Investigation of the Subunit and Lipid Interactions of the Mitochondrial Protein Import Machinery

描述（由申请人提供）：线粒体蛋白质组由大约1500种蛋白质组成，其中大部分编码于核DNA中，在细胞质核糖体上合成，翻译后靶向于细胞器。线粒体内膜的TIM23复合体介导大多数这些蛋白质的输入。蛋白质输入的初始阶段涉及由Tim23和Tim50亚基的可溶性结构域组成的受体复合物。该受体不仅协调线粒体靶向多肽的识别，而且还有助于维持内膜电位和线粒体酶的加工。尽管TIM23受体在细胞器生物发生和人类疾病中具有相关性，但其调控蛋白质进口早期步骤的分子机制尚不清楚。利用创新的模型膜结构和实验策略，结合先进的荧光光谱，分子建模和结构生物学技术，本研究计划将研究支撑TIM23受体运作的蛋白质和脂质相互作用网络。在Aim 1中，我们会

DESCRIPTION (provided by applicant): The mitochondrial proteome consists of about 1500 proteins, the majority of which are encoded in nuclear DNA, synthesized on cytosolic ribosomes, and post-translationally targeted to the organelle. The TIM23 complex of the mitochondrial inner membrane mediates the import of most of these proteins. The initial stages of protein import involve a receptor complex composed of the soluble domains of the Tim23 and Tim50 subunits. This receptor not only coordinates the recognition of mitochondria-targeted polypeptides, but also aids in the maintenance of the inner membrane potential and in the processing of mitochondrial enzymes. Despite its relevance in organelle biogenesis and human disease, the molecular mechanisms by which the TIM23 receptor regulates the early steps of protein import are poorly understood. Using innovations in model membrane constructions and an experimental strategy that combines advanced fluorescence spectroscopy, molecular modeling, and structural biology techniques, this research plan will investigate the network of protein and lipid interactions that underpin the operation of the TIM23 receptor. In Aim 1, we will analyze the dynamic associations between the Tim50 and Tim23 subunits under different conditions. With both in organello systems and soluble nanoscale lipid membrane models, we will employ site-specific crosslinking and fluorescence measurements to elucidate how the associations of these receptor subunits are regulated by substrate, membrane potential, and particular lipids. These results will resolve the molecular mechanisms by which the TIM23 receptor makes specific contact with targeted substrates and how the receptor gates the translocon channel for import. In Aim 2, we will investigate the lipid interactions of the receptor subunits. Based on our preliminary work indicating that specific sites on Tim23 and Tim50 bind to bilayers containing the mitochondria- specific lipid cardiolipin, we will analyze how interactio with this lipid may induce structural changes in these proteins and what implications this has for operation of the receptor. These results will define a novel paradigm for the role of protein-lipid interactions in regulating protein import, and offer insights into the molecular basis of heritable diseases linked to defects in mitochondrial lipid biogenesis. In Aim 3, we will employ a host of independent approaches to determine the high-resolution structure of the assembled Tim23-Tim50 receptor complex. The conformational changes of these cognate binding partners that occur upon formation of the receptor will be interpreted in the context of their interactions with lipids and substrate. This work will rigorously test and refine our unifying working model for the initial steps of TIM23 complex-mediated protein import. From a broader perspective, this research will give many critical insights into the regulation of cellular protein trafficking and mitochondrial biogenesis.