Structure and Function of the Exocyst Complex

描述(申请人提供)：真核细胞利用小的膜结合囊泡在亚细胞细胞器之间运输货物，并将货物输送到质膜进行分泌。囊泡运输和膜融合过程的适当功能和特异性对于维持细胞的完整性、生长、细胞运动和分泌活动(如激素释放和神经传递)至关重要。囊泡在质膜上的运输和融合需要许多必需的蛋白质，包括参与膜融合过程的SNARE蛋白和Sec1p，Rab和Rho GTP酶，以及一个称为外囊的大型复合体。胞囊复合体参与了许多不同的功能：胞吐作用部位的选择；分泌小泡与这些部位的物理连接；与细胞骨架和细胞周期蛋白的通讯；以及调节SNARE蛋白的特异性和组装。所有这些都没有在分子水平上得到很好的理解。我们的目标是将生化和生物物理技术与遗传学和细胞生物学方法相结合，以了解胞囊复合体的分子功能。我们选择研究模式生物酿酒酵母的胞外蛋白，以便利用现有的丰富的遗传和细胞生物学技术。为了实现这一目标，我们正在：(I)通过体外生化和生物物理研究来研究胞囊复合体内蛋白质-蛋白质的相互作用，并通过特定突变体的鉴定来分析胞囊在体内的功能；(Ii)表征胞囊在SNARE复合体组装中的作用；以及(Iii)鉴定和表征几种新的胞囊和SNARE复合体组装的调节因子。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。 公共卫生相关性：生命依赖于适当的细胞生长和发育，从单细胞真核细胞的细胞生长和分裂等简单过程，到大脑中神经元之间非常复杂的相互作用。细胞的生长、发育、运动和通讯绝对需要适当的囊泡靶向和膜融合。这些基本细胞生物学过程的一个重要组成部分是胞囊复合体，它从酵母到人类都是保守的。我们对胞囊及其膜融合蛋白调控的研究将有助于从分子水平上理解酵母胞囊复合体的功能。此外，我们的研究还将导致许多结构、试剂和想法的发展，这些将是研究外囊复合体如何调节所有真核细胞分泌的有价值的工具。

DESCRIPTION (provided by applicant): Eukaryotic cells utilize small membrane-bound vesicles to transport cargo between subcellular organelles, and to the plasma membrane for secretion. The proper function and specificity of the vesicular transport and membrane fusion processes are crucial for maintenance of cellular integrity, growth, cellular movement and secretory events such as hormone release and neurotransmission. Vesicle transport and fusion at the plasma membrane require many essential proteins, including the SNARE proteins and Sec1p that are involved in the membrane fusion process, the Rab and Rho GTPases, and a large complex called the exocyst. The exocyst complex has been implicated in a number of different functions: selection of the site of exocytosis; physical tethering of secretory vesicles to these sites; communicating with cytoskeletal and cell cycle proteins; and regulating the specificity and assembly of the SNARE proteins. None of these are well understood at the molecular level. Our aim is to combine biochemical and biophysical techniques with genetics and cell biological methods in order to understand the molecular functions of the exocyst complex. We have chosen to study the exocyst proteins from the model organism Saccharomyces cerevisiae so as to take advantage of the wealth of genetic and cell biological techniques available. To accomplish this goal, we are i) investigating the protein- protein interactions within the exocyst complex through biochemical and biophysical studies in vitro and analyzing the function of the exocyst in vivo through characterization of specific mutants; (ii) characterizing the role of the exocyst in SNARE complex assembly; and (iii) identifying and characterizing several novel regulators of the exocyst and SNARE complex assembly. 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. PUBLIC HEALTH RELEVANCE: Life depends upon proper cellular growth and development, from simple processes such as cell growth and division of unicellular eukaryotes, to very complicated interactions between the neurons in the brain. Cellular growth, development, movement and communication absolutely require proper vesicle targeting and membrane fusion. An essential component of these fundamental cell biological processes is the exocyst complex, which is conserved from yeast to man. Our studies of the exocyst and its regulation of the membrane fusion proteins will lead to a molecular understanding of the function of the yeast exocyst complex. In addition, our research will also lead to the development of many constructs, reagents and ideas that will be valuable tools for studying how the exocyst complex regulates secretion in all eukaryotic cells.