Fungal mitochondrial porins and DNA polymerases

线粒体是真核细胞中产生能量的关键膜结合细胞器。线粒体的功能依赖于两层线粒体膜间代谢物的交换。我们的研究调查了孔蛋白的结构，孔蛋白是一种允许小的水溶性分子穿过外膜的蛋白质。该分子也可能参与程序性细胞死亡的调控，在正常发育和包括癌症在内的疾病状态中扮演重要角色。因此，为了确定蛋白质如何执行这些功能，以及它如何与细胞其他部分相互作用，了解孔蛋白结构是必不可少的。为此，我们使用了在大肠杆菌中过表达的神经孢子菌的重组孔蛋白。不同的洗涤剂系统正在被评估为在没有膜的情况下折叠孔蛋白的手段，以便通过核磁共振波谱（NMR）等方法进行结构测定。此外，我们正在研究在合成后将蛋白质引导到线粒体的信号，并确定蛋白质与膜两侧其他蛋白质相互作用的区域，从而可能有助于调节其功能。我们研究的第二部分涉及线粒体DNA复制。线粒体含有对所有专需氧生物都必不可少的小基因组。在真菌中，复制这种DNA的酶，线粒体DNA聚合酶，包含一个未知的功能域。该研究将研究酵母蛋白中该结构域的功能，最终目的是了解DNA聚合酶进行有效的、过程合成的不同方式。总之，这些项目有助于围绕这些基本细胞器的维护及其与细胞其余部分的相互作用的知识体系。

Mitochondria are membrane-bound organelles that are key to energy production in eukaryotic cells. The function of mitochondria depends on the exchange of metabolites across the two mitochondrial membranes. Our research investigates the structure of porin, a protein that allows the passage of small, water soluble molecules across the outer membrane. This molecule may also be involved in the regulation of programmed cell death, an important player in normal development, and in disease states including cancer. Therefore, an understanding of porin structure is essential in order to determine how the protein carries out these functions, and how it interacts with the rest of the cell. To this end, we are using recombinant porin from Neurospora, that is overexpressed in Escherichia coli. Different detergent systems are being evaluated as the means to fold porin in the absence of membranes, to allow structural determination by methods like nuclear magnetic resonance spectroscopy (NMR). In addition, we are investigating the signals that direct the protein to mitochondria following its synthesis, and determining the regions of the protein that interact with other proteins on both sides of the membrane, and thereby may contribute to the regulation of its function.      The second part of our research deals with mitochondrial DNA replication. Mitochondria contain small genomes that are essential to all obligately aerobic organisms. In fungi, the enzyme that replicates this DNA, the mitochondrial DNA polymerase, contains a domain of unknown function. The proposed research will investigate the function of this domain in the yeast protein, with the ultimate goal of understanding the different ways in which DNA polymerases can carry out effective, processive synthesis. Together, these projects contribute to the body of knowledge surrounding the maintenance of these essential organelles, and their interactions with the rest of the cell.