Molecular Mechanisms underlying selection of cargo, scission, and coat release of intracellular vesicular carriers

在真核生物中，蛋白质包被的囊泡载体介导细胞膜和蛋白质的胞内运输。我们感兴趣的是这些囊泡形成和功能的结构和分子机制，它们对蛋白质和脂质货物的选择性结合，以及它们的脱包。我们之前已经通过重组外壳蛋白和定义的脂质体的重构研究，定义了形成COPI囊泡所必需和充分的最小蛋白质机制。尽管在形成COPI-、COPII-和网格蛋白包被囊泡（ccv）的各种被膜蛋白亚基中，基本结构元素彼此相似，但COPI被膜的整体结构与其他已建立的被膜不同。这种结构上的差异可能反映了将货物分子吸收到这些载体中的不同机制。此外，与分步骤形成的COPII和网格蛋白外壳不同，COPI外壳是一步就被招募到供体膜上的。然而，在所有类型的囊泡载体中，被膜募集和膜断裂的机制有一定的相似性。在对COPI外壳结构的深入了解的驱使下，在本申请中，我们建议研究i) COPI断裂反应的结构基础，以及是否存在适用于所有由小gtp结合蛋白催化的膜断裂事件的一般机制，ii)不同COPI和COPII外壳蛋白异构体对蛋白质和膜脂货物摄取的选择性。iii) gtpase活化蛋白如何催化COPI外壳的去除，并可能帮助涂层再循环到其可溶性形式。

In eukaryotes, protein-coated vesicular carriers mediate intracellular transport of membranes and proteins. We are interested in the structure and the molecular mechanisms that underlie the formation and function of such vesicles, their selective incorporation of protein and lipid cargo, and their un-coating. We have previously defined the minimal protein machinery that is necessary and sufficient to form COPI vesicles through reconstitution studies with recombinant coat proteins and defined liposomes. Whereas basic structural elements resemble each other in a variety of coat protein subunits that form COPI-, COPII-, and Clathrin-coated vesicles (CCVs), the overall structure of the COPI coat is different from the other established coats. This structural divergence might reflect different mechanisms for the uptake of cargo molecules into these carriers. Moreover, unlike the COPII and Clathrin coats that form stepwise, the COPI coat is recruited to the donor membrane in a single step. Yet, the mechanisms of coat recruitment, and possibly of membrane scission, share certain similarities among all classes of vesicular carriers. Driven by structural insight into the COPI coat, in this application we propose to investigate i) the structural basis for the COPI scission reaction, and whether a general mechanism exists that applies for all membrane scission events catalyzed by small GTP-binding proteins, ii) the selectivity of different COPI and COPII coat protein isoforms for the uptake of protein and membrane lipid cargo, and iii) how GTPase-activating proteins catalyze the removal of the COPI coat and possibly aid coatomer to be recycled to its soluble form.