Structure and Function of the Exocyst Complex

描述(申请人提供)：真核细胞运输货物在亚细胞器之间，并向质膜分泌，使用小的膜结合囊泡为载体。囊泡运输和膜融合过程的调节对细胞的形态、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的蛋白质，包括参与膜融合过程的SNARE蛋白和Sec1，Rab和Rho GTP酶，以及称为胞外囊泡的八聚体拴系复合体。虽然胞囊复合体参与了许多不同的功能，涉及识别、拴系和SNARs组装和融合的质量控制，但这些功能在分子水平上都没有被很好地理解。我们正在使用生化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。我们研究了模式生物酿酒酵母的胞外蛋白，以利用现有的丰富的遗传、细胞生物学和生化技术。我们的研究目标是：1)通过体外生化研究绘制外囊复合体的功能结构图，并分析突变体以测试体内外囊的功能；(2)利用电子显微镜、结晶学和分子建模来确定整个外囊复合体的三维结构；(3)在单分子水平上观察外囊系留小泡，以分析系留的需要；(4)剖析外囊和Sec1在SNARE复合体组装和膜融合中的作用。由于这些蛋白质从酵母到人类神经元都是保守的，这项研究将促进我们对所有真核细胞如何调节分泌和生长的了解。

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.