变体并准备 从突变体中提取胞浆，以检测胞浆是否有缺陷 体外导入FBPase。我们将检查FBPase是否导入到 囊泡受ATP或GTP水解酶的调节。