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

线粒体蛋白质输入机制的亚基和脂质相互作用的研究

• 批准号: 8802921
• 负责人: NATHAN N ALDER
• 金额: $ 29.44万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2019-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8802921
• 关键词: Address; Affect; Binding; Biogenesis; Calorimetry; Cardiolipins; Cell Proliferation; Cereals; Complex; Computer Simulation; Coupled; DNA; Defect; Disease; Energy Metabolism; Enzymes; Fluorescence; Fluorescence Spectroscopy; Functional disorder; Indium; Inner mitochondrial membrane; Investigation; Link; Lipid Bilayers; Lipid Binding; Lipids; Maintenance; Maps; Mass Spectrum Analysis; Measurement; Mediating; Membrane; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Molecular Models; Molecular Structure; NMR Spectroscopy; Nature; Nuclear; Organelles; Pathway interactions; Phospholipids; Play; Process; Protein Import; Protein translocation; Proteins; Proteome; Regulation; Research; Research Technics; Resolution; Ribosomes; Roentgen Rays; Role; Site; Solutions; Sorting - Cell Movement; Staging; Structure; System; Testing; Thermodynamics; Work; Yeasts; base; biophysical techniques; cell growth; cell growth regulation; crosslink; human disease; innovation; insight; lipid metabolism; membrane model; molecular dynamics; molecular modeling; nanodisk; nanoscale; novel; operation; polypeptide; protein transport; public health relevance; receptor; receptor binding; reconstitution; structural biology; voltage gated channel

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.

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，并在翻译后靶向细胞器。线粒体内膜的 TIM23 复合物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，而且有助于维持内膜电位和线粒体酶的加工。尽管 TIM23 受体与细胞器生物发生和人类疾病相关，但人们对 TIM23 受体调节蛋白质输入早期步骤的分子机制知之甚少。该研究计划将利用模型膜结构的创新以及结合先进荧光光谱、分子建模和结构生物学技术的实验策略，研究支撑 TIM23 受体运作的蛋白质和脂质相互作用网络。在目标 1 中，我们将 分析不同条件下 Tim50 和 Tim23 亚基之间的动态关联。在细胞器系统和可溶性纳米级脂质膜模型中，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到底物、膜电位和特定脂质的调节。这些结果将解决 TIM23 受体与目标底物特异性接触的分子机制，以及受体如何门控易位子通道的输入。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 Tim23 和 Tim50 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何影响。这些结果将为蛋白质-脂质的作用定义一个新的范例 调节蛋白质输入的相互作用，并提供对遗传性分子基础的见解 与线粒体脂质生物发生缺陷有关的疾病。在目标 3 中，我们将采用一系列独立的方法来确定组装的 Tim23-Tim50 受体复合物的高分辨率结构。这些同源结合配偶体在受体形成时发生的构象变化将在它们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针对 TIM23 复合物介导的蛋白质导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发生的调节提供许多重要的见解。