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转运出内质网（ER）而在早期分泌途径中发挥关键作用。 COPII 蛋白由 Sar1（一种 GTP 酶）、Sec23/24（货物选择和 GAP 活性）和 Sec13/31（促进外壳组装）组成。它们一起形成一层外壳，招募并集中货物，并逐渐使内质网膜变形为囊泡。最近，人们发现Sec13/31自组装成独特的立方八面体笼状结构。通过冷冻电子显微镜 (cryoEM) 和单粒子重建解析了这些结构，并深入了解了 COPII 包被囊泡组装的一些机制。这些初步研究得到了由 Sec13/31 与 Sec23/24 自组装形成的 COPII 涂层新结构的支持。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。当前的提案旨在通过四个具体目标解决有关 COPII 结构和组装的问题。目标 1 提议确定单个 COPII 涂层和 COPII 包被囊泡的结构。这些结构将揭示货物与 Sec23/24 相互作用以及 Sec23/24 与 Sec13/31 相互作用的方式，从而有助于了解货物指导适当大小的囊泡组装的机制。在目标2中，建议确定与Sar1复合的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 探讨了束缚的分子机制，其中建议确定与 TRAPPI 束缚蛋白 Bet3 复合的 COPII 外壳的结构。最后，目标 4 建议开发各种中间阶段中毒 COPII 笼组件的测定方法，并确定中间体的结构。总之，这些研究将进一步加深我们对 COPII 结构及其在细胞中组装方式的理解，从而推动囊泡运输领域的发展。反过来，这将有助于了解 COPII 蛋白在乳糜微粒滞留病和颅骨豆状缝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，例如如囊性纤维化。 公共健康相关性：COPII 蛋白参与分泌途径，这是人类等真核生物的关键和基本途径。乳糜微粒滞留病和颅缝豆状核发育不良这两种疾病与 COPII 蛋白的突变有关，而包括囊性纤维化在内的许多疾病都是由导致蛋白质滞留在 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 在这些疾病中发挥的作用，并可能有助于确定新的治疗靶点。