Structure and Function of the Exocyst Complex

项目摘要 真核细胞在亚细胞器之间运输货物，并运输到质膜上进行分泌， 使用小的膜结合囊泡作为载体。囊泡运输和膜融合的调节 这些过程对细胞形态、生长、运动和分泌至关重要，包括激素释放 和神经传递。这些过程需要许多必需蛋白质，包括SNARE 参与膜融合过程的蛋白质和Sec 1，Rab和Rho GTP酶，以及 称为外囊的八聚体束缚复合体。尽管外囊复合体与一种 涉及SNARE组件识别、系留和质量控制的多个不同功能， 融合，这些都没有在分子水平上得到很好的理解。我们采用多学科战略， 生物化学和生物物理技术，结合遗传学和细胞生物学方法，以 了解外囊复合物的分子结构和功能。我们研究外囊蛋白 从模式生物酿酒酵母利用丰富的遗传，细胞生物 和生物化学技术，但已扩展到其他生物体的外囊。我们的研究旨在： 1)通过体外生化研究绘制外囊复合物的功能组织图，并分析 突变体，以测试外囊在体内的功能;（2）确定整个外囊复合物的三维结构 使用电子显微镜，晶体学和分子建模;（3）观察外囊系链囊泡 在单分子水平上分析束缚的要求，以及（4）剖析外囊的作用 和Sec 1在SNARE复合物组装和膜融合中的作用。因为这些蛋白质是保守的 这项研究将推进我们对分泌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.