The mechanism of Mgm1-mediated mitochondrial inner membrane fusion.

描述(申请人提供)：线粒体的动力学和形态由平衡的分裂和融合事件控制，而分裂和融合事件是由高度保守的动力蛋白相关蛋白(DRPs)介导的。在许多与年龄相关的疾病中观察到的线粒体动力学和形态的改变反映了这些事件在细胞生理学中的重要性，并且人类融合DRP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，值得深入研究。以前利用酿酒酵母强大的遗传工具进行的工作表明，线粒体融合既需要自组装，也需要融合DRPs的GTPase活性。我们的研究目的是利用酵母线粒体内膜融合DRP的MGM1来确定融合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，我们提出了以下的具体目标：(1)利用脂类单层辅助二维结晶技术分析具有不同核苷酸状态的野生型和突变体的mGm1的结构和核苷酸依赖的构象变化。晶体将通过透射电子显微镜观察。然后，基于其他DRP的高分辨率结构的Mgm1的同源模型将被对接到从2D晶体数据计算的电子密度中。(2)通过建立体外实验来研究不同的膜融合事件，如膜系留、膜变形和脂质混合，以确定引起膜融合的MGM1的分子活性。将纯化的mGM1重组为脂质体，结合靶向突变，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。脂质体聚集、形态和融合依赖的荧光去猝灭的依赖于mGM1的变化将被用来分析mGm1介导膜融合的中间步骤。这些研究不仅将阐明线粒体融合的基本机制，还可能阐明与神经退行性疾病相关的DRP突变的潜在病理。 与公共卫生相关：在癌症、糖尿病、心脏病和神经退行性疾病等各种与年龄相关的疾病中，线粒体动力学和形态发生改变，表明线粒体功能障碍。事实上，线粒体融合动力蛋白相关蛋白的突变直接与两种不同的神经退行性疾病和中风有关。线粒体融合动力蛋白相关蛋白调节线粒体的动力学和形态。这里提出的研究将有助于阐明在各种疾病状态下被破坏的生理过程的机制，以及潜在地解释融合蛋白突变是如何导致疾病的。

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. PUBLIC HEALTH RELEVANCE: Mitochondrial dynamics and morphology are altered, indicating mitochondrial dysfunction, in a variety of age-related diseases such as cancer, diabetes, heart disease and neurodegenerative diseases. In fact, mutations in mitochondrial fusion dynamin-related proteins, which regulate mitochondrial dynamics and morphology, have been directly linked to two distinct neurodegenerative diseases and stroke. The research proposed here will help elucidate the mechanism of a physiological process disrupted in a variety of disease states as well as potentially explain how mutations in fusion proteins lead to disease.