The Structure and Assembly of the COPII Coat

COPII外套的结构和组装

• 批准号: 8310036
• 负责人: SCOTT M STAGG
• 金额: $ 25.98万
• 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/8310036
• 关键词: Address; Anderson syndrome; Basic Science; 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肪运输出内质网(ER)，在早期分泌途径中发挥关键作用。COPII蛋白由Sar1(GTP酶)、Sec23/24(货物选择和GAP活性)和Sec13/31(促进被膜组装)组成。在一起，它们形成了一层外套，聚集和浓缩货物，并逐渐使ER膜变形成小泡。最近，人们发现Sec13/31自组装成一种独特的立方八面体笼状结构。这些结构通过冷冻电子显微镜和单粒子重建得到了解决，并对COPII涂层囊泡组装的一些机制有了深入的了解。这些初步研究得到了由Sec13/31和Sec23/24自组装形成的COPII涂层的新结构的支持。这两种结构共同构成了剖析COPII蛋白在细胞中发挥其功能的机制的基础。目前的提案寻求通过四个具体目标解决关于COPII结构和组装的问题。目的1确定单个COPII包衣和包膜囊泡的结构。这些结构将阐明货物与Sec23/24和Sec23/24与Sec13/31相互作用的方式，从而有助于了解Cargo引导适当大小的囊泡组装的机制。在目标2中，建议确定与Sar1形成的络合物中COPII涂层的结构，这些研究将在分子水平上揭示Sar1如何参与启动COPII涂层的形成。目标3探索了拴系的分子机制，其中建议确定与TRAPPI系留蛋白Bet3形成的络合物中COPII外壳的结构。最后，目标4建议开发在不同中间阶段毒化COPII笼组件的分析方法，并确定中间体的结构。总之，这些研究将通过加深我们对COPII结构的理解以及它们如何在细胞中组装来推动囊泡运输领域。这反过来将有助于理解COPII蛋白在乳糜管滞留疾病和颅豆状结构发育不良等疾病中所起的作用，这些疾病分别由Sar1和Sec23/24的突变引起，以及表现为运输缺陷的疾病，如囊性纤维化。 与公共健康相关：COPII蛋白参与分泌途径，这是真核生物(如人类)的关键和基本途径。两种疾病，乳糜粒滞留症和颅豆状缝发育不良，与COPII蛋白的突变有关，包括囊性纤维化在内的一系列疾病是由导致蛋白质保留在内质网的突变引起的。了解Cargo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在这些疾病中所起的作用，并可能有助于确定治疗的新靶点。