GENETIC REGULATION OF PROTEASES IN YEAST

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 和受体介导的内吞作用。因为酵母菌对 基因、生化和分子分析，它提供了一种 用于功能和装配研究的优秀模型系统 这个重要的细胞器。这项研究的总体目标是 为了了解液泡水解酶在细胞周期中的作用 酵母菌的新陈代谢和分化，以了解这些 酶的活性是产生的，以了解活性是如何 这些酶中的一种被调节并整合到细胞中 功能，并了解管理 这些酶的基因。我们在这一时期的具体目标是1) 从基因上剖析蛋白酶A在影响 水解酶前体的加工2)描绘加工 蛋白B前体的途径并测试我们的 提出的路径是正确的，以及3)分析调控 影响碳分解代谢抑制和表达的回路 一组液泡蛋白水解酶。为了实现这些目标，我们将 体外诱变获得的突变体的分离和鉴定 克隆的蛋白酶。一种结构基因，并与 测序突变的表型变化。我们会 用动力学方法描述蛋白酶B的加工途径 使用抗体和工程衍生品进行的实验 应免除加工的结构基因 要求。为了分析监管，我们将把PRB1放在上游 SUC2和LacZ结构上游的调控序列 基因，并使用这些杂交基因来分离调节突变 并确定PRB1的顺式调控序列。

The yeast vacuole strongly resembles an animal cell lysosome, for it is an acid compartment, contains a set of hydrolases, is the final destination of ligands taken up by fluid phase endocytosis and receptormediated endocytosis. Since yeast is amenable to gentical, biochemical and molecular analyses, it affords an excellent model system for studies of the function and assembly of this important organelle. The overall goals of this research are to understand the role of the set of vacuolar hydrolases in the metabolism and differentiation of yeast, to understand how these enzyme activities are generated, to understand how the activities of these enzymes are regulated and integrated into cellular function and to understand the regulatory circuits that govern the genes for these enzymes. Our specific aims for this period are 1) to genetically dissect the role that protease A plays in effecting processing of hydrolase precursors 2) to delineate the processing pathway for the protease B precursor and test whether our proposed pathway is correct and 3) to analyze the regulatory circuit that effects carbon catabolite repression and expression of the set of vacuolar proteases. To achieve these goals we will isolate and characterize mutants obtained by in vitro mutagenesis of the cloned protease. A structural gene and relate the phenotypic changes to the sequenced mutations. We will delineate the protease B processing pathway by kinetic experiments using antibodies and by engineering derivatives of the structural gene that should be exempt from a processing requirement. To analyze regulation we will place PRB1 upstream regulatory sequences upstream of the SUC2 and lacZ structural genes and use these hybrid genes to isolate regulatory mutations and to define cis regulatory sequences of PRB1.