TRAFFICKING PATHWAYS TO THE CELL SURFACE IN YEAST

作为维持膜平衡和细胞器身份的一种机制，膜运输发生在所有真核细胞中。多种选择性途径强调了蛋白质运输的复杂性。在这十年中，对参与蛋白质运输的蛋白质的鉴定出现了爆炸式的增长。本文采用生物化学和遗传学相结合的方法来了解酿酒酵母的分选和转运。具体来说，我们分析了未能到达细胞表面的突变质膜蛋白（Pma1）的运输。突变的Pma1被传递到内吞/空泡途径，在那里它被降解。已分离出一系列sop （pma1的抑制因子）突变体，通过允许突变体pma1移动到质膜来阻止液泡降解。相应基因的鉴定揭示了新的SOP基因，这些基因被认为是调节内体系统的分类和运输。在Specific Aim 1中，提出了细胞分离和间接免疫荧光实验，通过确定突变体Pma1在不同的SOP突变体中是从高尔基体传播到细胞表面还是从内体传播到表面，来剖析新发现的SOP基因的作用机制。检测突变体Pma1从核内体到质膜的运动将有助于确定一种新的运输途径的存在。特异性靶2的实验集中在VPS8（被确定为sop突变体之一）在调节内体运输中的作用。特异性目标3提出了一个假设，即存在一种新的质量控制机制，可以识别和指导突变蛋白进入内体/空泡降解途径。实验将测试突变体Pma1的空泡传递是否是信号介导的，以及新的Sop蛋白是否参与了一种机制，通过这种机制，到达质膜的蛋白质从到达内体/空泡途径的蛋白质中分离出来。本提案中描述的工作对理解细胞内蛋白质在健康和疾病中的贩运具有重要意义。

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.