GENETICS OF ORGANELLE PROTEIN DELIVERY IN YEAST

真核细胞的一个显著特征是存在一系列的 胞内膜内有细胞器。 这种高度划分的 组织对于细胞的正常功能是必不可少的。 各种 特定的蛋白质、脂质和碳水化合物必须有效地 传递到这些细胞器。 这些分子的错误方向会导致 细胞和生物体功能障碍（例如，I-细胞疾病）。 分子 蛋白质被分选到不同细胞中的机制 目前，隔间基本上是未知的。 酵母为解决这些问题提供了一个很好的遗传系统。 酵母和高等真核生物似乎含有几乎相同的途径， 维持和组装这些复杂的细胞结构。 在这个建议中，重点将放在分析具体的机制， 蛋白质靶向三个酵母亚细胞区室。 代表 将研究蛋白质;分泌的蛋白质转化酶和α-因子， 液泡羧肽酶Y和线粒体ATP酶 β亚基 编码这些蛋白质的基因已经全部克隆到 酵母质粒。 为了便于分析，基因融合已经或将 在这些基因与大肠杆菌lacZ基因之间构建。杆菌 （编码β-半乳糖苷酶）或酵母的SUC 2基因（编码 转化酶）。 这些酶提供了有用的生化标记 用于跟踪由该基因编码的杂合蛋白的定位 融合 分析各种不同大小的融合， 一种是将基因分解成各种成分信号， 负责将这些分子有效地靶向它们的 各自的手机位置 此外，通过利用某些 通过这些杂合蛋白赋予酵母细胞的表型，它应该 可以分离定位缺陷突变体。 这种突变体可以 定义关键识别信号或参与的特定识别因子， 在交付过程中。 基因的克隆和表征， 通过这种突变分析鉴定的序列将提供更多的 参与维持的分子事件的精确图片 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.