MOLECULAR ANALYSIS OF SECRETORY PROTEIN TRANSLOCATION

分泌蛋白易位的分子分析

• 批准号: 2179386
• 负责人: DAVID I MEYER
• 金额: $ 32.64万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-09-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179386
• 关键词: affinity chromatography; binding proteins; cell free system; density gradient ultracentrifugation; endoplasmic reticulum; fungal genetics; gel filtration chromatography; gene expression; genetic transcription; immunologic techniques; ion exchange chromatography; laboratory mouse; laboratory rabbit; liposomes; membrane permeability; membrane proteins; molecular chaperones; molecular cloning; nucleic acid hybridization; posttranslational modifications; protein reconstitution; protein signal sequence; protein transport; secretory protein; yeasts

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.

蛋白质在粗糙的膜上的易位 内质网（ER）是分泌的第一步。 以前的 在此和其他实验室的工作已经确定了许多 参与的胞质和膜活性，蛋白质和基因 在易位。 拟议的研究将着重于生化 旨在获得更完整和统一的研究 理解前蛋白通过ER膜的通过， 依靠从纯化的能力重新构成该过程的能力 蛋白质和脂质。 该系统将继续是酵母 分子遗传学提供生化的体内验证 结果。 最初的努力将集中于确定蛋白质 定义了哪些活动。 其中包括：1）可饱和 在不存在的情况下，在体外观察到的膜蛋白结合 ATP（预蛋白受体）； 2）ATP水解的要求 在膜内（易位ATPase）。 Sec61-63基因的产物以及易位 ATPase，预蛋白受体和酵母对接蛋白将是 用于识别最小数量的组件数量 在重组系统中实现易位所必需的。 对此 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 有效重建所需的化学量和进一步 生化和遗传分析。 通过添加亲和力标签，重组 将使用前蛋白质和与易位相关的膜蛋白 作为“鱼钩”，可以从体外从酵母中恢复功能复合物 系统和完整的细胞。 这些复合物将对他们的 能够重建易位或其子接种之一的能力 在生化之前解剖之前的脂质体。 扮演的角色 然后，单个蛋白质将通过将其从中耗尽的测试 用于制备活性脂质体的提取物，并通过操纵 其基因在体内的表达。 相同的亲和力标记 预蛋白将以化学量的酵母裂解物进行翻译， 分离出参与后的未知胞质蛋白 酵母中的翻译易位。 当前对介导核糖体结合的蛋白质的研究将是 继续，重点是理解此类蛋白质的作用 （通常是核糖体结合）在易位过程中。 到 实现这一目标，假定的核糖体受体将从 用于重新构建的膜蛋白的提取物 脂质体中的易位。 这将有助于阐明 核糖体结合中给定的蛋白质，但更重要的是， 易位。 还将在酵母中研究核糖体结合 可以通过遗传手段来分析其在易位中的作用。