GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

酶加工和定位的遗传分析

• 批准号: 3277349
• 负责人: ELIZABETH W JONES
• 金额: $ 26.67万
• 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/3277349
• 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; oxygenases; plasmids; protein biosynthesis; transcription factor; 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.

酵母液泡非常类似于动物细胞溶酶体， 它是一个酸室，含有一组水解酶，是 通过液相内吞作用吸收的配体的最终目的地 和受体介导的内吞作用。 由于酵母菌是顺从于 遗传、生物化学、分子和细胞生物学分析， 提供了一个很好的模型系统的功能研究， 这个细胞器的组装。 本研究的总体目标是 来确定酵母的溶酶体样液泡 酵母菌是组装的，具有功能性， 酶活性被激活。 我们这一时期的具体目标 是1）从遗传学上剖析酶加工的途径， 定位酶的液泡2）遗传解剖 液泡酸化的机制，从而确定 酸性pH值是否触发蛋白酶原A的自激活， 启动蛋白水解活化循环，和3）开始遗传 分析了液泡的形成和组装机制， 本质上 为了实现这些目标，我们将分离和表征 由消除空泡的多效性突变定义的基因 水解酶，而不是分泌的蛋白质，并试图辨别 基因和基因产物的研究如何液泡酶是 被激活并有针对性 我们将分离并鉴定变种人 在使用染料的液泡酸化中有缺陷， 荧光是pH依赖性的。 这些突变所定义的基因 将被分离，以确定是否液泡ATP酶是 酸化剂以及除ATP酶以外的蛋白质是否 涉案 突变体，特别是条件突变体，缺乏液泡 将被分离，以便于识别基因和基因 产品所需的完整性，稳定性和形成的 空泡 相应的基因将被分离和研究。