Structure and Function of the Exocyst Complex

项目总结 真核细胞在亚细胞器之间运输货物，并将货物运送到质膜进行分泌， 使用小的膜结合的囊泡是载体。囊泡转运与膜融合的调控 过程对细胞的形态、生长、运动和分泌至关重要，包括激素的释放。 和神经传递。这些过程需要许多必需的蛋白质，包括SNARE 参与膜融合过程的蛋白质和Sec1，Rab和Rho GTP酶，以及 八聚体系留复合体称为胞囊。尽管外囊复合体与一种 圈套组件的识别、系绳和质量控制所涉及的不同功能的数量和 核聚变，这些都没有在分子水平上被很好地理解。我们正在使用一种多学科战略， 生化和生物物理技术，结合遗传学和细胞生物学方法，以便 了解胞囊复合体的分子结构和功能。我们研究胞囊蛋白 从模式生物酿酒酵母中利用丰富的遗传、细胞生物学 以及可用的生化技术，但已经扩展到从其他生物体中排出。我们的研究目标是： 1)通过体外生化研究绘制出胞囊复合体的功能组织图并进行分析 突变体在体内测试外囊的功能；(2)确定整个外囊复合体的三维结构 使用电子显微镜、结晶学和分子模拟；以及(3)观察外囊系留小泡 在单分子水平上分析拴系的需要，以及(4)剖析胞囊的作用 和Sec1在SNARE复合体组装和膜融合中。因为这些蛋白质是保守的 酵母对人类神经元的影响，这项研究将增进我们对分泌物和生长是如何调节的知识 在所有真核细胞中。

PROJECT SUMMARY 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 a 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, but have expanded to exocyst from other organisms. 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.