GENETIC REGULATION OF PROTEASES IN YEAST

酵母中蛋白酶的遗传调控

• 批准号: 3225935
• 负责人: ELIZABETH W JONES
• 金额: $ 18.62万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225935
• 关键词: Saccharomyces; aminoacid analog; cell cycle; cell differentiation; chemical cleavage; enzyme mechanism; eukaryote; fungal genetics; gel electrophoresis; linkage mapping; nucleic acid hybridization; nucleic acid sequence; peptidases; pleiotropism; protease inhibitor; protein metabolism; sporogenesis; structural genes; temperature sensitive mutant

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.

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 基因、生物化学和分子分析，它提供了一个 功能和装配研究的优秀模型系统 这个重要的细胞器。 本研究的总体目标是 为了了解空泡水解酶在细胞内的作用， 酵母的代谢和分化，以了解这些 酶的活动产生，以了解如何活动， 这些酶的调节和整合到细胞 功能，并了解管理的调节电路 这些酶的基因。 我们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）描述加工 蛋白酶B前体的途径，并测试我们是否 建议的途径是正确的，3）分析监管 影响碳分解代谢物抑制和表达的回路 一组液泡蛋白酶。 为了实现这些目标，我们将 分离和表征通过体外诱变获得突变体 克隆的蛋白酶。 一个结构基因， 表型变化的测序突变。 我们将 通过动力学描述蛋白酶B加工途径 实验使用抗体和工程衍生物的 应免于处理结构基因 要求。 为了分析调节，我们将PRB1置于上游 SUC2和lacZ结构上游的调节序列 基因，并使用这些杂交基因来分离调控突变 并确定PRB1的顺式调控序列。