Characterizing the mechanism of mitochondrial fission

线粒体是不断发生分裂和融合的动态细胞器。这些动态事件 调节线粒体的大小和形状，并在细胞生理中发挥关键作用，如分裂和融合 蛋白质参与了细胞凋亡的调节，而人类融合蛋白中的突变 与神经退行性疾病有关。在酵母中，至少有三种蛋白质是必需的。 线粒体分裂：DNM1、MDV1和FISL有趣的是，其中两种蛋白质的同源基因存在于 哺乳动物细胞表明，线粒体分裂的分子过程是保守的。 最近在酵母中的研究表明，动力蛋白相关的GTP酶DNM1在 通过自组装和构象变化进行裂变，并且DNM1结构中的这些变化可能是 受Mdv1和FIS1调节。详细了解DNM1、MDv1和FIS1如何组装成 一台有裂变能力的机器，以及一旦机器建成，蛋白质是如何共同发挥作用的 介导线粒体的收缩和最终分裂，线粒体的分裂事件将 在体外重组。这将包括纯化蛋白质的流体动力学分析，DNM1组装和 GTP酶在Mdv1和Fis1存在下的测定，以及在脂质双核上裂解机器的重建。 层次感。这些研究将定义最小的裂变机器，并确定每一次裂变的作用 蛋白质、核苷酸结合和流感病毒DNM1在分裂中。

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.