Genetics of Secretion in Yeast

酵母分泌的遗传学

• 批准号: 7246602
• 负责人: PETER Jay NOVICK
• 金额: $ 40.91万
• 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 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7246602
• 关键词: Actins; Binding; Biochemical; Bypass; Cell membrane; Complex; Coupled; Cytoplasm; Cytoskeleton; Cytosol; Event; Genes; Genetic; Golgi Apparatus; Growth; In Vitro; Integral Membrane Protein; Localized; Location; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Motor; Mutation; Myosin Type V; Nature; Nucleotides; Numbers; Pathway interactions; Peripheral; Plasmids; Process; Protein Overexpression; Proteins; Reaction; Regulation; Role; SNAP receptor; Secretory Component; Secretory Vesicles; Site; Specificity; Staging; Structural Genes; Structure; Surface; System; Testing; Transport Vesicles; Vesicle; Work; Yeasts; base; loss of function; mutant; plasma protein Z; programs; rab GTP-Binding Proteins; receptor; reconstitution; trafficking

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 Gl'Pase Sec4 and its exchange protein Sec2. Tethering requires a complex containing Sec3, Sec5, Sec6, Sec8, SeclO, SeclS, 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 Seel, 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 vescle that is recognized by SeclS and may provide specificity to the tethering process. 2) We will determine if activation by Sec4 mediates the binding of Seel 5 to Seel 0 or the interaction of a Secl5/SeclO 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 Seel 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.

我们的研究已经确定了三个系统，它们共同作用以介导分泌囊泡的极化递送， 它们与胞吐部位的局部束缚以及它们与质膜的融合。交付需要行动， V型肌球蛋白、GluSec 4及其交换蛋白Sec 2。栓系需要一个含有Sec 3的复合物， Sec 5、Sec 6、Sec 8、Sec 10、Sec 15、Exo 70和Exo 84，称为外囊。它与Sec 4-GTP在 细胞膜上的囊泡和Rhol。融合需要SNARE、Snc、Sso和Sec 9以及Seel， 与组装的SNARE复合物相互作用的蛋白质。这项建议的重点是外囊复合体，其 组装及其与分泌囊泡和质膜的相互作用。具体而言： 1)我们将使用生物化学的方法来确定分泌囊泡的组成部分， SeclS并且可以为绑定过程提供特异性。 2)我们将确定Sec 4的激活是否介导Seel 5与Seel 0的结合或Seel 5与Seel 0的相互作用。 Sec 15/Sec 10亚复合物与Sec 5，我们将在体外重建该组装步骤。 3)我们将使用一系列方法来分析外囊组装的过程。我们将确定 图4显示了存在于sec 4突变体中的亚复合物，并测试了RhoI和Rho 3对组装的影响。 4)我们将在体外重建囊泡束缚，并测试Sec 4-GTP和每种蛋白质的需求。 外囊的亚基在拴系反应中。 5)我们将使用生物化学的方法来确定质膜的成分，结合到 外囊。 6)我们将确定Seel或Sso过表达是否可以绕过任何外囊结构的缺失， 基因，并确定对表面生长的空间调节的影响。 7)我们将测试外囊在催化SNARE复合物组装中的作用，并寻找 外囊的特定亚基与组分SNARE的相互作用。