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。以前的工作已经证明，分泌和膜蛋白是通过由膜蛋白复合体形成的通道从细胞质跨膜转运的，在真核生物中称为Sec61p复合体，在细菌和古生物中称为SecY复合体。最近确定的古细菌SecY复合体的X射线结构为本提议提供了基础。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)，错误折叠的ER蛋白通过该途径被蛋白酶体降解。我们最近的实验已经确定了ERAD的大部分组件，如果不是全部的话，这为机械学研究铺平了道路。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.蛋白质传导通道如何与核糖体结合，以及它是如何被门控的？我们将使用电子冷冻显微镜来阐明通道如何在共翻译易位过程中与核糖体结合。我们将研究该通道是如何打开的，以及在蛋白质转运过程中小分子的膜屏障是如何保持的。2.SecY复合寡聚体的作用是什么？SecA是如何通过通道转运多肽的？我们将阐明SecY复合体的寡聚体在易位中的作用，研究SecA如何与SecY通道结合，并确定SecA如何通过通道移动多肽链。3.ERAD是否有不同的途径？我们将通过在酿酒酵母中筛选底物来测试不同ERAD途径(称为ERAD-L、-M和-C)的存在，这取决于蛋白质的错误折叠结构域是位于管腔、膜还是胞浆中。我们将测试易位和多泛素化的时间顺序，并确定一条途径是否主导于另一条途径。4.ERAD的分子机制是什么？我们将建立分析方法，概括ERAD与纯化酵母成分的亚反应，目标是开发一种模拟洗涤剂中逆转录转位的系统。我们将利用ERAD组分中的突变来阐明ERAD-L的机制，并将使用交联方法来鉴定可能形成逆转易位通道的候选蛋白质。蛋白质易位的机制具有重要的医学意义。已知的许多疾病中，突变会导致内质网中蛋白质的错误折叠，并随后在胞浆中降解。例如囊性纤维化和A1-抗胰蛋白酶缺乏症。该途径也被某些病毒和毒素劫持，更好的理解可能会导致允许干扰的新药出现。

DESCRIPTION (provided by applicant): The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. Previous work has demonstrated that secretory and membrane proteins are translocated from the cytosol across the membrane through a channel formed from a membrane protein complex, called the Sec61 p complex in eukaryotes and the SecY complex in bacteria and archae. The recently determined the Xray structure of an archaeal SecY complex provides the basis for the present proposal. In eukaryotes, there is a translocation pathway in the reverse direction, called ERAD (for ER associated degradation), by which misfolded ER proteins are degraded by the proteasome. Our recent experiments have led to the identification of most, if not all, ERAD components, which pave the way for mechanistic studies. Here, we will address key aspects of translocation with specific emphasis on the following questions: 1. How does the protein-conducting channel bind to the ribosome and how is it gated? We will use electron cryo-microscopy to elucidate how the channel binds the ribosome during co- translational translocation. We will investigate how the channel is opened and how the membrane barrier for small molecules is maintained during protein translocation. 2. What is the role of SecY complex oligomers and how does SecA move polypeptides through the channel? We will clarify the role of oligomers of SecY complexes in translocation, investigate how SecA binds to the SecY channel, and determine how SecA moves polypeptide chains through the channel. 3. Are there different ERAD pathways? We will test the existence of different ERAD pathways (called ERAD-L, -M, and -C), depending on whether the misfolded domain of a protein is located in the lumen, membrane, or cytosol, by screening for substrates in S. cerevisiae. We will test the temporal order of translocation and poly-ubiquitination, and determine whether one pathway is dominant over another. 4. What is the molecular mechanism of ERAD? We will establish assays that recapitulate sub-reactions of ERAD with purified yeast components with the goal to develop a system that mimics retro-translocation in detergent. Mutants in ERAD components will be employed to elucidate the mechanism of ERAD-L, and crosslinking methods will be used to identify candidate proteins that may form the retro-translocation channel. The mechanism of protein translocation is of great medical significance. A large number of diseases are known in which mutations cause the misfolding of proteins in the ER and their subsequent degradation in the cytosol. Examples include cystic fibrosis and a1-antitrypsin deficiency. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.