Protein Transport Across Membranes

蛋白质跨膜运输

• 批准号: 7937178
• 负责人: Tom A Rapoport
• 金额: $ 4.14万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937178
• 关键词: Address; Austria; Bacteria; Binding; Biological Assay; Boston; Classification; Collaborations; Complex; Coupled; Cryoelectron Microscopy; Cystic Fibrosis; Cytosol; Detergents; Development; Disease; Disulfides; Endoplasmic Reticulum; Endoplasmic Reticulum Degradation Pathway; Eukaryota; Goals; Joint Dislocation; Lead; Lipid Bilayers; Location; Mass Spectrum Analysis; Mediating; Medical; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Movement; Mutation; Pathway interactions; Pharmaceutical Preparations; Process; Protein C Inhibitor; Protein translocation; Proteins; Reaction; Ribosomes; Roentgen Rays; Role; Saccharomyces cerevisiae; Screening procedure; Structure; System; Testing; Toxin; Ubiquitination; Universities; Virus; Work; Yeasts; base; crosslink; design; insight; medical schools; multicatalytic endopeptidase complex; mutant; novel strategies; particle; polypeptide; protein complex; protein misfolding; protein transport; research study; secretory protein; small molecule

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.

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。先前的研究表明，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跨膜转运，膜蛋白复合物在真核生物中称为 Sec61 p 复合物，在细菌和古细菌中称为 SecY 复合物。最近确定的古细菌 SecY 复合体的 X 射线结构为当前的提议提供了基础。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），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鉴定出大多数（如果不是全部）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，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门控的？我们将使用电子冷冻显微镜来阐明通道在共翻译易位过程中如何结合核糖体。我们将研究通道如何打开以及如何在蛋白质易位过程中维持小分子的膜屏障。 2. SecY复合寡聚体的作用是什么？SecA如何使多肽通过该通道？我们将阐明 SecY 复合物寡聚物在易位中的作用，研究 SecA 如何与 SecY 通道结合，并确定 SecA 如何通过该通道移动多肽链。 3. 是否存在不同的 ERAD 途径？我们将通过筛选酿酒酵母中的底物来测试不同 ERAD 途径（称为 ERAD-L、-M 和 -C）的存在，具体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聚泛素化的时间顺序，并确定一种途径是否比另一种途径占主导地位。 4. ERAD的分子机制是什么？我们将建立重现 ERAD 与纯化酵母成分的子反应的测定方法，目的是开发一个模拟洗涤剂中逆转录转位的系统。 ERAD组分中的突变体将用于阐明ERAD-L的机制，并且交联方法将用于鉴定可能形成逆向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。已知许多疾病的突变导致内质网中蛋白质的错误折叠以及随后在胞质溶胶中的降解。例子包括囊性纤维化和α1-抗胰蛋白酶缺乏症。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。