ALTERNATIVE VACUOLAR TARGETING MECHANISMS IN YEAST

酵母中的替代液泡靶向机制

• 批准号: 6180783
• 负责人: DANIEL J. KLIONSKY
• 金额: $ 16.26万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 2000-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180783
• 关键词: Saccharomyces cerevisiae; affinity chromatography; aminopeptidase; biological models; biological signal transduction; electron microscopy; enzyme activity; fungal proteins; gel electrophoresis; green fluorescent proteins; immunoprecipitation; intracellular transport; lysosomes; membrane permeability; membrane proteins; molecular cloning; polymerase chain reaction; protein protein interaction; protein structure function; protein transport; vesicle /vacuole; yeast two hybrid system

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.

描述(改编自申请人的摘要)：真核细胞 含有各种离散的膜封闭细胞器。如此之高 分门别类的组织对于 牢房。这些亚细胞室中的每一个都有独特的结构和 由特定蛋白质赋予的功能特征， 脂类和/或组成它们的碳水化合物。溶酶体是主要的 负责哺乳动物细胞内降解的细胞器。它 包含许多可溶的水解酶以及各种 膜相关蛋白。泰·萨克斯病，伪赫勒 多发性营养不良和I细胞疾病是人类三十多种遗传基因中的三种。 已知的由于体内缺乏某些蛋白质而引起的疾病 溶酶体。这些疾病中的许多具体是由于不适当的 对这些蛋白质进行分类。这种缺陷分类和分类之间的关联 疾病状态强调了正确的蛋白质分类的重要性 细胞生理学。调查人员的长期目标是制定一项准确的 对分子事件参与识别、分类的理解 以及将蛋白质运输到酵母细胞中的溶酶体样空泡。这个 研究人员将使用酵母作为模型系统来研究这些问题，因为 用于蛋白质运输的途径似乎与那些 在动物细胞中，另外酵母菌也可以利用有用的基因 不适用于高等真核生物的方法。在这 提案中，调查人员将重点分析涉及的因素 膜相关液泡蛋白的传递。关于…的初步研究 碱性磷酸酶(ALP)表明它是一种膜蛋白，是 通过一种至少部分不同的机制传递到液泡 与可溶性液泡水解酶的作用不同。此外，空间上的 碱性磷酸酶靶向信号的位置表明分选的差异 可以反映与唯一排序组件的交互，例如 特定受体。研究人员将进一步描述空泡的特征 对碱性磷酸酶中的信号进行分类，并将尝试通过 一种生化和遗传方法的结合。此外， 研究人员将利用基因融合的方法来选择突变体 在这种蛋白质的定位上有缺陷。这些变种人应该 允许调查员定义分拣和运输的组件 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之前鉴定的突变体进行比较 可溶的液泡水解酶分类错误。排序的标识 碱性磷酸酶中的决定因素与参与的基因的特征 它的承认和交付应该加深我们对 运输过程。