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GENETIC REGULATION OF PROTEASES IN YEAST

酵母中蛋白酶的遗传调控

基本信息

批准号：
2602674
负责人：
ELIZABETH W JONES
金额：
$ 20.82万
依托单位：
CARNEGIE-MELLON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1977
资助国家：
美国
起止时间：
1977-08-01 至 1999-06-30
项目状态：
已结题

项目摘要

The yeast vacuole resembles an animal cell lysosome, for it is an acidic compartment, contains a complement of hydrolases, and is the final destination of ligands taken up by fluid phase and receptor-mediated endocytosis. Since yeast is amenable to genetical, biochemical and molecular analysis, it affords an excellent model system for studies of the function and assembly of this important organelle. These studies may contribute to our understanding of the mannose-6-phosphate independent pathway of lysosomal/vacuolar enzyme targeting and of translocation and chaperone functions (including intramolecular chaperones) required for lysosomal/vacuolar and secreted proteins that have a high charge density (like prostromelysin and procollagenase). The overall goals of this research are to understand the role of the set of vacuolar proteases in the metabolism and differentiation of yeast, how the activities of these enzymes are generated, regulated and integrated into cellular function, especially in relation to other regulatory circuits that also respond to glucose levels. Specific aims are: (1) To determine whether the acidic pH of the vacuole triggers the autocatalytic activation of the proteinase A (PrA) precursor in vivo, the step proposed to initiate the cascade that results in activation of all other hydrolase precursors. Synthesis and maturation of preproPrA will be followed kinetically. (2) To dissect genetically structure-function relationships of the proteinase B(PrB) precursor, including identification of regions responsible for its intramolecular chaperone function and for targeting to the vacuole. Mutations that compromise vacuolar targeting of a PRB1-SUC2 fusion will be selected and analyzed. (3) To identify gene products required for translocation of the highly charged PrB precursor into the lumen of the endoplasmic reticulum. Mutants that fails to translocate a PRB1-URA3 fusion protein will be selected and analyzed. (4) To continue studies on regulation of the vacuolar proteases, with an emphasis on PrB, since it is the most highly regulated of the protease complement. Mutants that fail to express or express constitutively PPB1-URA3, PRB1-lacZ and/or PRB1-SUC2 fusions will be selected or screened for an analyzed. Initially, the focus will be on a PrB (or protease) specific circuit. Analysis of integrating circuitry will follow.

酵母液泡类似于动物细胞溶酶体，因为它是酸性的室，含有补充的水解酶，并且是最终的流体相和受体介导的配体的目的地内吞作用。由于酵母易于遗传、生化和分子分析，它为研究提供了一个优秀的模型系统这个重要细胞器的功能和组装。这些研究可能有助于我们了解甘露糖-6-磷酸独立性溶酶体/液泡酶靶向和易位途径所需的伴侣功能（包括分子内伴侣）具有高电荷密度的溶酶体/液泡和分泌蛋白（如前体溶酶和前胶原酶）。本次活动的总体目标研究的目的是了解液泡蛋白酶组在酵母的代谢和分化，这些活性如何酶的产生、调节和整合到细胞功能中，特别是与其他也响应的调节电路有关血糖水平。具体目的是： (1) 确定是否呈酸性液泡的 pH 值触发蛋白酶的自催化激活体内的 (PrA) 前体，建议启动级联的步骤导致所有其他水解酶前体的激活。合成与 preproPrA 的成熟将在动力学上进行跟踪。 (2) 剖析蛋白酶 B (PrB) 的遗传结构与功能关系先驱，包括确定对其负责的区域分子内伴侣功能和靶向液泡。损害 PRB1-SUC2 融合的液泡靶向的突变将进行选择和分析。 (3) 鉴定所需的基因产物高电荷 PrB 前体易位到管腔中内质网。未能易位 PRB1-URA3 的突变体将选择并分析融合蛋白。（四）继续研究液泡蛋白酶的调节，重点是 PrB，因为它是蛋白酶补体中受调控程度最高的。突变体未能表达或组成型表达 PPB1-URA3、PRB1-lacZ 和/或将选择或筛选 PRB1-SUC2 融合体进行分析。最初，重点将放在 PrB（或蛋白酶）特定电路上。接下来将分析集成电路。