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

基本信息

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
项目类别：
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

项目摘要

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）：线粒体蛋白质组由大约1500种蛋白质组成，其中大多数是在核DNA中编码的，在胞质核糖体上合成，并在后的the骨后靶向靶向细胞器。线粒体内膜的TIM23复合物介导了大多数这些蛋白质的进口。蛋白质进口的初始阶段涉及由TIM23和TIM50亚基的可溶性结构域组成的受体复合物。该受体不仅可以协调对线粒体靶向多肽的识别，还可以帮助维持内膜电位和线粒体酶的加工。尽管它与细胞器生物发生和人类疾病相关，但TIM23受体调节蛋白质进口的早期步骤的分子机制知之甚少。使用模型膜构建体中的创新和实验策略，该策略结合了晚期荧光光谱，分子建模和结构生物学技术，该研究计划将研究蛋白质和脂质相互作用网络，这些蛋白质和脂质相互作用支持TIM23受体的运行。在AIM 1中，我们将在不同条件下分析TIM50和TIM23亚基之间的动态关联。在有机链系统和可溶性纳米级脂质膜模型中，我们将采用特定位点的交联和荧光测量，以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TIM23受体与靶向底物的特定接触的分子机制，以及受体门如何进口的转运通道。在AIM 2中，我们将研究受体的脂质相互作用亚基。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，以及该蛋白质对受体的操作有什么影响。这些结果将定义蛋白质脂质作用的新型范式调节蛋白质进口的相互作用，并提供对可遗传的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AIM 3中，我们将采用多种独立方法来确定组装的TIM23-TIM50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伴侣的构象变化将在与脂质和底物的相互作用的背景下解释。这项工作将严格测试并完善我们的统一工作模型，以tim23复合物介导的蛋白进口的初始步骤。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节提供许多关键见解。