Protein Transport Across Membranes

描述（由申请人提供）：这个项目的目标是从机械角度理解蛋白质是如何跨膜运输的。分泌蛋白和膜蛋白都是通过异三聚体膜蛋白复合物（真核生物中的Sec61p复合物和细菌和古细菌中的SecY复合物）形成的通道从细胞质溶胶转移到膜上的。我们已经确定了SecY复合物单独和与atp酶SecA相关时的x射线结构，这导致了新的见解，并为本提案的部分内容提供了基础。在真核生物中，存在一种反向易位途径，称为ERAD （ER相关降解），用于降解错误折叠的ER蛋白。我们已经确定了ERAD中涉及的大部分（如果不是全部的话）组成部分，为机制研究铺平了道路。在这里，我们将讨论易位的关键方面，并特别强调以下问题：蛋白质是如何协同翻译易位的？在这个过程中，小分子的膜屏障是如何维持的？基于在完整大肠杆菌细胞中生成共翻译易位中间体的新方法以及纯化核糖体/新生链/通道复合物的能力，我们将确定易位需要多少份SecY拷贝，并将使用电子显微镜阐明活性通道如何与核糖体结合。我们将研究通道如何在转运过程中维持小分子的膜屏障。2. 细菌翻译后易位的机制是什么？我们将阐明SecA通过通道移动多肽的机制。我们将探讨SecDFYajC复合体的未知作用，并测试其参与介导膜电位对易位的影响。3. ERAD的分子机制是什么？我们将探索一个腔内ERAD （ERAD- l）底物的路径，并确定它是如何被识别的。基于初步结果表明泛素连接酶Hrd1p的关键作用，我们将纯化该蛋白，并将其与伴侣蛋白一起重组。我们将开发一个纯化的组分系统，重述子反应甚至整个拉德- l过程。

DESCRIPTION (provided by applicant): The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Both secretory and membrane proteins are translocated from the cytosol across the membrane through a channel that is formed from a heterotrimeric membrane protein complex, the Sec61p complex in eukaryotes and the SecY complex in bacteria and archae. We have determined X-ray structures of the SecY complex alone and when associated with the ATPase SecA, which have led to new insights and provide the basis for part of the present proposal. In eukaryotes, there is a translocation pathway in the reverse direction, called ERAD (for ER associated degradation), which is used to degrade misfolded ER proteins. We have identified most, if not all, components involved in ERAD, paving the way for mechanistic studies. Here, we will address key aspects of translocation with specific emphasis on the following questions: 1. How are proteins cotranslationally translocated and how is the membrane barrier for small molecules maintained during the process? Based on a new method to generate cotranslational translocation intermediates in intact E. coli cells and the ability to purify ribosome/nascent chain/channel complexes, we will determine how many copies of SecY are required for translocation and will use electron microscopy to elucidate how the active channel binds to the ribosome. We will investigate how the channel maintains the membrane barrier for small molecules during translocation. 2. What is the mechanism of posttranslational translocation in bacteria? We will clarify the mechanism by which SecA moves polypeptides through the channel. We will address the unexplored role of the SecDFYajC complex and test its involvement in mediating the effect of a membrane potential on translocation. 3. What is the molecular mechanism of ERAD? We will probe the path of a luminal ERAD (ERAD-L) substrate and determine how it is recognized. Based on preliminary results that indicate a crucial role for the ubiquitin ligase Hrd1p, we will purify the protein, and reconstitute it together with its partner proteins. We will develop a purified component system that recapitulates subreactions or even the entire ERAD-L process.