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(申请人提供)：线粒体蛋白质组由大约1500个蛋白质组成，其中大部分编码在核DNA中，在胞质核糖体上合成，翻译后定位于细胞器。线粒体内膜的TIM23复合体介导了大部分这些蛋白质的输入。蛋白质输入的最初阶段涉及一个由TIM23和TIM50亚基的可溶性域组成的受体复合体。这种受体不仅协调线粒体靶向多肽的识别，而且帮助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，但它调节蛋白质输入的早期步骤的分子机制尚不清楚。利用模型膜结构的创新和结合先进的荧光光谱、分子建模和结构生物学技术的实验策略，这项研究计划将调查支撑TIM23受体运作的蛋白质和脂肪相互作用网络。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。在器官系统和可溶纳米级脂膜模型中，我们将使用特定位点的交联和荧光测量来阐明这些受体亚基的关联是如何受到底物、膜电位和特定脂类的调节的。这些结果将解决TIM23受体与目标底物进行特定接触的分子机制，以及受体如何控制转位蛋白通道的输入。在目标2中，我们将研究受体的脂类相互作用 亚单位。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类心磷脂的双层结合，我们将分析与这种脂类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-脂肪的作用定义一个新的范例 调节蛋白质输入的相互作用，并提供对可遗传性的分子基础的见解 与线粒体脂质生物生成缺陷有关的疾病。在目标3中，我们将使用许多独立的方法来确定组装的TIM23-Tim50受体复合体的高分辨率结构。在受体形成时发生的这些同源结合伙伴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的统一工作模型的初始步骤的TIM23复合体介导的蛋白质进口。从更广泛的角度来看，这项研究将为细胞蛋白运输和线粒体生物发生的调控提供许多关键的见解。