Assembly and Architecture of the Exocyst, a Membrane Tethering Complex

描述（由申请人提供）：胞吐作用是所有真核细胞中的基本细胞生物学过程，通过该过程分泌细胞内分子并调节膜蛋白和脂质的水平。这里提出的工作集中在外囊，一个多聚体，~ 800 kDa的蛋白质复合物，在极化胞吐的最后步骤中发挥作用。它有助于介导分泌囊泡和质膜之间的识别，并可能有助于SNARE复合物促进膜融合。它对于胰岛素刺激的Glut 4转运体的运输是重要的，并且作为RalA GT3的效应物，它与肿瘤发生相关。然而，很少有人知道外囊的结构或其组装是由小GTP酶和它们的监管机构严格控制的机制。在这里，我们将使用生物物理和生物化学的方法，探索关键的相互作用，促进外囊组装和外囊复合体的整体架构。我们的第一个目的是探索机制，其中rab GTdR Sec 4p，外囊装配的调节器，是由其鸟嘌呤交换因子（GEF）Sec 2 p激活。一旦被激活，Sec 4p将外囊组分Sec 15 p募集到囊泡膜。已经获得了Sec 4p和Sec 2 p的GEF结构域之间的复合物的良好质量的硒代甲硫氨酸取代的晶体。从Sec 4p/Sec 2 p复合物中获得的机制见解将是GEFS在大rab家族中激活rab GTP酶的第一个。我们的第二个目的是研究外囊亚基与外囊组装调节因子的相互作用。我们将调查的相互作用的小GTdR的Rho 3 p与其效应器，外囊组分Exo 70 p，和外囊亚基Sec 15 p，效应器的Sec 4p GTdR，和Sec 2 p，GEF Sec 4p之间的相互作用。这第二个相互作用被认为是确保GEF是维持在网站的外囊装配。最后，在我们的第三个目标中，我们将研究外囊的整体结构。将通过cryoEM和晶体学方法分析外囊亚复合物。各种外囊亚基如何相互作用的详细图片将有助于制定外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.