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）： 几乎所有线粒体蛋白均由核基因组编码，并在翻译后导入细胞器中。 TIM23 复合物介导前体蛋白穿过线粒体内膜 (IM) 的易位，以及蛋白质整合到 IM 中；然而，人们对这些过程的许多结构和机械方面知之甚少。在接下来的研究中，荧光和光反应探针将被纳入线粒体整合中间体和全长功能易位子组件新生链的特定位点。使用荧光光谱和光交联技术，将使用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。通过在定义的整合阶段直接监测分子环境、区室位置和特定底物区域的相邻蛋白质，这项工作将对 IM 蛋白质拓扑发生的所有阶段进行高分辨率分析。同样，通过将探针定位在 TIM23 易位子本身的特定区域内，这项工作将提供有关易位子动力学和与通道形成和底物输入相关的蛋白质相互作用以及门控机制的性质和身份的关键信息。除了提供整合过程的机制和结构见解之外，这种研究线粒体输入的新方法在线粒体致病研究的实际进展方面具有巨大的潜力。