Characterizing the mechanism of mitochondrial fission

线粒体是一种动态的细胞器，不断进行分裂和融合。这些动态事件 调节线粒体的大小和形状，并在细胞生理学中发挥关键作用，如分裂和融合 蛋白质与细胞凋亡的调节有关， 与神经退行性疾病有关在酵母S.至少有三种蛋白质是必不可少的 线粒体分裂：Dnm1，Mdv1和Fisl有趣的是，这些蛋白质中的两个的直系同源物存在于 哺乳动物细胞中，表明线粒体分裂的分子过程是很好的保守。 最近在酵母中的研究表明，动力蛋白相关的GTdom Dnm 1驱动膜收缩， 通过自组装和构象变化进行裂变，Dnm1结构的这些改变可能是 由Mdv1和Fis1调节。为了详细了解Dnm 1、Mdv1和Fis1如何组装成 一个有裂变能力的机器，一旦机器建成，蛋白质如何一起发挥作用， 介导线粒体的收缩和最终分裂，线粒体裂变事件将是 在体外重组。这将包括纯化蛋白质的流体动力学分析，Dnm 1组装和 在Mdv1和Fis1存在下的GTdR测定，以及在脂质双链体上的裂变机的重建， 层次。这些研究将定义最小的裂变机器，并将确定每个裂变的作用 蛋白质和核苷酸结合以及Dnm 1在分裂中的水解。

Mitochondria are dynamic organelles that continuously undergo fission and fusion. These dynamic events regulate the size and shape of mitochondria and play critical roles in cell physiology, as fission and fusion proteins have been implicated in the regulation of apoptosis and mutations in human fusion proteins have been linked to neurodegenerative diseases. In the yeast S. cerevisiae, at least three proteins are essential for mitochondrial fission: Dnm1, Mdv1, and Fisl Interestingly, orthologs of two of these proteins exist in mammalian cells indicating that the molecular processes underlying mitochondrial fission are well conserved. Recent studies in yeast suggest the dynamin related GTPase Dnm1 drives membrane constriction during fission by self-assembly and conformational changes, and that these alterations in Dnm1 structure may be regulated by Mdv1 and Fis1. To understand in mechanistic detail how Dnm1, Mdv1, and Fis1 assemble into a fission competent machine and, once the machine is constructed, how the proteins function together to mediate the constriction and ultimate division of mitochondria, mitochondrial fission events will be reconstituted in vitro. This will include hydrodynamic analysis of purified proteins, Dnm1 assembly and GTPase assays in the presence of Mdv1 and Fis1, and the reconstitution of the fission machine on lipid bi- layers. These studies will define the minimal fission machine and will determine the roles of each fission protein and of nucleotide binding and hvdrolvsis bv Dnm1 in fission.