Analysis of Mitochondrial Protein Integration Mechanisms

描述(由申请人提供)： 几乎所有的线粒体蛋白质都是由核基因组编码的，并在翻译后输入细胞器。TIM23复合体介导前体蛋白跨线粒体内膜(IM)的移位，以及蛋白质整合到IM中；然而，这些过程的许多结构和机制方面尚不清楚。在接下来的拟议研究中，荧光和光反应探针将被整合到线粒体整合中间体和全长功能易位子组件的新生链中的特定位置。利用荧光光谱和光交联技术，将使用一种独特的实验方法来解决TIM23介导膜蛋白整合的几个基本问题。通过在确定的整合阶段直接监测分子环境、隔室位置和特定底物区域的相邻蛋白质，这项工作将产生对IM蛋白拓扑发生的所有阶段的高分辨率分析。同样，通过在TIM23易位子本身的特定区域定位探针，这项工作将提供有关转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的关键信息，以及门控机制的性质和身份。除了为整合过程提供机制和结构上的见解外，这种研究线粒体输入的新方法在线粒体致病研究中具有很强的实用潜力。

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.