The Structure and Assembly of the COPII Coat

COPII外套的结构和组装

• 批准号: 7987056
• 负责人: SCOTT M STAGG
• 金额: $ 24.17万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7987056
• 关键词: Address; Anderson syndrome; Binding; Binding Sites; Biological Assay; Biological Transport; COPII-Coated Vesicles; Carrier Proteins; Cells; Coated vesicle; Complement; Complex; Coupled; Cryoelectron Microscopy; Cystic Fibrosis; Defect; Deuterium; Dideoxy Chain Termination DNA Sequencing; Disease; Dysplasia; Endoplasmic Reticulum; Eukaryota; Foundations; Fourier transform ion cyclotron resonance; Generations; Genome; Golgi Apparatus; Guanosine Triphosphate Phosphohydrolases; Human; Hydrogen; In Vitro; Individual; Kinetics; Light; Lipids; Mass Spectrum Analysis; Mediating; Medical Research; Membrane; Microsomes; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Mutation; Nucleotides; Pathway interactions; Play; Poisoning; Positioning Attribute; Proteins; Reaction; Recruitment Activity; Relative (related person); Role; Specificity; Staging; Structure; Techniques; Transport Vesicles; Vesicle; analytical ultracentrifugation; design; electron tomography; in vivo; insight; light scattering; new therapeutic target; novel; particle; protein function; public health relevance; reconstruction; self assembly; three dimensional structure

DESCRIPTION (provided by applicant): COPII proteins play a critical role in the early secretory pathway by transporting protein and lipid cargo out of the endoplasmic reticulum (ER). The COPII proteins consist of Sar1 (a GTPase), Sec23/24 (cargo selection and GAP activity), and Sec13/31 (promotes coat assembly). Together, these form a coat that recruits and concentrates cargo and gradually deforms the ER membrane into a vesicle. Recently, it was found that Sec13/31 self-assembles into a unique cuboctahedron cage-like structure. These structures were solved with cryo-electron microscopy (cryoEM) and single particle reconstruction and yielded insights into some of the mechanisms by which COPII coated vesicles are assembled. These initial studies were bolstered with a new structure of a COPII coat formed from the self-assembly of Sec13/31 with Sec23/24. Together, these two structures form a foundation for dissecting the mechanisms by which the COPII proteins perform their functions in the cell. The current proposal seeks to address questions about COPII structure and assembly through four specific aims. Aim 1 proposes to determine the structures of individual COPII coats and COPII coated vesicles. These structures will shed light on the ways that cargo interacts with Sec23/24 and Sec23/24 interacts with Sec13/31, and thus contribute to a picture of the mechanisms by which cargo directs the assembly of vesicles of the proper size. In aim 2, it is proposed to determine the structure of the COPII coat in complex with Sar1, and these studies will reveal at a molecular level how Sar1 is involved in initiating the formation of the COPII coat. The molecular mechanisms of tethering are explored in aim 3, where it is proposed to determine the structure of the COPII coat in complex with the TRAPPI tether protein Bet3. Finally, aim 4 proposes to develop assays for poisoning COPII cage assembly at various intermediate stages and to determine the structures of the intermediates. Together, these studies will drive the vesicle transport field by furthering our understanding of COPII structures and how they are assembled in the cell. This, in turn, will aid in the understanding of the role COPII proteins play in diseases like chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which result from mutations in Sar1 and Sec23/24 respectively, and diseases that manifest as transport defects such as cystic fibrosis. PUBLIC HEALTH RELEVANCE: The COPII proteins are involved in the secretory pathway, which is a critical and fundamental pathway in eukaryotes such as humans. Two diseases, chylomicron retention disease and cranio-lenticulo sutural dysplasia, are associated with mutations in COPII proteins, and a host of diseases including cystic fibrosis result from mutations that cause the proteins to be retained in the ER. Understanding of the mechanisms by which cargo proteins interact with the COPII coat and how COPII coat assembles will help us understand the role COPII plays in these diseases and may help in identifying novel targets for therapeutics.

描述（由申请人提供）：COPII蛋白通过将蛋白质和脂质货物运输出内质网（ER）在早期分泌途径中发挥关键作用。COPII蛋白由Sar 1（一种GTP酶）、Sec 23/24（货物选择和GAP活性）和Sec 13/31（促进外壳组装）组成。这些细胞共同形成一层外套，招募和集中货物，并逐渐使ER膜变形为囊泡。最近，人们发现Sec 13/31自组装成一个独特的立方八面体笼状结构。这些结构与冷冻电子显微镜（cryoEM）和单颗粒重建解决，并产生了一些COPII包被囊泡组装的机制的见解。这些初步研究得到了一种新结构的COPII涂层的支持，该涂层由Sec 13/31与Sec 23/24的自组装形成。这两种结构共同构成了剖析COPII蛋白在细胞中发挥功能的机制的基础。目前的建议试图通过四个具体目标解决有关COPII结构和组装的问题。目的1提出确定单个COPII涂层和COPII涂层囊泡的结构。这些结构将阐明货物与Sec 23/24相互作用和Sec 23/24与Sec 13/31相互作用的方式，从而有助于了解货物引导适当大小的囊泡组装的机制。在目标2中，建议确定与Sar 1复合的COPII涂层的结构，这些研究将在分子水平上揭示Sar 1如何参与启动COPII涂层的形成。在目标3中探索了拴系的分子机制，其中提出确定与TRAPPI拴系蛋白Bet 3复合的COPII外壳的结构。最后，目标4提出开发用于在各个中间阶段毒化COPII笼组件的测定法，并确定中间体的结构。总之，这些研究将通过进一步了解COPII结构以及它们如何在细胞中组装来推动囊泡运输领域。反过来，这将有助于理解COPII蛋白在乳糜微粒滞留病和颅-颅-缝-缝发育不良等疾病中的作用，这些疾病分别由Sar 1和Sec 23/24突变引起，以及表现为运输缺陷的疾病，如囊性纤维化。 公共卫生相关性：COPII蛋白参与分泌途径，这是真核生物如人类中的关键和基本途径。两种疾病，乳糜微粒滞留病和颅-颅缝发育不良，与COPII蛋白的突变有关，并且包括囊性纤维化在内的许多疾病是由导致蛋白质保留在ER中的突变引起的。了解货物蛋白与COPII外壳相互作用的机制以及COPII外壳如何组装将有助于我们了解COPII在这些疾病中的作用，并可能有助于确定新的治疗靶点。