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

描述（由申请人提供）：线粒体蛋白质组由约1500种蛋白质组成，其中大部分在核DNA中编码，在胞质核糖体上合成，并在细胞器后靶向细胞器。线粒体内膜的TIM 23复合物介导这些蛋白质中的大多数的输入。蛋白质输入的初始阶段涉及由Tim 23和Tim 50亚基的可溶性结构域组成的受体复合物。这种受体不仅协调识别靶向的多肽，而且还有助于维持内膜电位和线粒体酶的加工。尽管其在细胞器生物发生和人类疾病的相关性，TIM 23受体调节蛋白质输入的早期步骤的分子机制知之甚少。利用模型膜结构的创新和结合先进荧光光谱、分子建模和结构生物学技术的实验策略，该研究计划将研究支持TIM 23受体运作的蛋白质和脂质相互作用网络。在目标1中，我们 分析不同条件下Tim 50和Tim 23亚基之间的动态关联。在organello系统和可溶性纳米脂质膜模型中，我们将采用位点特异性交联和荧光测量来阐明这些受体亚基的关联如何受底物，膜电位和特定脂质的调节。这些结果将解决TIM 23受体与靶底物特异性接触的分子机制以及受体如何门控转运子通道进行输入。在目标2中，我们将研究受体的脂质相互作用 亚单位。基于我们的初步工作表明Tim 23和Tim 50上的特定位点与含有线粒体特异性脂质心磷脂的双层结合，我们将分析与这种脂质的相互作用如何诱导这些蛋白质的结构变化以及这对受体的操作有什么影响。这些结果将为蛋白质-脂质的作用定义一个新的范例 在调节蛋白质输入的相互作用，并提供深入了解遗传的分子基础 与线粒体脂质生物合成缺陷有关的疾病。在目标3中，我们将采用一系列独立的方法来确定组装的Tim 23-Tim 50受体复合物的高分辨率结构。在受体形成后发生的这些同源结合配偶体的构象变化将在其与脂质和底物相互作用的背景下进行解释。这项工作将严格测试和完善我们的TIM 23复合物介导的蛋白质输入的初始步骤的统一工作模型。从更广泛的角度来看，这项研究将为细胞蛋白质运输和线粒体生物合成的调节提供许多重要的见解。

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.