Characterizing the mechanism of mitochondrial fission

描述（由申请人提供）：线粒体是不断进行裂变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，并在细胞生理学中发挥关键作用，因为裂变和融合蛋白与细胞凋亡的调节有关，而人类融合蛋白的突变与神经退行性疾病有关。在酿酒酵母中，至少有三种蛋白质对线粒体分裂至关重要：Dnm1、Mdv1和Fis1。有趣的是，这两种蛋白质的同源物存在于哺乳动物细胞中，这表明线粒体分裂的分子过程是很保守的。最近在酵母中的研究表明，与动力蛋白相关的GTPase Dnm1在分裂过程中通过自组装和构象变化驱动膜收缩，并且这些Dnm1结构的改变可能受到Mdv1和Fis1的调节。为了详细了解Dnm1、Mdv1和Fis1如何组装成一个能产生裂变的机器，以及一旦这个机器被构建，这些蛋白质如何一起起作用来介导线粒体的收缩和最终分裂，线粒体裂变事件将在体外重建。这将包括纯化蛋白的流体动力学分析，在Mdv1和Fis1存在下的Dnm1组装和GTPase分析，以及在脂质双层上重建裂变机器。这些研究将定义最小的裂变机器，并将确定每个裂变蛋白的作用，以及核苷酸结合和Dnm1在裂变中的水解。

DESCRIPTION (provided by applicant): 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 Fis1 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 hvdrolysis bv Dnm1 in fission.