Genetics of Secretion in Yeast

酵母分泌的遗传学

• 批准号: 6913592
• 负责人: PETER Jay NOVICK
• 金额: $ 36.73万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6913592
• 关键词: biochemistry; cell component structure /function; cytogenetics; exocytosis; fungal genetics; fungal proteins; gene expression; gene mutation; guanine nucleotide binding protein; intracellular transport; membrane activity; protein protein interaction; secretory protein; vesicle /vacuole; yeasts

DESCRIPTION (provided by applicant): Our studies have defined three systems that work together to mediate the polarized delivery of secretor vesicles, their localized tethering to exocytic sites and their fusion with the plasma membrane. Delivery requires actin, a type V myosin, the GTPase Sec4 and its exchange protein Sec2. Tethering requires a complex containing Sec3, Sec5, Sec6, Sec8, SeclO, Secl5, Exo 70 and Exo84, termed the exocyst.It interacts with Sec4-GTP on the vesicles and Rhol on the plasma membrane. Fusion requires the SNAREs, Snc, Sso and Sec9 as well as Sec 1, a protein that interacts with the assembled SNARE complex. This proposal focuses on the exocyst complex, its assembly and its interactions with the secretory vesicles and plasma membrane. In specific: 1) We will use a biochemical approach to identify the component of the secretory vescie that is recognized by Secl 5 and may provide specificity to the tethering process. 2) We will determine if activation by Sec4 mediates the binding of Sec 15 to Sec 10 or the interaction of a Seci 5/Sec 10 subcomplex with Sec5 and we will reconstitute this assembly step in vitro. 3) We will analyze the process of exocyst assembly using a battery of approaches. We will identify the subcomplexes present in a sec4 mutant and test the effects of Rhol and Rho3 on assembly. 4) We will reconstitute vesicle tethering in vitro and test the requirements for Sec4-GTP and each of the subunits of the exocyst in the tethering reaction. 5) We will use a biochemical approach to identify the component of the plasma membrane that binds to the exocyst. 6) We will determine if Seci or Sso overexpression can bypass the deletion of any of the exocyst structural genes and determine the effects on the spatial regulation of surface growth. 7) We will test the role of the exocyst in catalyzing the assembly of the SNARE complex and look for interactions of specific subunits of the exocyst with component SNAREs.

描述(由申请人提供)：我们的研究定义了三个系统 它们共同调节分泌囊泡的极化传递， 它们与胞外部位的定位连接及其与血浆的融合 薄膜。传递需要肌动蛋白，一种V型肌球蛋白，GTPase Sec4及其 交换蛋白Sec2。系留需要一个包含Sec3，Sec5， Sec6，Sec8，Sec10，Sec15，Exo，Exo 70和Exo84，称为Exocys，它相互作用 囊泡上有Sec4-GTP，质膜上有Rho1。聚变需要 SNARES、SNC、SSO和Sec9以及与之相互作用的蛋白质Sec 1 组装好的圈套综合体。这项建议的重点是胞囊复合体，其 组装及其与分泌囊泡和质膜的相互作用。 具体地说： 1)我们将使用生化方法来鉴定 由Secl 5识别的分泌性囊泡，可提供特异性 系绳过程。 2)我们将确定Sec4的激活是否介导SEC 15与 SEC 10或SecI 5/SEC 10亚复合体与Sec5的相互作用，我们将 在体外重建这一组装步骤。 3)我们将使用一个电池来分析外囊组装的过程 接近了。我们将确定在sec4突变体中存在的亚复合体并进行测试 Rho3和Rho3对组装的影响。 4)我们将在体外重建小泡系留，并测试要求 用于连接反应中的Sec4-GTP和胞外囊的每个亚基。 5)我们将使用生化方法来鉴定血浆的成分 与外囊结合的膜。 6)我们将确定SECI或SSO过度表达是否可以绕过删除 任何外囊结构基因，并确定对空间的影响 对表面生长的调控。 7)我们将测试外囊在催化陷阱组装中的作用 复合体，并寻找胞外囊特定亚基与 组件陷阱。