Assembly and Architecture of the Exocyst, a Membrane Tethering Complex

描述(申请人提供)：胞吐作用是所有真核细胞中的一个基本的细胞生物学过程，通过这个过程，细胞内的分子被分泌，膜蛋白和脂类的水平被调节。这里提出的工作重点是胞囊，一个多聚体，~800 kDa的蛋白质复合体，在极化胞吐的最后几步发挥作用。它有助于调节分泌囊泡和质膜之间的识别，并可能帮助SNARE复合体促进膜融合。它对胰岛素刺激的GLUT4转运体的运输很重要，作为Rala GTP酶的效应因子，它与肿瘤的发生有关。然而，人们对胞囊的结构或其组装被小的GTP酶及其调节器严格控制的机制知之甚少。在这里，我们将使用生物物理和生化方法来探索促进胞囊组装和胞外复合体整体结构的关键相互作用。我们的第一个目的是探索Rab GTPase Sec4p被其鸟嘌呤交换因子(Global)Sec2p激活的机制。一旦被激活，Sec4p就会将外囊成分Sec15p招募到囊泡膜上。得到了质量较好的硒蛋氨酸取代的Sec4p与Sec2p的gef结构域之间的络合物晶体。从Sec4p/Sec2p复合体获得的机制见解将是第一批通过GEFS激活Rab大家族中的Rab GTP酶的研究之一。我们的第二个目标是研究胞囊亚单位与胞囊组装调节因子的相互作用。我们将研究小分子GTP酶Rho3p与其效应子Exo70p的相互作用，以及Sec4p GTPase的效应子Sec15p与Sec4p的环境基金Sec2p之间的相互作用。第二种相互作用被认为确保了全球环境基金维持在外囊组装地点。最后，在我们的第三个目标中，我们将研究外囊的整体结构。胞外包囊亚复合体将用低温电子显微镜和结晶学方法进行分析。对不同的胞囊亚单位如何相互作用的详细描述将有助于形成对胞囊组装和功能的理解。

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.