MOLECULAR ANALYSIS OF SECRETORY PROTEIN TRANSLOCATION

蛋白质穿过粗糙细胞膜的转移 内质网（ER）是分泌的第一步。 先前 在这个实验室和其他实验室的工作已经确定了一些 细胞质和膜活动，蛋白质和基因参与 在易位。 拟议的研究将集中在生物化学 旨在获得更完整和统一的研究 了解前蛋白通过ER膜的通道， 依靠从纯化的蛋白质中 蛋白质和脂质。 系统将继续发酵， 分子遗传学提供了生物化学 结果 最初的努力将集中在识别蛋白质， 哪些活动已经确定。 其中包括：（1）饱和 前蛋白结合的膜在体外观察到的情况下， ATP（前蛋白受体）;和2）ATP水解的需要 在膜内（易位ATP酶）。 SEC 61 -63基因的产物，以及 ATP酶、前蛋白受体和酵母对接蛋白将被 用于识别最少数量的组件 在重组系统中实现易位所必需的。 本 最后，将使用新的方法，其目标是确定参与者 在易位反应中，并同时产生 有效重构所需的化学量， 生化和遗传分析。 通过添加亲和标签，重组 前蛋白和易位相关的膜蛋白，将被使用 作为“鱼钩”，在体外从酵母中回收功能复合物 系统和完整的细胞。 这些复合物将被测试其 重建易位的能力，或它的一个子反应， 脂质体，然后进行生物化学分解。 发挥的作用 然后将通过从细胞中耗尽它们来测试单个蛋白质。 用于制备活性脂质体的提取物， 它们的基因在体内的表达。 同样的亲和标记 前蛋白将在酵母裂解物中，以化学量， 分离未知的胞质蛋白，这些蛋白参与了后 酵母中的翻译易位。 目前对介导核糖体结合的蛋白质的研究将是 继续，重点是了解这些蛋白质的作用， (and核糖体结合）。 到 实现这一点，假定的核糖体受体将被耗尽， 膜蛋白的提取物， 脂质体中的易位。 这将有助于阐明 一个给定的蛋白质在核糖体结合，但更重要的是， 易位 核糖体结合也将在酵母中进行研究， 它在易位中的作用可以通过遗传手段来分析。

The translocation of proteins across the membrane of the rough endoplasmic reticulum (ER) is the first step in secretion. Previous work in this and other laboratories has identified a number of cytosolic and membrane activities, proteins, and genes that participate in translocation. The proposed research will focus on biochemical studies that are designed to gain a more complete and unified understanding of the passage of a preprotein through the ER membrane, relying on the ability to reconstitute the process from purified proteins and lipids. The system will continue to be yeast, where molecular genetics provides in vivo verification of biochemical results. Initial efforts will concentrate on identifying proteins for which activities have been defined. These include: 1) the saturable preprotein binding to the membrane observed in vitro in the absence of ATP (preprotein receptor); and 2) the requirement for ATP hydrolysis within the membrane (translocation ATPase). The products of the SEC61-63 genes, as well as the translocation ATPase, the preprotein receptor and the yeast docking protein will be employed in the identification of the minimum number of components necessary to achieve translocation in a reconstituted system. To this end, new approaches will be used whose goal is to identify participants in the translocation reaction, and to simultaneously produce the chemical amounts needed for effective reconstitution and further biochemical and genetic analysis. By adding affinity-tags, recombinant preproteins and translocation-relevant membrane proteins, will be used as "fishhooks" to recover functional complexes from the yeast in vitro system and from intact cells. These complexes will be tested for their ability to reconstitute translocation, or one of its subreactions, in liposomes prior to being dissected biochemically. The roles played by individual proteins will then be tested by depleting them from the extracts used to prepare active liposomes, and by manipulating expression of their genes in vivo. The same affinity-tagged preproteins will be translated in yeast lysates, in chemical amounts, to isolate the unknown cytosolic proteins that are involved in post- translational translocation in yeast. Current studies on proteins that mediate ribosome binding will be continued, with an emphasis on understanding the role of such proteins (and ribosome binding in general) in the translocation process. To achieve this, putative ribosome receptors will be depleted from extracts of membrane proteins that are used to reconstitute translocation in liposomes. This will serve to elucidate the role of a given protein in ribosome binding, but more importantly, in translocation. Ribosome binding will also be investigated in yeast so that its role in translocation can be analyzed by genetic means.