The Role of Doa4 in Membrane Protein Trafficking

Doa4 在膜蛋白运输中的作用

• 批准号: 6904564
• 负责人: ALEXANDER AMERIK
• 金额: $ 15.6万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-03 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6904564
• 关键词: SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; affinity chromatography; enzyme activity; fungal proteins; gene interaction; membrane proteins; molecular cloning; peptidases; polymerase chain reaction; protein degradation; protein structure function; protein transport; site directed mutagenesis; ubiquitin; vesicle /vacuole; yeast two hybrid system

DESCRIPTION (provided by applicant): Proteolysis plays an important role in numerous cellular processes. Alteration in the abundance of critical regulatory proteins frequently leads to a variety of diseases including several forms of cancer. In eukaryotes, conjugation of ubiquitin to many short-lived proteins is an obligatory step in their degradation. The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is structurally related to the human tre-2 oncogene product. This enzyme is required for rapid degradation of ubiquitin-proteosome pathway substrates. In order to characterize Doa4 further, twelve extragenic suppressors of the doa4- 1 allele were isolated in a genetic screen aimed at the identification of spontaneous pseudorevertants. These involve seven different genes, of which six were cloned. Surprisingly, all six of these DID (Doa4-independent degradation) genes encode components of the vacuolar-protein-sorting (Vps) pathway. In particular, all of the proteins are class E Vps factors, which are necessary for maturation of a prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Genetic interaction between mutations in DOA4 and components of Vps pathway and physical interaction between corresponding proteins suggest that the enzyme is involved in protein sorting. Particularly, Doa4 may be responsible for deubiquitination of endocytosed membrane proteins prior to degradation in the vacuole. The main goal of this project is to characterize the relationship between the Vps pathway and the deubiquitinating enzyme Doa4. To achieve this aim, genetic and biochemical approaches will be used. Specifically: 1. Association of Doa4 with components of the Vps pathway will be studied by the methods of column chromatography and two-hybrid analysis. 2. Elements of the amino acid sequence of Doa4 involved in the interaction with components of the Vps pathway will be identified by site-directed mutagenesis. 3. Proteins necessary for suppression of the doa4 degradation defect by mutations of Vps factors will be identified by mutagenesis, using chemical and genetic approaches.

描述（由申请人提供）：蛋白水解在 许多细胞过程。 大量关键的改变 调节蛋白经常导致各种疾病 几种形式的癌症。 在真核生物中，泛素结合到许多 短寿命蛋白是其降解的强制性步骤。 这 酿酒酵母DOA4基因编码一种去泛素化酶 与人类TRE-2致癌基因产物结构相关。 这种酶是 快速降解泛素 - 蛋白酶途径底物所必需的。 在 为了进一步表征doa4，doa4-的十二个外部抑制剂 在旨在鉴定的遗传筛查中分离出1个等位基因 自发的伪逆想者。 这些涉及七个不同的基因，其中 六个被克隆。 令人惊讶的是，所有六个都做到了（独立于DOA4 降解）基因编码液泡 - 蛋白质分类（VPS）的成分 路径。 特别是，所有蛋白质都是E类VPS因素， 对于将prevacuolol隔室成熟到多孔中是必需的 然后与液泡融合的身体。 突变之间的遗传相互作用 在DOA4和VPS途径的组件和物理互动之间 相应的蛋白质表明该酶参与蛋白质分类。 特别是，doa4可能是对内吞的去泛素化的原因 在液泡中降解之前，膜蛋白。 主要目标 项目是为了表征VPS途径与 去泛素化酶DOA4。 为了实现这一目标，遗传和生化 将使用方法。 具体：1。doa4与 VPS途径的组件将通过列的方法研究 色谱和两性杂交分析。 2。氨基酸的元素 与VPS组件相互作用的DOA4序列 途径将通过定点诱变来识别。 3。蛋白质 通过VPS突变抑制DOA4降解缺陷所必需的 使用化学和遗传学将通过诱变来识别因素 方法。