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由核基因组编码，并在细胞分裂后输入细胞器。TIM 23复合物介导前体蛋白跨线粒体内膜（IM）的易位，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知之甚少。在下面提出的研究中，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性易位子组件的新生链中的特定位点。使用荧光光谱和光交联技术，一个独特的实验方法将被用来解决几个基本问题，关于TIM 23介导的膜蛋白整合。通过直接监测的分子环境，隔室的位置，和特定的底物区域的相邻蛋白质在定义的整合阶段，这项工作将产生一个高分辨率的分析IM蛋白拓扑发生的所有阶段。类似地，通过将探针定位在TIM 23转位子本身的特定区域内，这项工作将提供关于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的见解，这种研究线粒体进口的新方法具有很强的潜力，在线粒体致病性研究的实际进展。

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.