Determining the mechanism of mitochondrial outer membrane fusion

项目概要 该提案检查了线粒体融合蛋白，它催化线粒体束缚和外部 膜融合。线粒体形状是线粒体功能的核心，与线粒体的变化密切相关。 发育、压力和衰老过程中的细胞生理学。线粒体融合增加代谢产物， 防止线粒体过度降解，降低细胞对凋亡刺激的敏感性， 允许受损细胞器的功能补充。线粒体功能受损 细胞器连接性下降与神经退行性疾病和其他与年龄相关的疾病有关。 此外，编码线粒体融合机组件的基因突变会导致 周围神经病变、视神经萎缩、肌病和共济失调。线粒体分裂和线粒体 融合由保守的动力超家族蛋白介导。这个多样化的机械化学家族 GTPases 在整个过程中将自组装和构象变化与膜重塑事件结合起来 细胞。线粒体融合蛋白是线粒体外膜融合机器，必须在一个空间内顺式组装。 单膜，并反式穿过两个线粒体。分子细节，包括组成 这些组件、其形成的调节以及它们如何促进膜束缚和融合 不知道。我们利用线粒体融合蛋白功能的生化分析来解决这些知识空白。在 目标 1，我们将阐明控制线粒体融合蛋白组装成融合的变构调节机制 有能力的复合体。我们收集的功能变体和新颖方法将提供一个强大的工具 剖析每个线粒体融合蛋白功能域对顺式寡聚和反式复合物的贡献 形成。我们对线粒体融合蛋白 CMT2A 相关变体的研究表明，线粒体融合蛋白的分子缺陷 丝裂融合蛋白可以通过细胞内的胞质因子进行补偿。在目标 2 中，我们使用还原论方法 结合了我的实验室开发的细胞生物学、生物化学和重构测定法，以确定如何 细胞质线粒体融合蛋白效应子 Bax 改变线粒体融合蛋白组装来调节线粒体融合。这项研究 该计划利用了我们在线粒体生物学的细胞和生化分析方面的优势，并将产生 不仅对线粒体动力学而且对动力机制的基本见解 超家族蛋白质一般运作。

Project Summary This proposal examines the mitofusin proteins, which catalyze both mitochondrial tethering and outer membrane fusion. Mitochondrial shape is central to mitochondrial function and is closely linked to changes in cellular physiology during development, stress, and aging. Mitochondrial fusion increases metabolic production, protects against excessive degradation of mitochondria, reduces sensitivity of the cell to apoptotic stimuli and allows functional complementation of damaged organelles. Compromised mitochondrial function and decreased organelle connectivity are associated with neurodegeneration and other age-related diseases. Furthermore, mutations in the genes encoding components of the mitochondrial fusion machine cause peripheral neuropathy, optic atrophy, myopathy and ataxia. Both mitochondrial division and mitochondrial fusion are mediated by conserved dynamin superfamily proteins. This diverse family of mechanochemical GTPases couple self-assembly and conformational changes to membrane remodeling events throughout the cell. Mitofusin is the mitochondrial outer membrane fusion machine and must assemble both in cis within a single membrane, and in trans across two mitochondria. The molecular details, including of the composition of these assemblies, the regulation of their formation and how they contribute to membrane tethering and fusion are not known. We address these gaps in knowledge utilizing biochemical analyses of mitofusin function. In Aim 1, we will elucidate mechanisms of allosteric regulation that control assembly of mitofusin into fusion- competent complexes. Our collection of functional variants and novel approaches will provide a powerful tool in dissecting the contribution of each mitofusin functional domain to cis oligomerization and trans complex formation. Our work characterizing CMT2A-associated variants of mitofusin revealed that molecular defects of mitofusin can be compensated for by cytosolic factors in cells. In Aim 2, we use a reductionist approach that combines cell biology, biochemistry, and reconstituted assays developed in my lab to determine how the cytosolic mitofusin effector Bax alters mitofusin assembly to regulate mitochondrial fusion. This research program draws on our strengths in cellular and biochemical analysis of mitochondrial biology and will yield fundamental insights not only for mitochondrial dynamics but for the mechanisms through which dynamin superfamily proteins in general operate.