Insights into mitochondrial metabolism through structure and function analysis of outer and inner membrane transporters.

通过外膜和内膜转运蛋白的结构和功能分析深入了解线粒体代谢。

• 批准号: RGPIN-2016-05930
• 负责人: Court, Deborah
• 金额: $ 2.26万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683919
• 关键词: Insights; into; mitochondrial; metabolism; through

Membrane proteins, as determinants of mitochondrial structure and function, are the focus of this NSERC-funded program. In previous phases, we developed an understanding of the broad range of activities linked to mitochondrial porin, also known as the voltage-dependent anion-selective channel (VDAC), through investigation of strains of Neurospora crassa harbouring porin variants or lacking the protein. The first aim of the proposed research builds on this information and preliminary work to determine the role porin plays in the homeostasis of the mitochondrial outer membrane (MOM), in terms of lipid content, biophysical properties and protein complex formation. Information derived from this work will reveal the physical interconnections between lipid and protein in membranes that are critical to their function. The second aim is to determine the folded state of native porin and the sequence determinants thereof, building on the methods for isolation of highly purified porin from the N. crassa MOM that were developed in the last granting period. Detailed structural information is limited to vertebrate porins folded in detergent following expression in E. coli and the proposed structural studies will allow assessment of heterologously expressed porin as a model for the native form. In addition, structural analysis of a fungal porin and comparison with vertebrate porins will reveal how similar functions can be achieved by proteins of very low primary sequence similarity. The final aim of the proposed research is to identify and characterize protein(s) potentially involved in mitochondrial drug efflux and the regulatory systems that control them. Porin acts as a general pore, allowing small molecules access to the inner membrane and the transporters therein. This flow has the potential to allow toxins and antibiotics to enter mitochondria. Given the soil habitat of fungi, it is expected that antibiotics with mitochondrial targets will cross the inner membrane of the organelle. We hypothesized that processes similar to bacterial efflux exist for the removal of antibiotics from mitochondria, and recently we demonstrated that isolated N. crassa mitochondria can, in an energy-dependent manner, efflux ethidium bromide, a model compound used to assess bacterial drug efflux. The transport mechanisms used by eukaryotes to protect mitochondria in complex soil environments are important aspects of fungal biology and have implications for biocontrol in agriculture. Taken together, the information gleaned from the proposed studies will advance our understanding of mitochondrial membrane proteins as structural and transport molecules required for eukaryotic cell function. Through this work, graduate and undergraduate students will receive excellent training in membrane biology, important preparation for future careers in discovery and applied research in the life sciences.**

膜蛋白作为线粒体结构和功能的决定因素，是NSERC资助计划的重点。在以前的阶段，我们开发了一个广泛的活动与线粒体孔蛋白，也被称为电压依赖性阴离子选择性通道（VDAC）的理解，通过调查粗糙脉孢菌窝藏孔蛋白变体或缺乏蛋白质的菌株。拟议的研究的第一个目标建立在这些信息和初步工作，以确定在线粒体外膜（线粒体）的稳态中，在脂质含量，生物物理特性和蛋白质复合物的形成方面的作用孔蛋白。从这项工作中获得的信息将揭示膜中脂质和蛋白质之间的物理互连，这对它们的功能至关重要。第二个目的是确定天然孔蛋白的折叠状态及其序列决定簇，建立从N.上一个授予期开发的crassa MOM。 详细的结构信息仅限于脊椎动物孔蛋白在大肠杆菌中表达后在去污剂中折叠。大肠杆菌和拟议的结构研究将允许评估异源表达的孔蛋白作为天然形式的模型。此外，真菌孔蛋白的结构分析和与脊椎动物孔蛋白的比较将揭示相似的功能可以通过非常低的一级序列相似性的蛋白质来实现。拟议研究的最终目的是鉴定和表征可能参与线粒体药物外排的蛋白质以及控制它们的调控系统。孔蛋白作为一般的孔，允许小分子进入内膜和其中的转运蛋白。这种流动有可能使毒素和抗生素进入线粒体。鉴于真菌的土壤栖息地，预计具有线粒体靶点的抗生素将穿过细胞器的内膜。我们假设类似于细菌外排的过程存在于从线粒体中去除抗生素的过程中，最近我们证明了分离的N。crassa线粒体可以以能量依赖的方式外排溴化乙锭，溴化乙锭是用于评估细菌药物外排的模型化合物。真核生物在复杂土壤环境中保护线粒体的运输机制是真菌生物学的重要方面，对农业生物防治具有重要意义。总之，从拟议的研究中收集的信息将促进我们对线粒体膜蛋白作为真核细胞功能所需的结构和转运分子的理解。通过这项工作，研究生和本科生将获得膜生物学方面的优秀培训，为未来在生命科学领域的发现和应用研究做好重要准备。