GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

点击翻译按钮获取中文摘要

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.