Molecular Regulation of Exocytosis

项目摘要 胞吐作用对于所有真核细胞的存活、生长和功能都是重要的， 在许多生理过程中起着重要作用，从神经传递到 器官发生胞吐作用是由分泌囊泡的转运、对接和融合介导的 将货物运送到质膜上。外囊是一个进化上保守的八聚体 胞吐作用所必需的蛋白质复合物。外囊的功能障碍与糖尿病有关， 常染色体多囊肾疾病和癌症。我们最近解决了 通过冷冻电镜观察到完全组装的外囊复合物，并鉴定出两个四螺旋 束，称为“CorEx”，其潜在地介导外囊复合物的核心组装。在 这个建议，我们将首先研究的分子相互作用和高分辨率结构的 CorEx采取多管齐下的方法，结合了生物化学，生物物理学，晶体学， 和显微成像。我们还将确定外囊复合物在 使用体外重构脂质体系统的SNARE组装和膜融合。最后我们 将研究新发现的外囊和 EHD蛋白，其在从内体产生管状囊泡载体中起作用。 隔间我们将进一步研究RalA对exocyst-EHD相互作用的调节， 一种控制内体再循环至质膜的小GTdR， 生长因子信号传导我们的工作不仅有助于阐明胞吐作用的机制 在分子水平上，也揭示了一些疾病，如糖尿病和癌症。

Project Summary Exocytosis is important for the survival, growth and functions of all eukaryotic cells, and is centrally involved in many physiological processes ranging from neurotransmission to organogenesis. Exocytosis is mediated by transport, docking and fusion of secretory vesicles carrying cargos to the plasma membrane. The exocyst is an evolutionarily conserved octameric protein complex essential for exocytosis. Malfunction of the exocyst has been linked to diabetes, Autosomal Polycystic Kidney Diseases and cancer. We have recently solved the structure of the fully assembled exocyst complex by cryo-electron microscopy and identified two four-helix bundles, termed “CorEx”, that potentially mediate the core assembly of the exocyst complex. In this proposal, we will first study the molecular interactions and high-resolution structure of the CorEx taking a multipronged approach that combines biochemistry, biophysics, crystallography, and microscopic imaging. We will also determine the function of the exocyst complex in SNARE assembly and membrane fusion using in vitro reconstituted liposome systems. Finally, we will examine the functional implication of a newly identified interaction between the exocyst and EHD proteins, which function in the generation of tubular vesicular carriers from the endosomal compartments. We will further examine the regulation of the exocyst-EHD interaction by RalA, a small GTPase that controls endosomal recycling to the plasma membrane in response to growth factor signaling. Our work will not only help elucidate the mechanisms of exocytosis at the molecular level, but also shed light to a number of diseases such as diabetes and cancer.