Assembly and Architecture of the Exocyst, a Membrane Tethering Complex

描述（由申请人提供）：胞吐是所有真核细胞的基本细胞生物学过程，通过胞内分子分泌和膜蛋白和脂质水平调节。本文提出的研究重点是胞囊，这是一种多聚体，约800kDa的蛋白质复合物，在极化胞囊作用的最后步骤中起作用。它有助于介导分泌囊泡和质膜之间的识别，并可能协助SNARE复合体促进膜融合。它对于胰岛素刺激Glut4转运体的运输是重要的，并且作为RalA GTPase的效应物，它与肿瘤发生有关。然而，对于胞囊的结构以及胞囊的组装是如何被小的GTPases及其调控因子严格控制的机制，人们知之甚少。在这里，我们将使用生物物理和生化方法来探索促进囊胞组装和囊胞复合物整体结构的关键相互作用。我们的第一个目标是探索兔GTPase Sec4p（胞囊组装的调节剂）被其鸟嘌呤交换因子（GEF） Sec2p激活的机制。一旦被激活，Sec4p将囊泡成分Sec15p招募到囊泡膜上。在Sec4p和Sec2p的GEF结构域之间获得了高质量的硒代蛋氨酸取代晶体。从Sec4p/Sec2p复合物中获得的机制见解将是GEFS首次在大家族中激活兔GTPases。我们的第二个目标是研究胞囊亚基与胞囊组装调节因子的相互作用。我们将研究小GTPase Rho3p与其效应物外囊成分Exo70p的相互作用，以及外囊亚基Sec15p （Sec4p GTPase的效应物）与Sec4p的GEF Sec2p之间的相互作用。这第二种相互作用被认为确保了GEF在囊泡组装位点的维持。最后，在我们的第三个目标中，我们将研究囊泡的整体结构。囊泡亚复合物将通过低温电子显微镜和晶体学方法进行分析。各种囊胞亚基如何相互作用的详细图片将有助于形成对囊胞组装和功能的理解。

DESCRIPTION (provided by applicant): Exocytosis is a fundamental cell biological process in all eukaryotic cells, through which intracellular molecules are secreted and the levels of membrane proteins and lipids are regulated. The work proposed here focuses on the exocyst, a multimeric, ~800kDa protein complex that functions in the last steps of polarized exocytosis. It helps to mediate recognition between secretory vesicles and the plasma membrane and may assist the SNARE complex in promoting membrane fusion. It is important for the insulin stimulated trafficking of the Glut4 transporter and, as an effector of the RalA GTPase, it is relevant to tumorigenesis. Yet little is known of exocyst architecture or of the mechanisms by which its assembly is tightly controlled by small GTPases and their regulators. Here we will use biophysical and biochemical approaches in exploring key interactions that promote exocyst assembly and the overall architecture of the exocyst complex. Our first aim is to explore the mechanism by which the rab GTPase Sec4p, a regulator of exocyst assembly, is activated by its guanine exchange factor (GEF) Sec2p. Once activated, Sec4p recruits the exocyst component Sec15p to the vesicle membrane. Good quality, selenomethionine substituted crystals of a complex between Sec4p and the GEF domain of Sec2p have been obtained. The mechanistic insights obtained from the Sec4p/Sec2p complex will be among the first for the activation of rab GTPases in the large rab family by GEFS. Our second aim is to study the interactions of exocyst subunits with regulators of exocyst assembly. We will investigate the interactions of the small GTPase Rho3p with its effector, the exocyst component Exo70p, and the interactions between the exocyst subunit Sec15p, an effector of the Sec4p GTPase, and Sec2p, the GEF for Sec4p. This second interaction is believed to ensure that the GEF is maintained at sites of exocyst assembly. Finally, in our third aim, we will study the overall architecture of the exocyst. Exocyst subcomplexes will be analyzed by both cryoEM and by crystallographic methods. A detailed picture of how the various exocyst subunits interact will help to formulate an understanding of exocyst assembly and function.