A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outer-membrane tethering and fusion
A catalytic domain variant of mitofusin requiring a wildtype paralog for function uncouples mitochondrial outer-membrane tethering and fusion
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DOI：
10.1074/jbc.ra118.006347
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发表时间：
2019-05-17
影响因子：
4.8
通讯作者：
Hoppins, Suzanne
中科院分区：
文献类型：
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作者：
Engelhart, Emily A.;Hoppins, Suzanne
线粒体融合素 (Mfns) 是与动力相关的 GTP 酶，可介导线粒体外膜融合，这是线粒体和细胞健康所需的过程。在 Mfn1 和 Mfn2 旁系同源物中，位于保守链上 GTPase 结构域中的保守苯丙氨酸（Phe-202 (Mfn1) 和 Phe-223 (Mfn2)）是该结构域核心芳香族网络的一部分。为了深入了解人们对 Mfn 介导的膜融合知之甚少的机制，我们在这里描述了与夏科-玛丽-图斯综合征病因相关的线粒体融合蛋白突变体。通过对细胞中线粒体结构和体外线粒体融合的分析，我们发现Mfn1或Mfn2中Phe-202转化为亮氨酸会降低异型复合物与Mfn1和Mfn2的融合活性，并消除同型复合物的融合活性。使用免疫共沉淀和天然凝胶分析，我们进一步剖析了线粒体融合的步骤，并证明突变变体具有正常的束缚活性，但损害了高阶核苷酸依赖性组装。这里观察到的束缚与膜融合的缺陷耦合表明，束缚后需要核苷酸依赖性线粒体融合蛋白自组装来促进膜融合。
Mitofusins (Mfns) are dynamin-related GTPases that mediate mitochondrial outer-membrane fusion, a process that is required for mitochondrial and cellular health. In Mfn1 and Mfn2 paralogs, a conserved phenylalanine (Phe-202 (Mfn1) and Phe-223 (Mfn2)) located in the GTPase domain on a conserved strand is part of an aromatic network in the core of this domain. To gain insight into the poorly understood mechanism of Mfn-mediated membrane fusion, here we characterize a Mitofusin mutant variant etiologically linked to Charcot-Marie-Tooth syndrome. From analysis of mitochondrial structure in cells and mitochondrial fusion in vitro, we found that conversion of Phe-202 to leucine in either Mfn1 or Mfn2 diminishes the fusion activity of heterotypic complexes with both Mfn1 and Mfn2 and abolishes fusion activity of homotypic complexes. Using coimmunoprecipitation and native gel analysis, we further dissect the steps of mitochondrial fusion and demonstrate that the mutant variant has normal tethering activity but impaired higher-order nucleotide-dependent assembly. The defective coupling of tethering to membrane fusion observed here suggests that nucleotide-dependent self-assembly of Mitofusin is required after tethering to promote membrane fusion.