Mechanisms of COPII-Dependent Quality Control and ER Export

依赖COPII的质量控制和内质网输出机制 来自内质网(ER)的生物合成物质的分类和运输是真核生物的核心 细胞生理学。这一建议集中在保守的机制和机械分子，特别是 COPII泡囊外壳蛋白，负责数千种不同货物蛋白的受控出口 从急诊室。新合成的蛋白质受到质量控制(QC)的讯问，可能涉及 反复尝试伴侣介导的折叠，或以异常折叠形式为靶点，以供内质网破坏- 联合降级(ERAD)(1)。同样的情况是，QC决定是在ER出口地点做出的，因为 COPII外壳和相关的机械蛋白主动将错误折叠的货物从囊泡中排除(2，3)。这个 控制折叠货物与内质网伴侣蛋白和错误折叠蛋白分离的机制及其 对急诊室质量控制的贡献，还没有被很好地理解。这项研究将解决以下核心问题 COPII转运与细胞生理学和病理生理学有关。该提案的具体目的是：(目标 1)进行COPII·机械相互作用的结构-功能映射，并与之构建伪 COPII囊泡内部的原子模型。在之前工作的基础上，我们将完成所有12个主要项目的测绘 COPII囊泡的跨膜蛋白成分(22)。我们将在流通中识别ER出口主题 ER-Golgi受体蛋白Erv41/Erv46、Rer1和Erv29及其复合体的晶体结构 含有COPII蛋白。功能相关性将在COPII萌芽中的重组和整体 细胞实验；(目的2)研究COPII蛋白分选强制内质网滞留的机制，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 作为ER保留基础的机器(2)，利用在研究的初始阶段开发的一套工具- 特别是，一系列新的小分子结合COPII蛋白以降低内质网滞留的严谨性。 我们将调查保留和检索系统的能力，普遍定期审议中的保留细目-- 激活的细胞，我们将测试我们的工作模型，即伴侣复合体被排除在囊泡之外 因为它们太大，不能分割成与COPII相关的管腔阵列的空隙；以及(目标3) 通过强化分配来发展COPII货物吸收的新概念，作为主体流动模型的替代方案 急诊室出口。内质网到高尔基体的转运步骤对于真核细胞的生长和功能是必不可少的，因此 拟议中的研究对人类细胞生理学和对 蛋白质错配和蛋白质毒性。

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.