Analysis of Mitochondrial Protein Integration Mechanisms

线粒体蛋白整合机制分析

• 批准号: 6742195
• 负责人: NATHAN N ALDER
• 金额: $ 4.73万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6742195
• 关键词: crosslink; fluorescence spectrometry; fluorescent dye /probe; immunoprecipitation; membrane transport proteins; mitochondria; mitochondrial membrane; molecular assembly /self assembly; molecular dynamics; postdoctoral investigator; posttranslational modifications; protein protein interaction; protein quantitation /detection; protein structure function; protein transport; radiofluorescent probe; transposon /insertion element

DESCRIPTION (provided by applicant): Nearly all mitochondrial proteins are encoded by the nuclear genome and imported post-translationally into the organelle. The TIM23 complex mediates the translocation of precursor proteins across the inner membrane (IM) of mitochondria, as well as protein integration into the IM; however, many structural and mechanistic aspects of these processes are poorly understood. In the following proposed research, fluorescent and photoreactive probes will be incorporated into specific sites in nascent chains of mitochondrial integration intermediates and full-length functional translocon components. Using fluorescence spectroscopy and photocrosslinking techniques, a unique experimental approach will be used to address several fundamental issues regarding TIM23-mediated membrane protein integration. By directly monitoring the molecular environment, the compartmental location, and adjacent proteins of specific substrate regions during defined stages of integration, this work will yield a high-resolution analysis of all stages of IM protein topogenesis. Similarly, by positioning probes within specific regions of the TIM23 translocon itself, this work will provide key information regarding translocon dynamics and protein interactions associated with channel formation and substrate import, as well as the nature and identity of the gating mechanism. In addition to providing mechanistic and structural insights into the integration process, this novel method of studying mitochondrial import has strong potential for practical advances in mitochondrial pathogenic research.

描述（由申请人提供）： 几乎所有的线粒体蛋白均由核基因组编码，并在后译为细胞器中进口。 TIM23复合物介导了前体蛋白在线粒体的内膜（IM）上的易位，以及蛋白质整合到IM中。但是，这些过程的许多结构和机械方面知之甚少。在以下提议的研究中，将在线粒体整合中间体和全长功能转运组件的新生链中将荧光和光反应性探针合并到特定位点中。使用荧光光谱和光链接技术，将使用一种独特的实验方法来解决有关TIM23介导的膜蛋白整合的几个基本问​​题。通过直接监测定义整合阶段的特定底物区域的分子环境，隔室位置和相邻的蛋白质，这项工作将对IM蛋白质topogenesis的所有阶段进行高分辨率分析。同样，通过将探针定位在TIM23转运本身的特定区域中，这项工作将提供有关转运动力学和蛋白质相互作用与通道形成和底物导入以及门控机制的性质和身份相关的关键信息。除了为整合过程提供机械和结构见解外，这种新的研究线粒体进口的新方法具有强大的线粒体致病性研究实践进步的潜力。