GENETIC REGULATION OF PROTEASES IN YEAST

酵母液泡类似于动物细胞溶酶体，因为它是酸性的。 隔间，包含水解酶的补充物，是最后的 液体相和受体介导的配体的去向 内吞作用。由于酵母菌对遗传、生化和 分子分析，为分子生物学的研究提供了一个良好的模型系统 这种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-磷酸的非依赖性 溶酶体/空泡酶靶向和易位途径 必需的伴侣功能(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 (如原溶血素和原胶原酶)。这个项目的总体目标是 研究旨在了解一组液泡蛋白水解酶在 酵母的新陈代谢和分化，这些活性是如何 酶被产生、调节并整合到细胞功能中， 尤其是与其他也响应于 血糖水平。具体目标是：(1)确定酸性是否 液泡的pH触发了蛋白水解酶的自催化激活 A(PRA)前体在体内，该步骤建议启动级联即 导致所有其他水解酶前体的激活。综合和 前PrA的成熟将以动力学的方式进行。(2)解剖 蛋白水解酶B(PRB)的遗传结构与功能关系 前兆，包括确定对其负责的区域 分子内伴侣起作用，并定位于液泡。 破坏PRB1-SUC2融合的空泡靶向的突变将 被挑选和分析。(3)鉴定所需基因产物 高电荷的pRb前体转移到管腔内 内质网。未能将PRB1-URA3易位的突变体 将对融合蛋白进行筛选和分析。(四)继续研究 液泡蛋白水解酶的调节，重点是pRb，因为它 是最受调控的蛋白水解酶补体。变种人 未能表达或表达PPB1-URA3、PRB1-LacZ和/或 将选择或筛选PRB1-SUC2融合进行分析。 最初，重点将放在PRB(或蛋白水解酶)特定的回路上。 接下来是对集成电路的分析。

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.