The Structure and Assembly of the COPII Coat

COPII外套的结构和组装

基本信息

批准号：
8520327
负责人：
SCOTT M STAGG
金额：
$ 25万
依托单位：
FLORIDA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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/8520327
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

项目摘要

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从内质网（ER）运出蛋白质和脂质货物在早期分泌途径中起关键作用。 COPII蛋白由SAR1（GTPase），SEC23/24（货物选择和间隙活性）和SEC13/31（促进外套组装）组成。这些涂层形成了一件涂层，可募集和集中货物，并逐渐将ER膜变形为囊泡。最近，发现SEC13/31自组装成独特的cuboctohedron笼子状结构。这些结构用冷冻电子显微镜（冷冻）和单个粒子重建溶液，并对组装copii涂层囊泡组装的某些机制产生了见解。这些最初的研究用由SEC13/31的Sec23/24的自组装形成的COPII涂层的新结构增强。这两个结构一起构成了剖析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特定目标解决有关COPII结构和组装的问题。 AIM 1建议确定单个Copii涂层和Copii涂层囊泡的结构。这些结构将阐明货物与SEC23/24和SEC23/24相互作用的方式与SEC13/31相互作用，从而有助于货物指导适当尺寸的囊泡组装的机制图片。在AIM 2中，提议确定与SAR1复合物中的Copii外套的结构，这些研究将在分子水平上揭示SAR1如何参与启动Copii Coat的形成。在AIM 3中探索了绑扎的分子机制，在该AIM 3中提出了与the蛋白蛋白bet3中复合物中copii涂层的结构。最后，AIM 4提议在各个中间阶段开发中毒Copii Cage组件的测定法，并确定中间体的结构。这些研究一起将通过进一步了解COPII结构以及如何组装在细胞中来推动囊泡的传输场。反过来，这将有助于理解COPII蛋白在诸如乳糜微粒保留疾病和颅骨脑性核心核心异常增生等疾病中所起的作用，这分别是由SAR1和SEC23/24中的突变引起的，并且是表现为这些疾病，这些疾病表现为运输缺陷。作为囊性纤维化。公共卫生相关性：COPII蛋白与分泌途径有关，这是人类等真核生物的关键和基本途径。两种疾病，分别是乳糜微粒保留疾病和Cranio lenticulo缝合性发育不良，与COPII蛋白的突变有关，以及包括导致蛋白保留在ER中的蛋白质的突变引起的许多疾病，包括囊性纤维化。了解货物蛋白与COPII涂层相互作用的机制以及COPII大衣如何组装将帮助我们了解COPII在这些疾病中的作用，并可能有助于识别用于治疗疗法的新目标。