CYTOSOL/VESICLE/VACUOLAR PROTEIN DEGRADATION PATHWAY

细胞溶胶/囊泡/液泡蛋白降解途径

• 批准号: 6019529
• 负责人: HUI-LING CHIANG
• 金额: $ 22.79万
• 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/6019529
• 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、信号转导和细胞周期调控都很重要。 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 营养不良 一种新的蛋白质降解途径在酵母液泡已被发现， 建立在我们的实验室。它们的关键酶果糖-1.6- 生物磷酸酶（FBPase）从胞质溶胶靶向酵母 溶酶体（空泡）降解时，酿酒酵母是 补充了葡萄糖。我们的长期目标是了解FBPase 降解途径我们重组了这种葡萄糖调节的 使用半完整细胞、纯化的FBPase、ATP酶 再生系统和细胞质。FBPase靶向于细胞内的液泡， 重建系统。我们已经分离出33个VID（空泡输入， 在葡萄糖诱导的降解中有缺陷的突变体， FBPase。突变体分析导致了FBPase靶向于 胞质液到中间小泡，然后是液泡， 降解我们已经纯化了一种新的FBPase-associated囊泡， 同质性我们克隆了参与囊泡靶向的VID 24基因， 空泡Vid 24 p被合成并定位于囊泡。 我们的具体目标是：（1）将FBPase进口重组到 vid 24 -1突变体。我们将研究是否进口 FT 3确实靶向中间囊泡。我们会分开 vid 1-vid 13，其在胞质溶胶中积累FBPase， 分组。(2)VID基因的克隆。我们计划克隆VID基因 利用殖民地印迹法研究其表达， Vid蛋白的定位。作为替代方法，我们将 克隆与URA 3基因紧密连锁的VID 15基因， 染色体步移(3)纯化细胞溶质蛋白质， FBPase导入囊泡。我们将使用vid 1-vid 13突变体， 含有缺陷的胞质因子并添加分级分离的野生型 细胞质以鉴定补充突变细胞质的组分 缺损如果我们鉴定出这样的蛋白质，我们将制造突变体， 突变体的胞质溶胶，以测试胞质溶胶是否有缺陷， FBPase体外导入。我们将检查FBPase是否导入 囊泡由ATP或GTP水解调节。