GENETIC REGULATION OF PROTEASES IN YEAST

酵母液泡类似于动物细胞的溶酶体，因为它是酸性的。 隔室，含有水解酶的补充，并且是最终的 被流体相和受体介导的配体摄取的目的地 内吞作用 由于酵母是服从遗传，生化和 分子分析，它提供了一个很好的模型系统的研究 这一重要细胞器的功能和组装。 这些研究可能 有助于我们理解甘露糖-6-磷酸非依赖性 溶酶体/空泡酶靶向途径和易位途径， 需要伴侣功能（包括分子内伴侣） 具有高电荷密度的溶酶体/空泡和分泌蛋白 (like前基质分解素和前胶原酶）。 本项目的总体目标 研究的目的是了解一组液泡蛋白酶的作用， 酵母的代谢和分化，这些活动如何 酶被产生、调节并整合到细胞功能中， 特别是与其他调节电路有关， 葡萄糖水平 具体目的是：（1）确定是否为酸性 液泡的pH值触发蛋白酶的自催化激活 A（PrA）前体，该步骤提出启动级联反应， 导致所有其它水解酶前体的活化。 合成与 将动态地跟踪preproPrA的成熟。(2)解剖 蛋白酶B（PrB）的遗传结构与功能关系 前体，包括确定负责其 分子内伴侣功能和靶向液泡。 损害PRB1-SUC2融合体的空泡靶向的突变将 选择和分析。(3)为了鉴定基因产物， 高度带电的PrB前体易位到血管腔中， 内质网 不能易位PRB1-URA3的突变体 选择融合蛋白并进行分析。(4)继续研究 液泡蛋白酶的调节，重点是PrB，因为它 是最高度调节的蛋白酶补体。 的突变体 不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）特异性电路上。 积分电路的分析将随后进行。

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.