Protein translocation across the endoplasmic reticulum

跨内质网的蛋白质易位

• 批准号: 8017638
• 负责人: JAMES REID GILMORE
• 金额: $ 23.82万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-26 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8017638
• 关键词: Address; Anabolism; Animal Model; Binding; Binding Sites; Biochemical; Biological Assay; Cell Membrane Permeability; Cells; Complex; Consensus; Cryoelectron Microscopy; Cytosol; Defect; Disease; Docking; Endoplasmic Reticulum; Enzymes; Event; Funding; Genetic; Growth; Human; Integral Membrane Protein; Kinetics; Lateral; Link; Membrane; Membrane Proteins; Membrane Transport Proteins; Methods; Modeling; Modification; Molecular; Molecular Genetics; Movement; Organism; Pathway interactions; Post-Translational Protein Processing; Protein Biosynthesis; Protein translocation; Proteins; Quality Control; Reaction; Relative (related person); Reporter; Research; Research Personnel; Ribosomal RNA; Ribosomes; Roentgen Rays; Role; Rough endoplasmic reticulum; Saccharomyces cerevisiae; Saccharomycetales; Series; Signal Recognition Particle; Site; Specific qualifier value; Structure; Testing; Ubiquitin; Yeasts; crosslink; design; dolichyl-diphosphooligosaccharide - protein glycotransferase; glycosylation; human disease; image reconstruction; in vivo; insight; lysosomal proteins; mutant; polypeptide; protein folding; research study; rough endoplasmic reticulum membrane; signal recognition particle receptor

The research described in this proposal is directed towards elucidating the molecular mechanism by which nascent polypeptides are translocated across on integrated into the rough endoplasmic reticulum (PER) membrane. Particular emphasis will be placed upon (a) analyzing the signal recognition particle (SRP) and SRP receptor-dependent steps in the translocation reaction that culminate with the selective attachment of a ribosome-nascent chain complex (RNC) to the translocation channel and (b) analyzing the function of the translocation channel during nascent polypeptide transport and membrane protein integration. One objective of the first specific aim is to characterize the interaction between the SRP receptor (SR) and the ribosome by indentifying the minimal SR domain that interacts with the ribosome. The binding site for the SR on the large ribosomal subunit will be determined using a combination of biochemical methods and cryoelectron microscopy. A second objective is to understand the role of cytoplasmic loops of Sec61p in the SRP- dependent cotranslational translocation pathway. The objective of the second specific aim is to investigate the in vivo kinetics of membrane protein integration in the yeast Saccharomyces cerevisiae using a series of ubiquitin-translocation assay (UTA) reporters. We will test the hypothesis that the lateral movement of a nascent transmembrane span within the protein translocation channel is a slow event relative to the protein synthesis elongation rate. These experiments should provide insight into the in vivo dynamics of the ribosome-channel junction. Several crucial questions about the mechanism of protein translocation were raised by the recently solved X-ray crystal structure of the M. jannaschii SecYEb complex. Yeast molecular genetics methods will be used to test current models of protein translocation by conducting a structure- function analysis of the auxiliary Sshlp translocation channel. The three research objectives outlined above address poorly understood aspects of the protein translocation reaction. The accurate and efficient biosynthesis of integral membrane proteins, secreted proteins and lysosomal proteins is an essential function in human cells, as well as in simple model organisms like budding yeast. Defects in the modification or folding of proteins in the rough endoplasmic reticulum are responsible for a growing list of human diseases that are termed "ER-quality control" diseases.

该提案中描述的研究旨在阐明分子机制 新生的多肽在整合到粗糙的内质网中被易位（per） 膜。 （a）分析信号识别粒子（SRP）和 易位反应中的SRP受体依赖性步骤，与A的选择性附着达到顶点 核糖体 - 脱链链复合物（RNC）到易位通道，（b）分析该功能 新生多肽转运和膜蛋白整合过程中的易位通道。一个目标 第一个具体目的是表征SRP受体（SR）与核糖体之间的相互作用 缩进与核糖体相互作用的最小SR结构域。大型SR的绑定位点 核糖体亚基将使用生化方法和冷冻电子的组合确定 显微镜。第二个目标是了解Sec61p的细胞质环在SRP-中的作用 依赖性共转运途径。第二个特定目的的目的是调查 使用一系列酿酒酵母中膜蛋白整合的体内动力学使用一系列 泛素转移测定法（UTA）记者。我们将检验以下假设 蛋白质易位通道内的新生跨膜跨度相对于蛋白质而言是一个缓慢的事件 合成伸长率。这些实验应提供对体内动力学的见解 核糖体通道交界处。关于蛋白质易位机制的几个关键问题是 由Jannaschii M. secyeb复合物的最近解决的X射线晶体结构提出。酵母分子 遗传学方法将用于通过进行结构 - 辅助SSHLP易位通道的功能分析。上面概述的三个研究目标 解决蛋白质易位反应的方面知之甚少。 整合膜蛋白，分泌蛋白和溶酶体的准确有效的生物合成 蛋白质是人类细胞以及简单模型生物（如萌芽酵母）的重要功能。 粗糙内质网中蛋白质修饰或折叠的缺陷是负责 越来越多地称为“ ER质量控制”疾病的人类疾病清单。