GENETICS OF ORGANELLE PROTEIN DELIVERY IN YEAST

真核细胞的一个显著特征是存在一系列 细胞内膜包裹细胞器。这是高度划分的 组织对于细胞的正常运作是必不可少的。品种繁多 特定蛋白质、脂肪和碳水化合物的含量必须是有效的 送到这些细胞器。这些分子的误导可能会导致 细胞和生物体故障(例如，i-cell病)。分子 蛋白质被分选到不同细胞中的机制 目前，这些隔间基本上是未知的。 酵母为解决这些问题提供了一个极好的遗传系统。 酵母和高等真核生物似乎包含几乎相同的途径 维护和组装这些复杂的细胞结构。 在这项建议中，将重点分析具体的机制 蛋白质靶向三个酵母亚细胞隔间。代表 将对蛋白质进行研究；分泌蛋白质转化酶和α-因子， 液泡型羧基肽酶Y和线粒体ATPase β亚基。编码这些蛋白质的基因都已被克隆到 酵母质粒。为了便于分析，基因融合已经或将会 在这些基因和大肠杆菌的lacZ基因之间构建 (编码β-半乳糖苷酶)或酵母的SUC2基因(编码 对于酶转化酶)。这些酶提供了有用的生化标签。 用于跟踪该基因编码的杂交蛋白的定位 融合。 对各种不同大小的融合类型的分析应该能够 一种是将基因分解成各种成分信号，这些信号是 负责有效地将这些分子定向到他们的 各自的蜂窝位置。此外，通过利用某些 由这些杂交蛋白赋予酵母细胞的表型，它应该 有可能分离出定位缺陷突变体。这样的突变体可能 定义关键识别信号或参与的特定接收器 在交付过程中。基因和基因的克隆与鉴定 通过这种突变分析确定的序列将提供更多 精确的分子事件图，涉及到维持 细胞器的结构和功能。

A distinguishing feature of eukaryotic cells is the presence of an array of intracellular membrane enclosed organelles. This highly compartmentalized organization is essential to the normal functioning of the cell. A variety of specific proteins, lipids, and carbohydrates must be efficiently delivered to these organelles. Misdirection of these molecules can lead to cell and organism malfunction (e.g., I-cell disease). The molecular mechanisms by which proteins are sorted into the different cellular compartments are at present largely unknown. Yeast provide an excellent genetic system for addressing these questions. Yeast and higher eukaryotes appear to contain nearly identical pathways for the maintenance and assembly of these complex cellular structures. In this proposal focus will be put on analyzing the mechanism of specific protein targeting to three yeast subcelluar compartments. Representative proteins will be studied; the secreted proteins invertase and Alpha-factor, the vacuolar carboxypeptidase Y, and the mitochondrial ATPase Beta-subunit. The genes coding for these proteins have all been cloned on yeast plasmids. To facilitate the analysis, gene fusions have been or will be constructed between these genes and either the lacZ gene of E. coli (codes for the enzyme Beta-galactosidase) or the SUC2 gene of yeast (codes for the enzyme invertase). These enzymes provide useful biochemical tags for following the localization of the hybrid proteins encoded by the gene fusions. Analysis of a variety of different size classes of fusions should enable one to dissect a gene into the various component signals that are responsible for the efficient targeting of these molecules to their respective cellular locations. In addition, by exploiting certain phenotypes conferred to the yeast cell by these hybrid proteins, it should be possible to isolate localization defective mutants. Such mutants could define critical recognition signals or specific recrptors that participate in the delivery process. Cloning and characterization of the genes and sequences identified by this mutational analysis will provide a more precise picture of the molecular events involved in the maintenance of cellular organelle structure and function.