GENETIC ANALYSIS OF ENZYME PROCESSING AND LOCALIZATION

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 分子分析和由于基本的生物过程已经 酵母在真核生物中几乎被疯狂地保守，它提供了一种极好的 模型系统是研究这一重要部件的功能和装配 细胞器。这些研究可能有助于我们理解 溶酶体/空泡酶的甘露糖-6-磷酸非依赖性途径 靶向和膜组件。它们可以使人们理解 某些病理中出现的溶酶体蛋白水解酶的错误分选 像乳腺癌这样的疾病。这项研究的总体目标是 在分子细节上了解蛋白质是如何定位于液泡的， 细胞器是如何组装的，以及它是如何作为消化和 动态平衡细胞器。突变体无法正确分类液泡水解酶 (Pep7和Pep12)或酸化(Vph1)或组装(Pep5)液泡 分离并克隆相应的基因。其中的具体目标是 建议1)确定管腔所需的Pepl2p是否 水解酶的靶向性，是在高尔基体和液泡膜上，无论是它 通过在两个细胞器之间循环来发挥作用，无论这种循环是 对它的功能至关重要，它的功能是什么，还有什么其他蛋白质 它在执行其功能时与之交互，以确定 腔内水解酶靶向所需的Pep7p位于 细胞核，它是否发挥转录因子的功能，它是什么基因？ 调控是否存在，以及受调控基因的功能是什么3) 确定VPH1和STV1是否编码细胞器特异性ATPase 亚单位，细胞器是什么，是否有其他成员 基因家族，如果是的话，克隆和研究它们，并确定 Vph1p在液泡膜中的拓扑结构4)确定结构域 形成液泡所需的Pep5p的结构，以 确定在组装液泡的过程中与之相互作用的其他蛋白质， 并确定这些蛋白质功能的丧失是否会产生类似的 表型5)恢复编码液泡钙的基因(S) 转运蛋白，进行突变，并用它们来探测 细胞内钙稳态中的转运体和细胞器。方法将 包括靶向突变、抑制子选择和分析， 亚细胞分离、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.