SORTING AND TRANSPORT OF YEAST MEMBRANE PROTEINS

这项研究的总体目标是开发一种机制， 了解膜蛋白在酵母中的分选和组装 酿酒酵母 最近的研究结果表明， 进入酵母的膜蛋白的默认区室 分泌途径 为了解释这些结果，液泡默认模型 已经被提议了。 该模型指出，ER的膜蛋白 而高尔基体则含有阻止它们运输到 液泡，而质膜蛋白具有阳性分选 信息，这确保它们不会被运送到液泡。 在酵母高尔基体膜蛋白DPAP A上的保留信号已经被证实是 这些信息将用于设计基因工程。 筛选以鉴定酵母高尔基体保留器的组分。 具体目标#1和#2侧重于确定组件 所需的保留膜蛋白在酵母高尔基体，和 分子遗传学和生物化学表征这些成分。 将分离出不能保留高尔基体膜的酵母突变体 蛋白质，GAL 4双杂交遗传系统将用于鉴定 编码直接与DPAP A高尔基体相互作用的蛋白质的基因 保留信号 确定的基因将被测序， 构建等位基因以测试组分的体内功能，和 高尔基体保留机制的生化特征。 具体目标 #3将测试空泡默认模型的第二部分， 调查定向信息的性质， FUS 1编码的质膜蛋白质粘附到酵母细胞表面。 基因 FUS 1基因和编码酵母高尔基体的基因之间的融合 将构建膜蛋白KEX 1，以定位FUS 1的任何区域 能够重定向缺乏高尔基体保留的KEX 1蛋白的蛋白 信号传递到酵母细胞膜。 本建议的第二个重点是对 组装和靶向多亚基膜蛋白复合物， 酵母液泡膜H+-ATP酶。 大部分的液泡H+- ATP酶亚基基因克隆，该工具现在可以开始 解决这个复杂的液泡膜的生物合成问题 蛋白 具体目标#4旨在分离和表征 空泡H ~+-ATP酶缺陷酵母突变体（vma）的克隆和鉴定 VMA基因，并研究VMA蛋白的功能。 我们 将集中在VMA 6，VMA 21和VMA 22基因及其编码 蛋白质，因为这些因素是绝对需要的组装 的膜和外周部门的酵母液泡H+- ATP酶复合体 最后一个具体目标（#5）的重点是调查 酵母液泡H+-ATP酶的生物合成、组装和靶向 膜复合体 单个VMA蛋白在组装中的作用 液泡膜H ~+-ATP酶的膜区功能将被 评估。 研究的长期目标是用液泡H+- ATP酶是发展的途径的机械理解 组装这个非常复杂的膜蛋白。

The overall goal of this research is to develop a mechanistic understanding of membrane protein sorting and assembly in the yeast Saccharomyces cerevisiae. Recent results indicate that the vacuole is the default compartment for membrane proteins that enter the yeast secretory pathway. To explain these results the vacuolar default model has been proposed. This model states that membrane proteins of the ER and Golgi contain retention signals preventing their transport to the vacuole, whereas plasma membrane proteins have positive sorting information, which ensures that they are not transported to the vacuole. The retention signal on the yeast Golgi membrane protein DPAP A has been characterized, and this information will be used to design genetic screens to identify components of the yeast Golgi retention apparatus. Specific aims #1 and 2 are focused on the identification of components required for retention of membrane proteins in the yeast Golgi, and the molecular genetic and biochemical characterization of these components. Yeast mutants will be isolated that fail to retain a Golgi membrane protein, and the GAL4 two-hybrid genetic system will be used to identify genes encoding proteins that directly interact with the DPAP A Golgi retention signal. The genes identified will be sequenced, disruption alleles constructed to test the in vivo function of the components, and the Golgi retention machinery biochemically characterized. Specific aim #3 will test the second part of the vacuolar default model by investigating the nature of the targeting information that directs the FUS1-encoded plasma membrane protein to the yeast cell surface. Gene fusions between the FUS1 gene and the gene encoding the yeast Golgi membrane protein, KEX1, will be constructed to map any region of the FUS1 protein capable of redirecting KEX1 protein lacking its Golgi retention signal to the yeast plasma membrane. A second focus of this proposal is the mechanistic analysis of the assembly and targeting of a multisubunit membrane protein complex, the yeast vacuolar membrane H+-ATPase. With a majority of the vacuolar H+- ATPase subunit genes cloned, the tools are now available to begin to address issues of the biosynthesis of this complex vacuolar membrane protein. Specific aim #4 is designed to isolate and characterize vacuolar H+-ATPase deficient yeast mutants (vma), clone and characterize the VMA genes, and to investigate the function of the VMA proteins. We will focus on the VMA6, VMA21, and VMA22 genes and their encoded proteins, since these factors are absolutely required for the assembly of both the membrane and peripheral sectors of the yeast vacuolar H+- ATPase complex. The final specific aim (#5) is focused on investigating the biosynthesis, assembly, and targeting of the yeast vacuolar H+-ATPase membrane complex. The roles of individual VMA proteins in the assembly and function of the membrane sector of the vacuolar H+-ATPase will be assessed. The long-term objective of the studies with the vacuolar H+- ATPase is to develop a mechanistic understanding of the pathway of assembly for this very complex membrane protein.