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

膜蛋白作为线粒体结构和功能的决定因素，是NSERC资助的这个项目的重点。在前几个阶段，我们通过研究含有孔蛋白变体或缺乏孔蛋白的粗糙脉孢霉菌株，对与线粒体孔蛋白相关的广泛活动有了了解，也被称为电压依赖阴离子选择通道(VDAC)。拟议研究的第一个目标建立在这些信息和初步工作的基础上，以确定孔蛋白在脂质含量、生物物理性质和蛋白质复合体形成方面在线粒体外膜(MOM)的动态平衡中所起的作用。从这项工作中获得的信息将揭示膜中脂类和蛋白质之间的物理相互联系，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因素，建立在上一次授权期开发的从粗面新月夜蛾中分离高纯度孔蛋白的方法。详细的结构信息仅限于在大肠杆菌中表达后在洗涤剂中折叠的脊椎动物孔蛋白，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，对真菌孔蛋白的结构分析以及与脊椎动物孔蛋白的比较将揭示初级序列相似性很低的蛋白质如何实现类似的功能。这项拟议研究的最终目标是确定和表征可能参与线粒体药物外流的蛋白质(S)以及控制它们的调控系统。孔蛋白就像一个普通的孔，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。考虑到真菌在土壤中的栖息地，预计带有线粒体靶标的抗生素将穿过细胞器的内膜。我们假设存在类似于细菌外排的过程来从线粒体中移除抗生素，最近我们证明了分离的粗毛线虫线粒体可以能量依赖的方式外排出溴化乙锭，这是一种用于评估细菌药物外排的模型化合物。真核生物在复杂的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，对农业中的生物防治具有重要意义。综上所述，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运输分子的理解。通过这项工作，研究生和本科生将接受膜生物学方面的出色培训，为未来生命科学发现和应用研究的职业生涯做好重要准备。

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.