Protein translocation across the endoplasmic reticulum

跨内质网的蛋白质易位

基本信息

批准号：
8112657
负责人：
JAMES REID GILMORE
金额：
$ 35.44万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-04-01 至 2014-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8112657
关键词：
Address Amino Acids Anabolism Animal Model Autosomal Dominant Polycystic Kidney Binding Binding Sites Biochemical Biological Biological Assay Cells Collaborations Complex Conserved Sequence Cryoelectron Microscopy Defect Disease Endoplasmic Reticulum Event Genes Genetic Health Human Hydrogen Bonding In Vitro Integral Membrane Protein Kinetics Lateral Link Location Malignant neoplasm of prostate Mediating Membrane Membrane Proteins Methanococcus Methods Modeling Modification Molecular Molecular Conformation Movement Mutagenesis Mutation Organism Pathology Pathway interactions Peptide Signal Sequences Physiological Protein Biosynthesis Protein translocation Proteins Quality Control Reaction Research Resolution Ribosomes Role Rough endoplasmic reticulum Saccharomycetales Signal Recognition Particle Site Structure Testing Work Yeasts base human disease in vitro Assay in vivo insight lysosomal proteins mutant novel overexpression polypeptide protein folding public health relevance rough endoplasmic reticulum membrane secretory protein signal recognition particle receptor signal sequence receptor yeast protein

项目摘要

DESCRIPTION (provided by applicant): The research described in this proposal is directed towards elucidating the molecular mechanism by which nascent polypeptides are translocated across and integrated into the rough endoplasmic reticulum membrane. Particular emphasis will be placed upon (a) analyzing structural domains in the Sec61 complex that are required for cotranslational gating of the translocation channel by a ribosome-nascent chain (RNC) complex, (b) obtaining additional structures of yeast protein translocation channels by cryoelectron microscopy, and (c) analyzing the role of the Sec62/Sec63 complex in the integration of multi- spanning membrane proteins. The structure of the Methanococcus jannaschii SecYE2 complex provides an excellent model for the closed conformation of a eukaryotic protein translocation channel. Amino acid residues in Sec61p that are important for the cotranslational translocation pathway will be identified by structure-guided mutagenesis of RNC-contact sites and cytoplasmically exposed Sec61 segments. The role of contact sites in RNC-mediated channel gating will be evaluated using a combination of cell biological and biochemical assays. Insertion of a nascent polypeptide into the signal sequence-binding site of the Sec61 complex is thought to require partial separation of the lateral gate of the translocation channel. The effect of Sec61 lateral gate mutations on the in vivo kinetics and fidelity of translocation channel gating will be analyzed. Cotranslational and posttranslational protein translocation channels will be isolated from wild type and mutant cells for cryoelectron microscopy. RNC-channel complexes will be prepared to obtain mid-resolution structures of translocation intermediates. Although the Sec62/Sec63 complex has a well-described role as a signal sequence receptor for yeast proteins that are translocated by a posttranslational pathway, there is a growing body of evidence that suggests that the Sec62/Sec63 complex may enhance the fidelity of membrane protein integration. Yeast strains that have mutations in the subunits of the Sec62/Sec63 complex will be assayed for the ability to integrate single-spanning and multi-spanning membrane proteins. These studies may provide insight into the in vivo role of the Sec62/Sec63 complex in mammalian organisms. 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. PUBLIC HEALTH RELEVANCE: The biosynthesis of secretory proteins, lysosomal proteins and integral membrane proteins is of fundamental importance for human health. Recent genetic evidence shows that mutations in the human Sec63 gene can cause autosomal dominant polycystic kidney disease, while overexpression of human Sec62 may be linked to prostate cancer. Defects in the biosynthesis, modification and folding of proteins in the rough endoplasmic reticulum is responsible for a growing number of pathologies that are collectively referred to as "ER quality control" diseases. Our studies, which are directed towards understanding the molecular mechanism of protein translocation and membrane protein integration, will provide insight into these crucial events in protein biosynthesis.

描述（由申请人提供）：本提案中描述的研究旨在阐明新生多肽的分子机制，并将其整合到粗糙的内质网膜中。将特别强调（a）分析Sec61复合物中的结构结构域，这是通过核糖体 - 脱粒链（RNC）复合物对转运通道进行旋转通道所需的，（b）获得酵母蛋白易位通道的其他结构，通过冷冻蛋白质易位频率启动，以及（c）分析Sec62/sec62/septim 662/c复杂的作用。蛋白质。 Jannaschii secye2复合物的甲烷球菌的结构为真核蛋白易位通道的闭合构型提供了极好的模型。 SEC61P中对共转运易位途径很重要的氨基酸残基将通过RNC接触位点的结构引导诱变和细胞质暴露于SEC61节段来识别。接触位点在RNC介导的通道门控的作用将使用细胞生物学和生化测定的组合进行评估。将新生多肽插入SEC61复合物的信号序列结合位点被认为需要部分分离易位通道的横向门。 SEC61侧栅突变对体内动力学的影响和易位通道门控的保真度。从野生型和突变细胞中分离出旋转和翻译后蛋白易位通道以进行冷冻电子显微镜。 RNC通道复合物将准备好获得易位中间体的中分辨率结构。尽管SEC62/SEC63复合物作为酵母蛋白的信号序列受体的作用很好，而酵母菌蛋白是由翻译后途径易位的，但越来越多的证据表明SEC62/SEC63复合物可以增强膜蛋白整合的忠诚度。在SEC62/SEC63复合物亚基中具有突变的酵母菌菌株将被测定，以便整合单跨度和多跨膜膜蛋白的能力。这些研究可能会洞悉Sec62/Sec63复合物在哺乳动物生物中的体内作用。上面概述的三个研究目标涉及蛋白质易位反应的方面知之甚少。整合性膜蛋白，分泌蛋白和溶酶体蛋白的准确有效生物合成是人类细胞以及像萌芽酵母这样的简单模型生物中的重要功能。粗糙内质网中蛋白质修饰或折叠的缺陷是导致越来越多的人类疾病列表，这些疾病被称为“ ER质量控制”疾病。公共卫生相关性：分泌蛋白，溶酶体蛋白和整体膜蛋白的生物合成对人类健康至关重要。最近的遗传证据表明，人类SEC63基因中的突变会引起常染色体显性肿瘤肾脏疾病，而人类SEC62的过表达可能与前列腺癌有关。粗糙内质网中蛋白质的生物合成，修饰和折叠的缺陷负责越来越多的病理，这些病理被共同称为“ ER质量控制”疾病。我们的研究致力于理解蛋白质易位和膜蛋白整合的分子机制，将为蛋白质生物合成中的这些关键事件提供洞察力。