GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

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，因为基本的生物过程已经 酵母几乎在真核生物中被过度保守， 模型系统的功能和组装的研究，这一重要的 细胞器 这些研究可能有助于我们了解 溶酶体/空泡酶的甘露糖-6-磷酸非依赖性途径 靶向和膜组装。 它们可以帮助理解 溶酶体蛋白酶的错误分类发生在一些病理性的 比如乳腺癌 本研究的总体目标是 了解蛋白质是如何在分子上定位到液泡的， 细胞器是如何组装的，它是如何作为一个消化和 稳态细胞器不能正确分选液泡水解酶的突变体 (pep7和pep 12）或酸化（vph 1）或组装（pep 5）的液泡。 分离并克隆相应的基因。 具体目标是 建议是1）确定Pepl 2 p是否，这是内腔所需的 水解酶靶向，是在高尔基体和液泡膜，无论是 通过在两个细胞器之间循环来发挥作用，无论这种循环是 它的功能是什么，以及其他蛋白质 它在执行其功能时与之交互，以确定是否 Pep 7 p是内腔水解酶靶向所需的，它位于 核，它是否作为转录因子发挥作用，它是什么基因， 调节基因的功能是什么3） 以确定VPH 1和STV 1是否编码细胞器特异性ATP酶 亚单位，细胞器是什么，是否有其他成员的 基因家族，如果有的话，克隆和研究它们，并确定 Vph 1 p在液泡膜中的拓扑结构4）以确定结构域 Pep 5 p的结构，这是形成液泡所必需的， 鉴定在组装液泡时与其相互作用的其他蛋白质， 并确定这些蛋白质的功能丧失是否会产生类似的 表型5）以恢复编码液泡Ca 2+的基因 转运蛋白，使突变，并用它们来探测的作用， 细胞内Ca ~（2+）稳态中的转运蛋白和细胞器。 方法将 包括定向诱变、抑制子选择和分析， 亚细胞分级、生物化学和免疫荧光。

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 and since fundamental biological processes have been almost obsessively conserved across eukaryotes, yeast offers 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 membrane assembly. They may enable understanding of the missorting of lysosomal proteases that occurs in some pathologic conditions like breast cancer. The overall goals of this research are to understand in molecular detail how proteins are targeted to the vacuole, how the organelle is assembled, and how it functions as a digestive and homeostatic organelle. Mutants unable to properly sort vacuolar hydrolases (pep7 and pep12) or to acidify (vph1) or assemble (pep5) the vacuole were isolated and the corresponding genes cloned. The specific aims in this proposal are 1) to determine whether Pepl2p, which is required for lumenal hydrolase targeting, is in both Golgi and vacuolar membranes, whether it functions by cycling between the two organelles, whether this cycling is essential for its function, what its function is, and what other proteins it interacts with as it carries out its functions 2) to determine whether Pep7p, which is required for lumenal hydrolase targeting, is in the nucleus, whether it functions as a transcription factor, what genes it regulates if it does and what the functions of the regulated genes are 3) to determine whether VPH1 and STV1 encode organelle specific ATPase subunits, what the organelles are, whether there are other members of the gene family, and if so, clone and study them, and to determine the topology of Vph1p in the vacuolar membrane 4) to determine the domain structure of Pep5p, which is required for formation of vacuoles, to identify other proteins with which it interacts in assembling the vacuole, and determine whether loss of function of these proteins gives similar phenotypes 5) to recover the gene(s) encoding the vacuolar Ca2+ transporter, make mutations, and use them to probe the role of the transporter and organelle in cellular Ca2+ homeostasis. Methods will include targeted mutagenesis, suppressor selection and analysis, subcellular fractionation, biochemistry, and immunofluorescence.