Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase.
Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase.
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DOI：
10.1074/jbc.m114.563031
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发表时间：
2014-05-23
期刊：
影响因子：
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通讯作者：
Kittler JT
中科院分区：
文献类型：
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作者：
Birsa N;Norkett R;Wauer T;Mevissen TE;Wu HC;Foltynie T;Bhatia K;Hirst WD;Komander D;Plun-Favreau H;Kittler JT
背景：MIRO是一种参与线粒体运输的线粒体蛋白。结果：在Parkin中，线粒体损伤以依赖Ser-65的方式驱动Miro的快速泛素化；然而，Miro的降解被延迟。结论：Miro的泛素化，而不是其降解，可能是线粒体停止和清除的信号。意义：线粒体运输机制的紊乱可能与帕金森病有关。线粒体转运在使线粒体分布与局部能量产生和钙缓冲需求相匹配方面起着重要作用。在这里，我们证明了在人多巴胺能神经母细胞瘤细胞中，线粒体膜外膜(OMM)蛋白Miro1是PINK1/Parkin线粒体质量控制系统的底物。此外，在含有PARK2基因(编码Parkin)致病突变的帕金森病(PD)患者来源的成纤维细胞中，受损线粒体上的Miro1翻转发生了变化。通过分析Miro1泛素化的动力学，我们进一步证明线粒体损伤触发OMM上快速的(在几分钟内)和持久的Lys-27型泛素化，依赖于PINK1和Parkin。然后，在泛素化开始的2-3小时内，在较慢的时间尺度上看到Miro1的蛋白酶体降解。我们发现，多巴胺能神经母细胞瘤细胞中的Miro泛素化不依赖于Ser-156处的Miro1磷酸化，但依赖于最近发现的Parkin中被PINK1磷酸化的Ser-65残基。有趣的是，我们发现Miro1可以稳定OMM上的Parkin的磷酸化突变版本，这表明Miro也是Parkin受体复合体的一部分。此外，我们证明了Parkin中的Ser-65在调节啮齿动物皮质神经元线粒体损伤时MIRO水平方面起关键作用。我们的结果为PINK1/Parkin对MIRO介导的线粒体运输机制的泛素化依赖的调控提供了新的见解，也表明这一调控的破坏可能与帕金森病的发病有关。
Background: Miro is a mitochondrial protein involved in mitochondrial trafficking. Results: Mitochondrial damage drives rapid Miro ubiquitination in a manner dependent on Ser-65 in Parkin; however, Miro degradation is delayed. Conclusion: Ubiquitination of Miro, rather than its degradation, could act as a signal for mitochondrial arrest and clearance. Significance: Disruption of the mitochondrial transport machinery could be implicated in Parkinson disease. Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2–3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.