Protein Transport Across Membranes

描述（由申请人提供）：本项目的目标是了解蛋白质如何跨膜转运的机制。先前的工作已经证明，分泌和膜蛋白从胞质溶胶跨膜通过由膜蛋白复合物形成的通道易位，所述膜蛋白复合物在真核生物中被称为Sec 61 p复合物，在细菌和古细菌中被称为SecY复合物。最近确定的古SecY复杂的X射线结构提供了本建议的基础。在真核生物中，有一个反向的易位途径，称为ERAD（ER相关降解），通过该途径，错误折叠的ER蛋白被蛋白酶体降解。我们最近的实验已经鉴定了大多数（如果不是全部的话）ERAD组分，这为机理研究铺平了道路。在这里，我们将解决易位的关键方面，特别强调以下问题：1。蛋白质传导通道是如何与核糖体结合的，它是如何被门控的？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如何结合核糖体。我们将研究通道是如何打开的，以及如何在蛋白质易位过程中维持小分子的膜屏障。2. SecY复合物寡聚体的作用是什么？SecA如何通过通道移动多肽？我们将阐明SecY复合物的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.是否有不同的ERAD途径？我们将通过筛选S中的底物来测试不同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否位于管腔、膜或胞质溶胶中。啤酒。我们将测试易位和多聚泛素化的时间顺序，并确定一种途径是否占主导地位。4. ERAD的分子机制是什么？我们将建立重现ERAD与纯化酵母组分的子反应的测定法，目的是开发一种模拟洗涤剂中逆易位的系统。ERAD组分中的突变体将用于阐明ERAD-L的机制，交联方法将用于鉴定可能形成反向易位通道的候选蛋白。蛋白质移位的机制具有重要的医学意义。已知大量疾病中突变导致ER中蛋白质的错误折叠及其随后在胞质溶胶中的降解。例子包括囊性纤维化和α 1-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.