Structural studies of the exocyst

描述（由申请人提供）：分泌囊泡从反式高尔基体出芽，并沿沿着细胞骨架元件移动到活性分泌部位。外囊是一种~800 kDa的异源八聚体复合物，被认为在膜融合之前，当囊泡与质膜对接时，其在囊泡与其靶膜之间的识别中起作用。胞吐作用与许多其他蛋白质相互作用沿着胞吐途径，因此，其架构/结构的知识将是理解胞吐作用的分子机制的核心。分离或表达整个外囊的尝试一直不成功。相反，我们将研究exocyst亚复合物，这应该是更容易分离的结构研究所需的数量，但应产生有关亚基排列的信息（目的1）。将监测任何确定的亚复合物在凝胶过滤上的溶解度，并进行结晶试验（目的2）。天然晶体和/或重原子取代晶体将用于定相。一旦确定了结构，我们将研究蛋白质如何与其他外囊亚基相互作用，以推断整体外囊结构（目的3）。通过结构域分析，将鉴定亚复合物的亚基结合伴侣，并缩小结合伴侣相互作用区域。将进行突变分析，以更精确地定义与其他外囊亚基相互作用所需的亚复合物中的表面。此外，将进行类似的实验以确定GTP酶的活化形式如何与外囊亚基相互作用，这将提供关于外囊-膜相互作用的信息。这些功能研究将使我们能够构建外囊结构模型。公共卫生相关性：胞吐途径或胞吐的功能障碍与许多心脏疾病有关，如贫血、高血压和糖尿病。外囊对于转铁蛋白受体再循环（在血红蛋白缺乏性贫血中受损）、ANP和其他激素的分泌（在高血压中增加）以及胰岛素和GLUT 4运输的分泌（在糖尿病中有缺陷）至关重要。了解外囊的结构将有助于阐明外囊在囊泡束缚中的功能，并可能深入了解这些心脏和血液疾病的病因。

DESCRIPTION (provided by applicant): Secretory vesicles bud from the trans-Golgi and move along cytoskeletal elements to sites of active secretion. The exocyst is an ~800 kDa hetero-octameric complex that is thought to function in recognition between the vesicle and its target membrane, as the vesicle is docking to the plasma membrane, prior to membrane fusion. The exocyst interacts with numerous other proteins along the exocytic pathway, so that knowledge of its architecture/structure will be central in understanding the molecular mechanisms underlying exocytosis. Attempts to isolate or express the entire exocyst have been unsuccessful. Instead we will study exocyst subcomplexes, which should be easier to isolate in quantities required for structural studies and which should nevertheless yield information regarding subunit arrangement (Aim 1). Any subcomplexes identified will be monitored for solubility on gel filtration and subject to crystallization trials (Aim 2). The native crystals and/or heavy atom substituted crystals will be used for phasing. Once the structure has been determined, we will study how the proteins interact with other exocyst subunits, to infer overall exocyst architecture (Aim 3). Subunit binding partners to the subcomplex will be identified, and binding partner interaction regions will be narrowed down, through domain analysis. Mutational analysis will be performed to more precisely define surfaces in the subcomplex required for interaction with other exocyst subunits. Additionally, similar experiments will be performed to determine how the activated form of GTPases interacts with exocyst subunits, which will give information regarding exocyst-membrane interactions. These functional studies will allow us to construct a model for exocyst architecture. Public Health Relevance: Malfunctions in the exocytic pathway or the exocyst are relevant to a number of cardiac diseases, such as anemia, hypertension, and diabetes. The exocyst is critical for transferrin receptor recycling (which is impaired in hemoglobin-deficient anemia), secretion of ANP and other hormones (which are increased in hypertension), and for secretion of insulin and GLUT4 trafficking (which are defective in diabetes). A structural understanding of the exocyst will help clarify the function of the exocyst in vesicle tethering, and might give insight into the etiologies of these heart and blood diseases.