Protein Transport Across Membranes

蛋白质跨膜运输

• 批准号: 8258798
• 负责人: Tom A Rapoport
• 金额: $ 49.73万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8258798
• 关键词: ATP phosphohydrolase; Address; Amino Acids; Austria; Back; Bacteria; Binding; Biochemical; Biological Assay; Boston; Cells; Chronic; Collaborations; Complex; Couples; Cryoelectron Microscopy; Cysteine; Cystic Fibrosis; Cytosol; Development; Disease; Electron Microscopy; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Escherichia coli; Eukaryota; Fingers; Goals; Inflammatory; Lead; Lipid Bilayers; Mediating; Medical; Membrane; Membrane Potentials; Membrane Proteins; Methods; Modification; Molecular; Molecular Chaperones; Mutation; Pathway interactions; Peptide Signal Sequences; Peptides; Pharmaceutical Preparations; Process; Protein C Inhibitor; Protein translocation; Proteins; Protons; Reagent; Ribosomes; Roentgen Rays; Role; Saccharomyces cerevisiae; Site; Slide; Stretching; Structure; System; Testing; Therapeutic Intervention; Toxin; Translating; Universities; Virus; Work; base; crosslink; design; in vivo; insight; mutant; particle; periplasm; polypeptide; protein complex; protein misfolding; protein transport; public health relevance; reconstitution; research study; secretory protein; single-molecule FRET; small molecule; ubiquitin ligase

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.

描述（由申请人提供）：该项目的目标是从机械角度理解蛋白质如何跨膜运输。分泌蛋白和膜蛋白均通过异三聚体膜蛋白复合物（真核生物中的 Sec61p 复合物以及细菌和古细菌中的 SecY 复合物）形成的通道从细胞质跨膜转运。我们已经确定了 SecY 复合物单独以及与 ATPase SecA 结合时的 X 射线结构，这带来了新的见解，并为当前提案的一部分提供了基础。在真核生物中，存在一条相反方向的易位途径，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 ERAD 中涉及的大多数（如果不是全部）成分，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，特别强调以下问题： 1. 蛋白质如何共翻译易位以及在此过程中如何维持小分子的膜屏障？基于在完整大肠杆菌细胞中生成共翻译易位中间体的新方法以及纯化核糖体/新生链/通道复合物的能力，我们将确定易位需要多少个 SecY 拷贝，并将使用电子显微镜来阐明活性通道如何与核糖体结合。我们将研究通道如何在易位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 通过该通道移动多肽的机制。我们将探讨 SecDFYajC 复合物尚未探索的作用，并测试其在介导膜电位对易位的影响中的参与。 3. ERAD的分子机制是什么？我们将探测管腔 ERAD (ERAD-L) 底物的路径并确定它是如何被识别的。基于表明泛素连接酶 Hrd1p 至关重要的作用的初步结果，我们将纯化该蛋白质，并将其与其伙伴蛋白质一起重建。我们将开发一个纯化的组分系统，概括子反应甚至整个 ERAD-L 过程。 公共健康相关性：蛋白质易位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症疾病的治疗干预。许多疾病，包括囊性纤维化和α1-抗胰蛋白酶缺乏症，都是由导致 ER 蛋白错误折叠及其随后在细胞质中降解的突变引起的。该通路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。