TRAFFICKING PATHWAYS TO THE CELL SURFACE IN YEAST

酵母细胞表面的运输途径

• 批准号: 6613439
• 负责人: AMY Y CHANG
• 金额: $ 8.95万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6613439
• 关键词: Animalia; Golgi apparatus; Saccharomyces cerevisiae; analytical ultracentrifugation; chimeric proteins; density gradient ultracentrifugation; endocytosis; fungal proteins; genetic mapping; guanine nucleotide binding protein; immunofluorescence technique; intracellular transport; membrane proteins; protein transport; site directed mutagenesis; suppressor mutations; temperature sensitive mutant; vesicle /vacuole

Membrane trafficking occurs in all eukaryotic cells as a mechanism to maintain membrane balance and organellar identity. The complexity of protein trafficking is underscored by multiple and selective pathways. During this decade there has been an explosion in the identification of proteins involved in protein transport. The approach taken in this proposal is to use combined biochemical and genetic methods in Saccharomyces cerevisiae to understand sorting and transport. Specifically, we have analyzed the trafficking of a mutant plasma membrane protein (Pma1) which fails to arrive at the cell surface. Mutant Pma1 is delivered instead to the endocytic/vacuolar pathway where it is degraded. A collection of sop (suppressor of pma1) mutants has been isolated which prevents vacuolar degradation by allowing mutant Pma1 to move to the plasma membrane. Identification of the corresponding genes has revealed novel SOP genes which are believed to regulate sorting and trafficking in the endosomal system. In Specific Aim 1, cell fractionation and indirect immunofluorescence experiments are proposed to dissect the mechanism of action of the newly-discovered SOP genes by determining whether mutant Pma1 travels from Golgi to cell surface, or endosome to surface in different sop mutants. Detection of mutant Pma1 movement from endosome to plasma membrane will help to establish the existence of a novel traffic pathway. Experiments in Specific Aim 2 focus on the role of VPS8 (identified as one of the sop mutants) in the regulation of endosomal traffic. Specific Aim 3 addresses the hypothesis that there is a novel quality control mechanism which recognizes and directs mutant proteins to the endosomal/vacuolar pathway for degradation. Experiments will test whether vacuolar delivery of mutant Pma1 is signal-mediated, and whether the novel Sop proteins are involved in a mechanism by which proteins destined for the plasma membrane are sorted from proteins destined for the endosomal/vacuolar pathway. The work described in this proposal has implications for understanding intracellular protein trafficking in health and disease.

膜运输发生在所有真核细胞中，作为维持膜平衡和细胞器特性的机制。多种选择性途径凸显了蛋白质运输的复杂性。 在这十年中，参与蛋白质运输的蛋白质的鉴定呈爆炸式增长。 该提案采用的方法是在酿酒酵母中结合使用生化和遗传学方法来了解分选和运输。 具体来说，我们分析了无法到达细胞表面的突变质膜蛋白（Pma1）的运输。 突变体 Pma1 被传递到内吞/液泡途径，并在那里被降解。 已分离出一系列 sop（pma1 抑制子）突变体，它们通过允许突变体 Pma1 移动到质膜来防止液泡降解。 相应基因的鉴定揭示了新的 SOP 基因，这些基因被认为调节内体系统中的分选和运输。 在具体目标1中，提出了细胞分级分离和间接免疫荧光实验，通过确定不同sop突变体中突变体Pma1是否从高尔基体移动到细胞表面，或从内体移动到表面，来剖析新发现的SOP基因的作用机制。检测突变 Pma1 从内体到质膜的运动将有助于确定新交通途径的存在。 具体目标 2 中的实验重点关注 VPS8（被确定为 sop 突变体之一）在内体运输调节中的作用。 具体目标 3 提出了这样的假设：存在一种新的质量控制机制，可以识别突变蛋白并将其引导至内体/液泡途径进行降解。 实验将测试突变体 Pma1 的液泡传递是否是信号介导的，以及新型 Sop 蛋白是否参与将前往质膜的蛋白质与前往内体/液泡途径的蛋白质分选的机制。 该提案中描述的工作对于理解健康和疾病中的细胞内蛋白质运输具有重要意义。