A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division
A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division
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DOI：
10.1074/jbc.m110.142430
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发表时间：
2010-10-15
影响因子：
4.8
通讯作者：
Blackstone, Craig
中科院分区：
文献类型：
--
作者：
Chang, Chuang-Rung;Manlandro, Cara Marie;Blackstone, Craig
线粒体在细胞内动态地融合和分裂，并且融合和分裂的适当平衡对于正常的线粒体功能、形态和分布是必要的。Drp 1是哺乳动物细胞中线粒体分裂所需的动力蛋白相关的GT3。它具有四个不同的结构域：GTP结合、中间、插入B和GTP酶效应子。在患有小头畸形、异常脑发育、视神经萎缩和乳酸血症的新生儿中报道了Drp 1中间结构域内的致死突变（A395 D）（Waterham，H. R.，Koster，J.，货车鲁尔蒙德角W.，穆耶，宾夕法尼亚州，万德斯河J.，和伦纳德，J. V.（2007）N. Engl. 356，1736-1741）。患者来源的成纤维细胞内的线粒体明显延长，但这些发现背后的分子机制尚未得到证实。由于中间结构域对于一些发动蛋白超家族蛋白的自组装特别重要，我们测试了这一假设，即A395 D突变和另外两个中间结构域突变（G350 D，G363 D）对Drp 1四聚体，高阶组装和功能很重要。虽然四聚体似乎基本上是完整的，这些突变中的每一个都损害了高阶组装和组装依赖性刺激的Drp 1 GTdR活性。此外，突变体Drp 1蛋白表现出受损的定位到线粒体，这表明，这种高阶组装是重要的线粒体招聘，保留，或两者兼而有之。这些中间结构域突变体的过表达显着抑制细胞中的线粒体分裂。因此，Drp 1 A395 D致死性缺陷可能导致Drp 1在线粒体上的高阶组装受损，导致分裂减少、线粒体延长和线粒体细胞分布改变。
Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736-1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.