Protein Transport Across Membranes

描述(申请人提供)：这个项目的目标是从机械的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一个通道从细胞质中跨膜转运的，该通道由异源三聚体膜蛋白复合体、真核生物中的Sec61p复合体和细菌和古生物中的SecY复合体组成。我们单独确定了SecY复合体的X射线结构，并确定了与ATPase SecA相关的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，有一条反向易位途径，称为ERAD(内质网相关降解)，用于降解错误折叠的内质网蛋白。我们已经确定了ERAD中涉及的大部分(如果不是全部)组成部分，为机械论研究铺平了道路。在这里，我们将讨论易位的关键方面，并特别强调以下问题：1.蛋白质是如何共翻译易位的，在这个过程中小分子的膜屏障是如何维持的？基于一种在完整的大肠杆菌细胞中产生共翻译易位中间体的新方法和纯化核糖体/新生链/通道复合体的能力，我们将确定转位需要多少拷贝的SecY，并将使用电子显微镜来阐明活性通道如何与核糖体结合。我们将研究该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通过该通道运输多肽的机制。我们将讨论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？我们将探索管腔ERAD(ERAD-L)底物的路径，并确定它是如何被识别的。根据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，并将其与其伙伴蛋白一起重组。我们将开发一个纯化的组分系统，它概括了子反应甚至整个ERAD-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.