ALTERNATIVE VACUOLAR TARGETING MECHANISMS IN YEAST

描述（改编自申请人摘要）：真核细胞 包含各种离散的膜封闭细胞器。 这种高度 划分的组织对组织的正常运作至关重要。 牢房 这些亚细胞区室中的每一个都具有独特的结构和 由特定蛋白质赋予的功能特性， 脂质和/或包含它们的碳水化合物。 溶酶体是主要的 哺乳动物细胞中负责细胞内降解的细胞器。 它 含有许多可溶性水解酶以及多种 膜相关蛋白。 泰萨克斯病，假Hurler 多发性营养不良和I细胞疾病是30多种人类遗传病中的三种。 已知由某些蛋白质缺乏引起的疾病， 溶酶体 这些疾病中有许多是由不适当的 对这些蛋白质进行分类。 这种分类缺陷和 疾病状态强调了正确的蛋白质分选在 细胞生理学 研究人员的长期目标是开发一种精确的 了解参与识别、分选 以及将蛋白质运输到酵母细胞中的溶酶体样液泡。 的 研究人员将使用酵母作为模型系统来研究这些问题， 用于蛋白质转运的途径似乎与 在动物细胞中，此外，酵母适合于有用的遗传修饰。 这些方法不适用于高等真核生物。 在这 建议，调查人员将重点分析涉及的因素， 膜相关液泡蛋白的递送。 初步研究 碱性磷酸酶（ALP）表明它是一种膜蛋白， 通过至少部分不同的机制传递到液泡 与可溶性液泡水解酶所用的不同。 此外，空间 ALP靶向信号的位置表明， 可以反映与独特的分类组件的交互， 特异性受体 研究者将进一步表征空泡 在ALP中分选信号，并将尝试通过 生物化学和遗传学方法的结合。 此外该 研究者将利用基因融合方法来选择突变体， 在这种蛋白质的定位上有缺陷。 这些变种人应该 允许调查员定义分类和运输的组成部分 识别这种蛋白质并将其定位到液泡膜上的装置。 这些新的突变体将与先前鉴定的突变体进行比较， 错配可溶性液泡水解酶。 排序的一个标识 ALP中的决定簇，再加上参与ALP的基因的表征， 它的承认和交付应进一步加深我们对 运输过程。

DESCRIPTION (Adapted from the applicant's abstract): Eukaryotic cells contain a variety of discrete membrane-enclosed organelles. This highly compartmentalized organization is essential to the normal functioning of the cell. Each of these subcellular compartments has unique structural and functional characteristics which are conferred by particular proteins, lipids and/or carbohydrates that comprise them. The lysosome is the major organelle responsible for intracellular degradation in mammalian cells. It contains numerous soluble hydrolases as well as a variety of membrane-associated proteins. Tay Sachs disease, pseudo-Hurler polydystrophy and I cell disease are three of over thirty human genetic disorders which are known to result from the absence of certain proteins in the lysosome. Many of these diseases specifically result from the improper sorting of these proteins. This correlation between sorting defects and disease states underscores the importance of correct protein sorting in cell physiology. The investigators long-term goal is to develop a precise understanding of the molecular events involved in the recognition, sorting and transport of proteins to the lysosome-like vacuole in yeast cells. The investigator will use yeast as a model system to study these problems since the pathways used for protein transport appear to be very similar to those in animal cells, and in addition yeast are amenable to useful genetic approaches which are not as applicable to higher eukaryotes. In this proposal, the investigators will focus on analyzing factors involved in delivery of membrane-associated vacuolar proteins. Initial studies of alkaline phosphatase (ALP) indicate that it is a membrane protein that is delivered to the vacuole by a mechanism that is at least in part different from that used by soluble vacuolar hydrolases. Furthermore, the spatial location of the ALP targeting signal suggests that differences in sorting may reflect an interaction with unique sorting components such as a specific receptor. The investigator will further characterize the vacuolar sorting signal in ALP and will attempt to identify the ALP receptor through a combination of biochemical and genetic approaches. In addition, the investigator will utilize a gene fusion approach to select mutants which are defective in the localization of this protein. These mutants should allow the investigator to define components of the sorting and transport apparatus that recognize and target this protein to the vacuole membrane. These new mutants will be compared with previously identified mutants which missort soluble vacuolar hydrolases. An identification of the sorting determinant in ALP coupled with a characterization of the genes involved in its recognition and delivery should further our understanding of the transport process.