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。先前的工作已经证明，分泌蛋白和膜蛋白通过膜蛋白复合物形成的通道从细胞质转运穿过膜，真核生物称为Sec61 p复合物，细菌和古细菌称为SecY复合物。最近确定的一个古细菌SecY复合物的x射线结构为本建议提供了基础。在真核生物中，存在一种反向易位途径，称为ERAD（内质网相关降解），通过该途径，错误折叠的内质网蛋白被蛋白酶体降解。我们最近的实验已经确定了大多数（如果不是全部的话）ERAD成分，这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：蛋白质传导通道是如何与核糖体结合的，又是如何进行门控的？我们将使用电子冷冻显微镜来阐明在共翻译易位过程中通道是如何与核糖体结合的。我们将研究通道是如何打开的，以及在蛋白质易位过程中如何维持小分子的膜屏障。2. SecA复合物低聚物的作用是什么？ SecA如何通过通道移动多肽？我们将阐明SecY复合物低聚物在易位中的作用，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有不同的途径吗？通过筛选酿酒酵母中的底物，我们将测试不同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否位于管腔，膜或细胞质中。我们将测试易位和多泛素化的时间顺序，并确定一种途径是否优于另一种途径。4. ERAD的分子机制是什么？我们将建立实验，概括ERAD与纯化酵母成分的亚反应，目的是开发一个系统，模仿洗涤剂中的逆转录。ERAD组分的突变体将被用来阐明ERAD- l的机制，并将使用交联方法来鉴定可能形成逆转录易位通道的候选蛋白。蛋白质易位的机制具有重要的医学意义。已知的大量疾病中，突变导致内质网中蛋白质的错误折叠及其随后在细胞质中降解。例子包括囊性纤维化和a1-抗胰蛋白酶缺乏症。这条通路也会被某些病毒和毒素劫持，更好的理解可能会导致新的药物允许干扰。

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.