The mechanism of Mgm1-mediated mitochondrial inner membrane fusion.

描述（由申请人提供）：线粒体动力学和形态受平衡的裂变和融合事件控制，这些事件由高度保守的动力蛋白相关蛋白（DRP）介导。这些事件在细胞生理学中的重要性反映在许多年龄相关疾病中观察到的线粒体动力学和形态学改变，并且人融合DRP中的突变与神经退行性疾病和中风直接相关。此外，膜融合是DRPs的一种新的膜重塑功能，值得详细研究。以前的工作利用强大的遗传工具，在S。酿酒酵母的研究表明，线粒体融合需要融合DRP的自组装和GTP酶活性。本研究的目的是利用酵母线粒体内膜融合DRP基因Mgm1，探讨融合DRP功能和线粒体膜融合的分子机制。我们将利用开发的工具来研究Mgm1生物化学和重组膜融合。因此，提出了以下具体目标：（1）使用脂质单层辅助的Mgm 1野生型和具有代表不同核苷酸状态的核苷酸类似物的突变体的2D结晶来分析Mgm 1的结构和核苷酸依赖性构象变化。将通过透射电子显微镜观察晶体。然后，基于其他DRP的高分辨率结构的Mgm1同源模型将对接到从2D晶体学数据计算的电子密度中。(2)通过开发体外试验来研究不同的膜融合事件，如膜束缚、膜变形和脂质混合，确定引起膜融合的Mgm1的分子活性。纯化的Mgm1重组到脂质体中结合靶向诱变将允许分析自组装、GTP结合和水解以及膜结合对膜融合事件的贡献。Mgm1依赖的脂质体聚类，形态和融合依赖的荧光去猝灭的变化将被用来分析Mgm1介导的膜融合的中间步骤。这些研究不仅将阐明线粒体融合的基本机制，而且将潜在地阐明与神经退行性疾病相关的DRP突变的潜在病理学。

DESCRIPTION (provided by applicant): Mitochondrial dynamics and morphology are controlled by balanced fission and fusion events, which are mediated by highly conserved dynamin-related proteins (DRPs). The importance of these events in cellular physiology is reflected by the altered mitochondrial dynamics and morphology observed in many age-related diseases, and that mutations in human fusion DRPs have been directly linked to neurodegenerative diseases and stroke. Furthermore, membrane fusion is a novel membrane remodeling function of DRPs and warrants detailed investigation. Previous work utilizing the powerful genetic tools available in S. cerevisiae suggest that both self-assembly and GTPase activity of fusion DRPs are required for mitochondrial fusion. The objective of our proposed research is to use Mgm1, the yeast mitochondrial inner membrane fusion DRP, to determine the molecular mechanism of fusion DRP function and mitochondrial membrane fusion. We will take advantage of the tools developed to study Mgm1 biochemistry and reconstituted membrane fusion. Thus, the following specific aims are proposed: (1) Analyze the structure of Mgm1 and nucleotide-dependent conformational changes using lipid monolayer-assisted 2D crystallization of Mgm1 wild type and mutants with nucleotide analogs that represent distinct nucleotide states. Crystals will be observed by transmission electron microscopy. A homology model for Mgm1 based on high-resolution structures of other DRPs will then be docked into the electron density calculated from the 2D crystallographic data. (2) Determine the molecular activities of Mgm1 that give rise to membrane fusion by developing in vitro assays to study distinct membrane fusion events such as membrane tethering, membrane deformation, and lipid mixing. Purified Mgm1 reconstituted into liposomes in conjunction with targeted mutagenesis will allow the analysis of the contribution of self-assembly, GTP binding and hydrolysis, and membrane binding to membrane fusion events. Mgm1-dependent changes in liposome clustering, morphology, and fusion-dependent fluorescence dequenching will be used to analyze intermediate steps of Mgm1- mediated membrane fusion. These studies will not only illuminate the basic mechanism of mitochondrial fusion, but will potentially elucidate the underlying pathology of DRP mutations linked to neurodegenerative diseases.