Regulation of mitochondrial fission

描述：(申请人提供) 线粒体执行基本的细胞功能，这些功能受 细胞器的形态和动力学。拟议工作的目标是 了解基本的和复杂的拓扑结构所涉及的机制 线粒体分裂的过程，使用的是真核生物酿酒酵母。在……里面 酵母，我们已经证明线粒体的分裂是由 动力蛋白相关的GTP酶，Dnmlp，集中在点状结构中 线粒体膜收缩和分裂发生的部位。Human和C. 优雅的Dnm1p同源物也被证明控制线粒体分裂。 因此，阐明酵母线粒体分裂的机制将是 与人类细胞的裂变有关，最终将帮助我们了解 线粒体形态和拷贝数的变化如何影响 疾病的发展。 到目前为止，线粒体分裂机器的其他组件还没有 已经上报了。我们已经分离出三个新的核基因，这些基因需要 线粒体分裂，线粒体分裂的MDV1-MDV3。Mdvlp是一个 预测的可溶性胞浆蛋白，包含至少三个不同的 区域：一个新的NH2-末端区域；预测将形成一个 螺旋线圈结构；以及包含7个WD重复序列的C-端区。四 多组数据表明，Mdvlp与Dnm1p相互作用，介导线粒体 分裂：1)mdv1和dnm1等位基因遗传互作；2)双杂交分析 揭示了Mdvlp和Dmnlp之间的强烈相互作用，3)Mdvlp定位于 与线粒体相关的点状结构，类似于Dnmlp的模式， 4)Mdvlp定位于点状结构，而不是定位于 线粒体膜，需要Dnm1p功能。在dmdv1细胞中，Dnm1p是 与线粒体膜上的点状结构有关，但这些 结构不能调节裂变。因此，我们的数据表明， Mdvlp在裂变过程中的作用相对较晚，以刺激 依赖Dnmlp的线粒体膜收缩和/或分裂。这个 这项拨款中提出的实验将检验这一假说，并确定 利用细胞学、细胞学和生物化学相结合 遗传和生化方法。具体地说，我们将描述这一步骤 Mdvlp在裂变过程中的作用，决定了结构特征 线粒体分裂所需的mdv1p，mdv1p与Dnm1p的相互作用， 以及mdv1p在Delta DNM1细胞中的线粒体定位，鉴定和 鉴定mdvlp结合伙伴，克隆和鉴定新的分裂 组件。从长远来看，我们的目标是确定分子机制 通过在体外重组这一过程来实现线粒体的分裂。

DESCRIPTION:(provided by applicant) Mitochondria perform essential cellular functions that are influenced by the morphology and dynamics of the organelle. The goal of the proposed work is to understand the mechanisms involved in the fundamental and topologically complex process of mitochondrial fission, using the model eukaryote, S. cerevisiae. In yeast, we have shown that mitochondrial fission is mediated by the dynamin-related GTPase, Dnmlp, which is concentrated in punctate structures at sites where mitochondrial membrane constriction and fission occur. Human and C. elegans Dnm1p homologs also have been shown to control mitochondrial fission. Thus, elucidating the mechanism of mitochondrial fission in yeast will be relevant to fission in human cells and will ultimately help us to understand how changes in mitochondrial morphology and copy number contribute to the development of diseases. To date, additional components of the mitochondrial fission machinery have not been reported. We have isolated three novel nuclear genes required for mitochondrial fission, MDV1-MDV3 for Mitochondrial Division. Mdvlp is a predicted soluble cytosolic protein, containing at least three distinct regions: a novel NH2-terminal region; a middle region predicted to form a coiled-coil structure; and a C-terminal region containing 7 WD repeats. Four sets of data suggest that Mdvlp interacts with Dnm1p to mediate mitochondrial fission: 1)mdvl and dnm1 alleles genetically interact, 2) two-hybrid analysis reveals a strong interaction between Mdvlp and Dmnlp, 3) Mdvlp localizes to punctate structures associated with mitochondria, a pattern similar to Dnmlp's, and 4) localization of Mdvlp to punctate structures, but not to the mitochondrial membrane, requires Dnm1p function. In dmdv1 cells, Dnm1p is associated with punctate structures on mitochondrial membranes, but these structures are not able to mediate fission. Our data, therefore, suggest that Mdvlp functions relatively late in the fission process to stimulate Dnmlp-dependent mitochondrial membrane constriction and/or fission. The experiments proposed in this grant will test this hypothesis and determine the role of additional fission components using a combination of cytological, genetic and biochemical approaches. Specifically, we will characterize the step at which Mdvlp acts in the fission process, determine the structural features of Mdv1p required for mitochondrial fission, Mdv1p's interaction with Dnm1p, and the mitochondrial localization of Mdv1p in delta dnm1 cells, identify and characterize Mdvlp binding partners and, clone and characterize novel fission components. In the long term, our goal is to determine the molecular mechanism of mitochondrial fission by reconstituting this process in vitro.