Structural studies of the exocyst

描述(申请人提供)：分泌囊泡从反高尔基体萌发，并沿着细胞骨架元素移动到活跃的分泌部位。外囊是一个~800 kDa的异八聚体复合体，被认为在囊泡与其靶膜之间的识别中发挥作用，因为在膜融合之前，囊泡与质膜对接。胞囊与许多其他蛋白质沿着胞吐途径相互作用，因此对其结构/结构的了解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都没有成功。相反，我们将研究胞外囊亚复合体，这应该更容易分离出结构研究所需的数量，但仍应提供有关亚基排列的信息(目标1)。任何确定的亚络合物都将在凝胶过滤上监测其溶解性，并进行结晶试验(目标2)。将使用天然晶体和/或重原子取代晶体进行位相。一旦确定了结构，我们将研究蛋白质如何与其他胞囊亚基相互作用，以推断整个胞囊结构(目标3)。通过结构域分析，将确定亚单位与亚复合体的结合伙伴，并缩小结合伙伴相互作用区域。将进行突变分析，以更精确地定义与其他胞外囊亚基相互作用所需的亚复合体中的表面。此外，还将进行类似的实验，以确定激活形式的GTP酶如何与胞外亚基相互作用，这将提供关于胞外-膜相互作用的信息。这些功能研究将使我们能够构建一个外囊结构的模型。公共卫生相关性：胞外途径或外囊的功能障碍与许多心脏疾病有关，如贫血、高血压和糖尿病。胞囊对于转铁蛋白受体循环(在血红蛋白缺乏性贫血中受损)、心钠素和其他激素的分泌(在高血压时增加)以及胰岛素和GLUT4运输(在糖尿病中有缺陷)的分泌至关重要。对外囊的结构了解将有助于阐明外囊在囊泡捆绑中的作用，并可能有助于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.