Characterizing the mechanism of mitochondrial fission

线粒体裂变机制的表征

基本信息

批准号：
7316866
负责人：
Laura L Lackner
金额：
$ 4.88万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-12-01 至 2009-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7316866
关键词：
Affect Apoptosis Binding Biological Assay Build-it Cell physiology Cellular biology Centrifugation Chemistry Chimeric Proteins Complex Constriction procedure Data Dynamin Eukaryota Eukaryotic Cell Event Fellowship Gel Chromatography Guanosine Triphosphate Phosphohydrolases Human Hydrolysis In Vitro Individual Institutes Link Lipid Bilayers Lipids Malignant Neoplasms Mammalian Cell Mediating Membrane Mitochondria Molecular Mutation Names Neurodegenerative Disorders Nucleotides Organelles Orthologous Gene Outer Mitochondrial Membrane Play Process Property Proteins Regulation Resolution Role Saccharomyces cerevisiae Shapes Structure Sucrose Surface System Therapeutic Work Yeasts inhibitor/antagonist insight intermolecular interaction protein function proteoliposomes reconstitution self assembly size small molecule small molecule libraries tool

项目摘要

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.

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，如裂变和融合蛋白质与细胞凋亡的调节有关，并且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，至少三种蛋白质是必需的线粒体裂变：Dnm1、Mdv1 和 Fisl 有趣的是，其中两种蛋白质的直系同源物存在于哺乳动物细胞表明线粒体裂变的分子过程非常保守。最近对酵母的研究表明，与动力相关的 GTPase Dnm1 在酵母过程中驱动膜收缩。通过自组装和构象变化进行裂变，Dnm1 结构中的这些变化可能是受 Mdv1 和 Fis1 监管。详细了解 Dnm1、Mdv1 和 Fis1 如何组装成具有裂变能力的机器，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，线粒体裂变事件将体外重构。这将包括纯化蛋白质的流体动力学分析、Dnm1 组装和在 Mdv1 和 Fis1 存在的情况下进行 GTPase 测定，以及在脂质双链上重建裂变机层。这些研究将定义最小裂变机并将确定每个裂变的作用蛋白质和核苷酸结合以及裂变中 Dnm1 的水解。