Mechanisms of COPII-Dependent Quality Control and ER Export

依赖 COPII 的质量控制和 ER 导出机制

基本信息

批准号：
10318982
负责人：
JONATHAN D GOLDBERG
金额：
$ 49.6万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-02-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318982
关键词：
Address Affect Architecture Basic Science Binding COPII-Coated Vesicles Capsid Proteins Cell physiology Cells Complex Consensus Crowding Crystallization Dependence Disease Endoplasmic Reticulum Eukaryotic Cell Exclusion Extravasation Functional disorder Goals Golgi Apparatus Human Integral Membrane Protein KDEL receptor Measures Mediating Minority Modeling Molecular Molecular Chaperones Oxidation-Reduction Pathway interactions Phase Play Process Protein Array Protein Family Protein Sortings Proteins Quality Control Readiness Reporting Research Residencies Retrieval Role Series Signal Transduction Site Sorting - Cell Movement Structural Models Structure System Testing Vesicle Work attenuation cell growth driving force experimental analysis experimental study macromolecule misfolded protein novel programs protein transport proteostasis proteotoxicity public health relevance receptor reconstitution segregation small molecule synergism theories tool trafficking uptake vesicle transport

项目摘要

Mechanisms of COPII-Dependent Quality Control and ER Export The sorting and transport of biosynthetic cargo from the endoplasmic reticulum (ER) is central to eukaryotic cell physiology. This proposal is focused on the conserved mechanisms and machinery molecules, in particular the COPII vesicular coat proteins, responsible for the controlled export of thousands of distinct cargo proteins from the ER. Newly synthesized proteins are subject to quality control (QC) interrogations that may involve repeated attempts at chaperone-mediated folding, or that target aberrantly folded forms for destruction by ER- associated degradation (ERAD) (1). It is also the case that QC decisions are made at ER exit sites, as the COPII coat and associated machinery proteins actively exclude misfolded cargo from vesicles (2, 3). The mechanisms that control segregation of folded cargo from ER chaperones and misfolded proteins, and their contribution to ER quality control, are not well understood. This research will address central questions of COPII trafficking relevant to cell physiology and pathophysiology. The specific aims of the proposal are to: (Aim 1) carry out a structure-function mapping of COPII·machinery interactions, with which to construct a pseudo- atomic model of the COPII vesicle interior. Building on prior work we will complete the mapping for all 12 major transmembrane protein constituents of COPII vesicles (22). We will identify ER export motifs on the circulating ER-Golgi receptor proteins Erv41/Erv46, Rer1 and Erv29, and determine their crystal structures in complex with COPII protein. Functional relevance will be tested definitively in COPII budding reconstitutions and whole cell experiments; (Aim 2) investigate the mechanism of COPII protein sorting that imposes ER retention, and explore its contribution to QC. We will test the complex interplay of cargo, chaperones and COPII-associated machinery that underlies ER retention (2), exploiting a set of tools developed in the initial phase of research— in particular, a novel series of small molecules that bind COPII protein to reduce the stringency of ER retention. We will investigate the capacity of the retention and retrieval systems, the breakdown of retention in UPR- activated cells, and we will test our working model that chaperone complexes are excluded from vesicles because they are too large to partition into the interstices of a COPII-associated luminal array; and (Aim 3) develop the new idea of COPII cargo uptake by enhanced partitioning as an alternative to the bulk flow model for ER exit. The ER-to-Golgi transport step is essential for eukaryotic cell growth and function, thus the proposed studies have considerable relevance to human cell physiology and to understanding diseases of protein mistrafficking and proteotoxicity.

依赖 COPII 的质量控制和 ER 导出机制内质网 (ER) 生物合成物质的分类和运输是真核生物的核心该提案的重点是保守机制和机器分子，特别是。 COPII 囊泡外壳蛋白，负责数千种不同货物蛋白的受控输出来自 ER 的新合成蛋白质需要接受质量控制 (QC) 审查，其中可能涉及反复尝试伴侣介导的折叠，或针对异常折叠形式以通过 ER- 进行破坏相关降解 (ERAD) (1) QC 决策也是在 ER 出口站点做出的。 COPII 外壳和相关机械蛋白主动排除囊泡中错误折叠的货物 (2, 3)。控制折叠货物与内质网伴侣和错误折叠蛋白质分离的机制及其对 ER 质量控制的贡献尚未得到充分理解。这项研究将解决以下核心问题。与细胞生理学和病理生理学相关的 COPII 贩运该提案的具体目标是：（目的） 1）进行COPII·机器交互的结构-功能映射，以此构建伪 COPII 囊泡内部的原子模型在之前的工作的基础上，我们将完成所有 12 个主要的映射。 COPII 囊泡的跨膜蛋白成分 (22) 我们将鉴定循环中的 ER 输出基序。 ER-高尔基体受体蛋白 Erv41/Erv46、Rer1 和 Erv29，并确定其复杂的晶体结构与 COPII 蛋白的功能相关性将在 COPII 出芽重建和整体中进行明确测试。细胞实验；（目标 2）研究 COPII 蛋白质分选导致 ER 保留的机制，以及探索其对 QC 的贡献。我们将测试货物、伴侣和 COPII 相关的复杂相互作用。 ER 保留机制 (2)，利用在研究初始阶段开发的一套工具—— 特别是一系列新型小分子，可结合 COPII 蛋白以降低 ER 保留的严格性。我们将调查保留和检索系统的能力、UPR 保留的分解情况激活的细胞，我们将测试我们的工作模型，伴侣复合物被排除在囊泡之外因为它们太大而无法划分到 COPII 相关的管腔阵列的间隙中；并且（目标 3）通过增强分区开发 COPII 货物吸收的新思路，作为散装流模型的替代方案 ER 到高尔基体的转运步骤对于真核细胞的生长和功能至关重要，因此拟议的研究与人类细胞生理学和了解疾病具有相当大的相关性蛋白质错误运输和蛋白质毒性。