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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566745
• 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区室或通过分泌途径分泌。此外，真核细胞从其环境中接收重要线索，这些线索通过内吞途径传递。分泌和内吞途径中蛋白质（货物）的运输依赖于细胞膜上蛋白质外壳的组装和分解。涂层成分招募货物，产生高度弯曲的膜区域，促进囊泡分裂并招募脱涂层和其他调节因子。迄今为止，已描述了三种主要类型的囊泡在不同细胞区室之间移动货物：网格蛋白、COPI（外壳蛋白复合物 I）和 COPII（外壳蛋白复合物 II）包被的囊泡。尽管这些囊泡在不同的细胞区室之间运输，并且它们的外壳由不同的蛋白质组成，但据推测，外壳组装和货物招募的基本设计原理是相似的，但在顺序上有明显的差异，以允许运输的特异性。该假设基于 COPII 和网格蛋白包被的囊泡之间的相似性。然而，人们对 COPI 组件的实际结构和完全组装的 COPI 囊泡的结构知之甚少。 这里提出的研究旨在进一步了解 COPI 囊泡组装的基本原理，使用高分辨率分子冷冻电子显微镜（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。为此，我们将确定纯化分离的整个 COPI 复合物 (CM7) 以及子复合物 CM3（涂层形成复合物）和 CM4（涉及的适配器样复合物）的 3D 结构。这将有助于了解亚基与涂层复合物的排列，并提供关键信息以了解复合物如何发挥作用和受到调节。我们还将研究促进涂层组装和涂层聚合的体外条件。将测试在体内显示对囊泡形成效率有影响的因素的影响。这些包括脂质、膜的脂质成分、含二赖氨酸的肽、Arf1 和各种 ArfGAP。一旦在体外获得低聚物，将使用单粒子和/或电子断层扫描和子体积平均来确定其3D结构，以便了解不同的涂层聚合物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。总而言之，这些研究将为理解 COPI 包被囊泡组装所涉及的分子机制提供结构基础，并强调 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 亚基如何相互作用以组装成大型寡聚复合物，检测组装过程中发生的任何构象变化，并确定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、冷冻电子显微镜和 3DEM 重建的机会，使用麦吉尔电子显微镜研究 (FEMR) 和分子动力学设施提供的最先进的仪器。当前的提议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。