Investigation of the assembly mechanisms of COPI-coated vesicles using cryo-electron microscopy

使用冷冻电子显微镜研究 COPI 包被的囊泡的组装机制

• 批准号: RGPIN-2014-04798
• 负责人: Rouiller, Isabelle
• 金额: $ 2.55万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629389
• 关键词: Investigation; assembly; mechanisms; COPI; coated

In eukaryotic cells, proteins are mainly synthesized in the endoplasmic reticulum to be transported to the correct cellular compartment or secreted through the secretory pathway. Additionally, eukaryotic cells receive important clues from their environment, clues that are transmitted via the endocytosis pathway. The transport of proteins (cargo) within the secretory and endocytic pathways relies on the assembly and disassembly of protein coats onto cellular membranes. The coat components recruit cargo, generate highly curved membrane areas, facilitate vesicle scission and recruit uncoating and other regulatory factors. To date, three major types of vesicles have been described moving cargo between different cellular compartments: clathrin-, COPI (coat protein complex I)- and COPII (coat protein complex II)-coated vesicles. Despite the fact that these vesicles traffic between different cellular compartments and that their coats are composed of different proteins, it has been hypothesized that the basic design principles of assembly of the coat and cargo recruitment are similar but with distinct differences in ordered to allow specificity of transport. This hypothesis was based on similarities between COPII- and clathrin-coated vesicles. However, little is known regarding the actual structure of COPI components and the structure of the fully-assembled COPI vesicles. The research proposed here seeks to further our understanding of the underlying principles of COPI vesicle assembly using high-resolution molecular cryo-electron microscopy (single particle and electron tomography image analysis) combined with molecular modeling using Molecular Dynamics. To this end, we will determine the 3D structure of purified isolated entire COPI complex (CM7) and the sub-complexes CM3 (coat forming complex) and CM4 (adaptor-like complex involved). This will allow understanding of the subunits arrangement with the coatamer complex and provide critical information to understand how the complex may function and be regulated. We will also study the conditions in vitro that promote coat assembly and coatamer polymerization. Influence of factors, shown to have an impact in vivo on the efficiency of vesicles formation will be tested. These include lipids, lipid composition of the membrane, dilysine containing peptides, Arf1 and various ArfGAPs. Once oligomers are obtained in vitro, their 3D structures will be determined using single particle and/or electron tomography and sub-volumes averaging in order to understand how different coatamer assemble into a protein coat and to determine the different subunit interactions involved in this process. Taken together, these studies will provide the structural basis for understanding the molecular mechanisms involved in the assembly of COPI-coated vesicles as well as highlight the similarities and differences between COPI-, COPII- and clathrin-coated vesicles assembly mechanisms. In particular, these studies will reveal how the different COPI subunits interact with each other to assemble into large oligomeric complexes, detect any conformation changes that occur during assembly and define the intermolecular contacts responsible of assembly. The interdisciplinary nature of these projects will provide trainees the opportunity to learn biochemical techniques, cryo-electron microscopy and 3DEM reconstruction using state-of-the-art instrumentation available at the McGill Facility for Electron Microscopy Research (FEMR) and Molecular Dynamics. The current proposal fits within the long-term objectives of my research program that are to understand the molecular mechanisms and structural principles involved in the interaction of protein and membrane leading to membrane penetration, scission and fusion.

在真核细胞中，蛋白质主要在内质网中合成，然后转运到正确的细胞腔室或通过分泌途径分泌。此外，真核细胞从环境中获得重要的线索，这些线索通过内吞作用途径传递。蛋白质（货物）在分泌和内吞途径中的运输依赖于蛋白质外壳在细胞膜上的组装和拆卸。涂层成分吸收货物，产生高度弯曲的膜区，促进囊泡分裂和吸收脱涂层等调节因素。迄今为止，已经描述了三种主要类型的囊泡在不同的细胞室之间移动货物：网格蛋白-，COPI（外壳蛋白复合体I）-和COPII（外壳蛋白复合体II）-包被囊泡。尽管这些囊泡在不同的细胞间运输，并且它们的外壳由不同的蛋白质组成，但有假设认为，外壳组装和货物招募的基本设计原则是相似的，但在运输特异性方面存在明显差异。这一假设是基于COPII-和网格蛋白包被囊泡之间的相似性。然而，关于COPI组分的实际结构和完全组装的COPI囊泡的结构知之甚少。本文提出的研究旨在利用高分辨率分子低温电子显微镜（单粒子和电子断层扫描图像分析）结合分子动力学的分子建模，进一步了解COPI囊泡组装的基本原理。为此，我们将确定纯化分离的整个COPI复合物（CM7）及其子复合物CM3（外壳形成复合物）和CM4（所涉及的适配器样复合物）的三维结构。这将有助于了解涂层复合物的亚基排列，并为了解复合物的功能和调节方式提供关键信息。我们还将研究促进涂层组装和涂层聚合的体外条件。影响因素，显示有影响囊泡形成的效率在体内将进行测试。这些包括脂质、膜的脂质组成、含二赖氨酸肽、Arf1和各种arfgap。一旦在体外获得低聚物，将使用单粒子和/或电子断层扫描和亚体积平均来确定它们的3D结构，以便了解不同的涂层如何组装成蛋白质涂层，并确定该过程中涉及的不同亚基相互作用。综上所述，这些研究将为理解COPI包被囊泡组装的分子机制提供结构基础，并突出COPI-、COPII-和网格蛋白包被囊泡组装机制之间的异同。特别是，这些研究将揭示不同的COPI亚基如何相互作用，组装成大的寡聚复合物，检测组装过程中发生的任何构象变化，并定义负责组装的分子间接触。这些项目的跨学科性质将为学员提供学习生化技术、低温电子显微镜和使用麦吉尔电子显微镜研究设施（FEMR）和分子动力学中最先进的仪器进行3DEM重建的机会。目前的提案符合我的研究计划的长期目标，即了解蛋白质和膜相互作用导致膜渗透，分裂和融合的分子机制和结构原理。