TRAFFICKING PATHWAYS TO THE CELL SURFACE IN YEAST

说明： 膜循环发生在所有真核细胞中，作为一种机制 维持细胞膜平衡和细胞器识别。在哺乳动物细胞中，一种 内涵体和质膜之间的循环途径是 许多内化的膜蛋白和脂类。然而，相对较少的 气泡输送机械的部件，用于运输 通过内体途径发生的情况还知之甚少。一个主要目标 这项建议的目的是使用生化和遗传相结合的方法来 酿酒酵母定义内体到质膜的运输 途径，并对内体循环的分子机制进行剖析。我们的 方法是分析突变的质膜蛋白的运输 (Pma1)，它不能到达细胞表面而被传递 通向胞内/血管膜途径。Pma1抑制子的集合 (SOP)突变体已经被分离出来，它们将突变体Pma1重新路由到血浆 薄膜。在具体目标1中，细胞分级和间接 建议进行免疫荧光实验，以确定某些SOP是否 突变体通过允许突变体pma1移动到细胞表面来抑制pma1 在它进入内噬系统之后。 在特定目的2中的实验集中在VPS8，被鉴定为sop突变体， 以及它在调节内体运输中的作用。具体目标3描述 三个新发现的编码新基因的SOP基因的拟议工作 与哺乳动物受体同源的膜蛋白。实验将会 测试这些基因产物SOP3、SOP4和SOP5是否参与了 去往质膜运输的蛋白质的机制 从去往内体/液泡的蛋白质中分离出来的高尔基体 路径。这项提案中描述的工作对以下几个方面有影响 涉及细胞内蛋白质运输缺陷的疾病状态。

DESCRIPTION: Membrane recycling occurs in all eukaryotic cells as a mechanism to maintain membrane balance and organellar identify. In mammalian cells, a recycling pathway between the endosome and plasma membrane is taken by many internalized membrane proteins and lipids. However, relatively few of the components of the vesicular transport machinery by which transport through the endosomal pathway occurs is poorly understood. One major goal of this proposal is to use combined biochemical and genetic approaches in Saccharomyces cerevisiae to define an endosome-to-plasma membrane traffic pathway and to dissect the molecular mechanism of endosomal recycling. Our approach is to analyze the trafficking of a mutant plasma membrane protein (Pma1) which fails to arrive at the cell surface and is delivered instead to the endocytic/vasuolar pathway. A collection of suppressor of pma1 (sop) mutants have been isolated which re-route mutant Pma1 to the plasma membrane. In Specific Aim 1, cell fractionation and indirect immunofluorescence experiments are proposed to determine whether some sop mutants suppress pma1 by allowing mutant Pma1 to move to the cell surface after it has entered the endosomal system. Experiments in Specific Aim 2 focus on VPS8, identified as an sop mutant, and its role in regulation of endosomal traffic. Specific Aim 3 describes proposed work in three newly-discovered SOP genes which encode novel membrane protein which homology to mammalian receptors. Experiments will test whether these gene products, SOP3, SOP4, and SOP5, are involved in a mechanism by which proteins destined for plasma membrane delivery are sorted at the Golgi from proteins destined for the endosomal/vacuolar pathway. The work described in this proposal has implications for a number of disease states involving defects in intracellular protein trafficking.