Protein Transport Across Membranes

描述（由申请人提供）：该项目的目标是了解蛋白质如何跨膜转运的机制。分泌蛋白和膜蛋白都从胞质溶胶通过由异源三聚体膜蛋白复合物（真核生物中的Sec 61 p复合物和细菌和古细菌中的SecY复合物）形成的通道跨膜转运。我们已经确定了单独的SecY复合物的X射线结构，以及与ATP酶SecA结合时的X射线结构，这带来了新的见解，并为本提案的一部分提供了基础。在真核生物中，有一个反向的易位途径，称为ERAD（ER相关降解），用于降解错误折叠的ER蛋白。我们已经确定了大多数，如果不是全部，ERAD中涉及的组件，为机制研究铺平了道路。在这里，我们将解决易位的关键方面，特别强调以下问题：1。蛋白质是如何协同转运的，在这个过程中小分子的膜屏障是如何维持的？基于一种新的在完整大肠杆菌中产生共翻译易位中间体的方法。大肠杆菌细胞和纯化核糖体/新生链/通道复合物的能力，我们将确定有多少拷贝的SecY易位所需的，并将使用电子显微镜来阐明活性通道如何结合到核糖体。我们将研究在转运过程中通道如何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。我们将解决的SecDFYajC复杂的未开发的作用，并测试其参与介导的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。基于初步结果表明泛素连接酶Hrd 1 p的关键作用，我们将纯化蛋白质，并将其与其伴侣蛋白一起重组。我们将开发一个纯化的组分系统，重现子反应，甚至整个ERAD-L过程。 公共卫生相关性：蛋白质易位的机制具有重要的医学意义。抑制信号序列结合的药物可用于慢性炎性疾病的治疗干预。许多疾病，包括囊性纤维化和α 1-抗胰蛋白酶缺乏症，是由导致ER蛋白错误折叠及其随后在胞质溶胶中降解的突变引起的。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. 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. PUBLIC HEALTH RELEVANCE: The mechanism of protein translocation is of great medical importance. Drugs that inhibit signal sequence binding can be used for therapeutic intervention in chronic inflammatory diseases. A large number of diseases, including cystic fibrosis and a1-antitrypsin deficiency, are caused by mutations that result in the misfolding of ER proteins and their subsequent degradation in the cytosol. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference.