Structure and Function of the Exocyst Complex

描述（由申请人提供）：真核细胞在亚细胞细胞器之间运输货物，并向质膜分泌，使用小的膜结合囊泡作为载体。囊泡运输和膜融合过程的调节对细胞形态、生长、运动和分泌，包括激素释放和神经传递至关重要。这些过程需要许多必需的蛋白质，包括参与膜融合过程的SNARE蛋白和Sec1， Rab和Rho gtpase，以及称为胞囊的八聚体系结复合体。尽管胞囊复合体涉及许多不同的功能，包括SNARE组装和融合的识别、系住和质量控制，但在分子水平上，这些功能都没有得到很好的理解。我们正在使用生物化学和生物物理技术的多学科策略，结合遗传学和细胞生物学方法，以了解胞囊复合体的分子结构和功能。我们研究模式生物酿酒酵母的胞囊蛋白，以利用丰富的遗传、细胞生物学和生物化学技术。我们的研究目标是：1)通过体外生化研究，绘制出胞囊复合物的功能组织，并分析突变体，以检测胞囊在体内的功能；(2)利用电子显微镜、晶体学和分子模型确定整个囊胞复合物的三维结构；(3)在单分子水平上观察囊泡系留囊泡，分析系留要求；(4)剖析囊泡和Sec1在SNARE复合物组装和膜融合中的作用。因为这些蛋白质从酵母到人类神经元都是保守的，这项研究将推进我们对所有真核细胞的分泌和生长是如何调节的认识。

DESCRIPTION (provided by applicant): Eukaryotic cells transport cargo between subcellular organelles, and to the plasma membrane for secretion, using small membrane-bound vesicles are carriers. The regulation of vesicular transport and membrane fusion processes are crucial for cellular morphology, growth, movement and secretion, including hormone release and neurotransmission. Many essential proteins are required for these processes, including the SNARE proteins and Sec1 that are involved in the membrane fusion process, the Rab and Rho GTPases, and an octameric tethering complex called the exocyst. Although the exocyst complex has been implicated in a number of different functions involved in recognition, tethering and quality control of SNARE assembly and fusion, none of these are well understood at the molecular level. We are using a multidisciplinary strategy of biochemical and biophysical techniques, combined with genetics and cell biological methods, in order to understand the molecular architecture and function of the exocyst complex. We study the exocyst proteins from the model organism Saccharomyces cerevisiae to take advantage of the wealth of genetic, cell biological and biochemical techniques available. Our studies aim to: 1) map the functional organization of the exocyst complex through biochemical studies in vitro and analyze mutants to test the function of the exocyst in vivo; (2) determine the 3D structure of the entire exocyst complex using electron microscopy, crystallography and molecular modeling; and (3) watch the exocyst tether vesicles at the single molecule level to analyze the requirements for tethering, and (4) dissect the role of the exocyst and Sec1 in SNARE complex assembly and membrane fusion. Because these proteins are conserved from yeast to human neurons, this research will advance our knowledge of how secretion and growth are regulated in all eukaryotic cells.