Structural studies of the exocyst

描述（由申请人提供）：分泌囊泡从反式高尔基体萌发并沿着细胞骨架元件移动到活跃分泌部位。囊泡是一种约800 kDa的异八聚体复合物，被认为在囊泡与质膜对接时，在膜融合之前，在囊泡与其靶膜之间起识别作用。胞囊在胞吐途径上与许多其他蛋白质相互作用，因此了解其结构对理解胞吐的分子机制至关重要。分离或表达整个囊泡的尝试均未成功。相反，我们将研究胞囊亚复合物，它应该更容易分离出结构研究所需的数量，并且应该提供有关亚基排列的信息（目的1）。确定的任何亚配合物将被监测凝胶过滤的溶解度，并进行结晶试验（目标2）。原生晶体和/或重原子取代晶体将用于相位。一旦结构确定，我们将研究蛋白质如何与其他囊胞亚基相互作用，以推断整体囊胞结构（目的3）。通过结构域分析，确定亚基与亚复合物的结合伙伴，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囊胞亚基相互作用所需的亚复合物的表面。此外，将进行类似的实验来确定激活形式的GTPases如何与囊泡亚基相互作用，这将提供有关囊泡-膜相互作用的信息。这些功能研究将使我们能够构建一个囊胞结构模型。公共卫生相关性：胞外通路或胞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。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GLUT4运输的分泌（在糖尿病中有缺陷）至关重要。对囊泡结构的了解将有助于阐明囊泡在囊泡栓系中的功能，并可能对这些心脏和血液疾病的病因有深入的了解。

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.