单颗粒冷冻电镜研究TRAPPII复合体的结构与功能

囊泡转运是细胞内物质运输的主要方式，分为出芽、移动、拴系和融合四个步骤。.由拴系蛋白介导的拴系是指转运小泡与其受体之间的初始、远距离相互作用，这一步被认为对于建立囊泡靶向转运的特异性是必不可少的。拴系蛋白TRAPP家族蛋白具有鸟嘌呤核苷酸交换功能，可以将Ypt/Rab GTP酶从非激活状态转换成激活状态，进而招募下游的效应因子调控囊泡运输。酵母TRAPPII是酵母TRAPP家族中最大的成员，分子量约为1MDa，调控高尔基体内部和内吞体到晚期高尔基体之间的囊泡运输，它的缺陷引起多种人类疾病。然而对其工作机制的研究尚显不足。目前完整TRAPPII的高分辨率结构信息非常匮乏，限制了对其作用机制的认识。本项目拟利用先进的单颗粒冷冻电镜技术对从酵母体内直接纯化的完整TRAPPII复合物的结构进行解析，并在此基础上结合交联质谱、突变分析、酵母遗传学等实验，揭示TRAPPII复合体的组装方式和作用机制。

Vesicular trafficking mediates the transport of proteins and lipids between different membrane-bound compartments. Each vesicle transport reaction can be divided into four essential steps that include vesicle budding, transport, tethering, and fusion. Tethering refers to the initial, long-range interaction between a transport vesicle and its acceptor compartment, which is believed to be essential for establishing the specificity of vesicle targeting and is mediated by tethering factors. TRAPP family proteins are tethering factors shown to act as guanine nucleotide exchange factors, which can convert Ypt/Rab GTP enzymes from inactivated state to activated state. In yeast, TRAPPII is the largest member of the yeast TRAPP family, with a molecular weight of about 1MDa. TRAPPII functions in intra-Golgi and endosome-to-Golgi traffic, and its defects are involved in many human diseases. However, the molecular mechanisms of TRAPPII in the tethering events are only partially understood because of the lack of the high-resolution structural information of the intact TRAPPII. The purpose of this proposal is to reveal the high-resolution structure of the intact TRAPPII purified directly from yeast by using single-particle cryo-electron microscopy. And on the basis of the structure, we will study the assembly of TRAPPII, as well as the mechanisms TRAPPII uses to exchange nucleotides and tether membranes, combined with the chemical cross-linking of proteins coupled with mass spectrometry analysis, the mutation analysis, methods in yeast genetics and so on.