GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

酶加工和定位的遗传分析

• 批准号: 3277341
• 负责人: ELIZABETH W JONES
• 金额: $ 22.53万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277341
• 关键词: Saccharomyces; antibody formation; carboxypeptidase; enzyme mechanism; fungal genetics; gel electrophoresis; gene complementation; gene expression; gene mutation; genetic mapping; genetic promoter element; molecular cloning; molecular genetics; nucleic acid hybridization; plasmids; protein biosynthesis; ultracentrifugation; vesicle /vacuole

The yeast vacuole strongly resembles an animal cell lysosome, for it is an acid compartment, contains a set of hydrolases, and is the final destination of ligands taken up by fluid phase endocytosis and receptor-medicated endocytosis. Since yeast is amenable to genetical, biochemical, molecular and cell biological analyses, it affords an excellent model system for studies of the function and assembly of this organelle. The overall goals of this research are to determine how the lysosome-like vacuole of the yeast Saccharomyces is assembled, made functional, and its contained enzymatic activities activated. Our specific aims for this period are 1) to genetically dissect the pathway of enzyme processing and localization for enzymes of the vacuole 2) to genetically dissect the mechanism of acidification of the vacuole and thereby determine whether acid pH acts to trigger autoactivation of proprotease A to initiate the proteolytic activation cycle and 3) to begin a genetic analysis of the mechanism of formation and assembly of the vacuole, per se. To achieve these goals we will isolate and characterize genes defined by pleiotropic mutations that eliminate vacuole hydrolases but not secreted proteins and try to discern from studies of the genes and gene products how the vacuolar enzymes are activated and targeted. We will isolate and characterize mutants defective in acidification of the vacuole using dyes whose fluorescence is pH-dependent. The genes defined by these mutations will be isolated to determine whether the vacuolar ATPase is the acidification agent and whether proteins other than the ATPase are involved. Mutants, especially conditional ones, that lack vacuoles will be isolated to facilitate identification of genes and gene products required for integrity, stability and formation of the vacuole. The corresponding genes will be isolated and studied.

酵母液泡与动物细胞溶酶体非常相似，因为 它是一个酸室，包含一组水解酶，并且是 液相内吞作用所吸收的配体的最终目的地 和受体介导的内吞作用。 由于酵母适合 遗传、生化、分子和细胞生物学分析， 为功能和功能的研究提供了一个优秀的模型系统 该细胞器的组装。 本研究的总体目标是 确定酵母的溶酶体样液泡如何 酵母菌被组装起来，具有功能性，并且它所包含的 酶活性被激活。 我们这一时期的具体目标 1）从基因角度剖析酶加工的途径以及 液泡酶的定位 2) 进行基因解剖 液泡酸化机制，从而确定 酸性 pH 值是否会触发蛋白酶 A 的自动激活 启动蛋白水解激活循环并 3) 开始遗传 分析液泡形成和组装的机制， 本身。 为了实现这些目标，我们将隔离并表征 由消除液泡的多效性突变定义的基因 水解酶而不是分泌蛋白，并尝试辨别 基因和基因产物的研究 液泡酶是如何存在的 激活并有针对性。 我们将分离并表征突变体 使用染料的液泡酸化有缺陷 荧光是 pH 值依赖性的。 这些突变定义的基因 将被分离以确定液泡ATP酶是否是 酸化剂以及除 ATP 酶之外的蛋白质是否 涉及。 缺乏液泡的突变体，尤其是条件突变体 将被分离以利于基因和基因的鉴定 完整性、稳定性和形成所需的产品 液泡。 相应的基因将被分离和研究。