GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

酵母液泡非常类似于动物细胞溶酶体，因为 它是一个酸性隔间，含有一组水解酶，是 液体相内吞作用所摄取的配体的最终目的地 和受体药物内吞作用。因为酵母菌对 遗传、生化、分子和细胞生物学分析，它 为功能和功能研究提供了一个很好的模型系统 细胞器组装这种细胞器的组件这项研究的总体目标是 为了确定酵母菌的溶酶体样空泡 酵母菌被组装，使其具有功能，并含有 酶活性被激活。我们在这一时期的具体目标 1)从基因上剖析酶的加工途径和 液泡酶的定位2)遗传解剖 液泡的酸化机制，从而确定 酸性pH是否触发了蛋白酶A的自动激活 启动蛋白分解激活循环和3)开始基因 分析了液泡的形成和组装机制， 本身就是。为了实现这些目标，我们将隔离和描述 消除液泡的多效性突变所定义的基因 水解酶，但不分泌蛋白质，并试图区分 基因和基因产物液泡酶的研究 激活并锁定目标。我们将分离并鉴定突变株 使用染料使液泡酸化有缺陷的 荧光是与pH有关的。由这些突变定义的基因 将被分离以确定空泡ATPase是否是 酸化剂和ATPase以外的蛋白质是否 牵涉其中。突变体，尤其是缺乏液泡的条件性突变体 将被分离出来，以便于鉴定基因和基因 完整性、稳定性和成型所需的产品 液泡。相应的基因将被分离和研究。

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.