Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke National Institutes of Health, Bethesda, MD, USA.
Autophagy. 2010 Nov;6(8):1090-106. doi: 10.4161/auto.6.8.13426.
Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.
线粒体随着衰老而受损，由此导致的线粒体功能障碍与许多疾病有关，包括帕金森病。我们最近的研究表明，与隐性帕金森病相关的 E3 泛素连接酶 Parkin，在其从细胞质易位到线粒体后，会导致线粒体自噬显著增加和线粒体分布发生变化。研究 Parkin 如何诱导这些变化可能有助于深入了解导致受损线粒体隔离和消除的机制。我们报告说，在 Parkin 从细胞质易位到线粒体后，Parkin（而非致病性突变体）促进线粒体底物的 K63 连接多泛素化，并募集泛素和 LC3 结合蛋白 p62/SQSTM1 到线粒体。在募集后，p62/SQSTM1 通过其 PB1 结构域介导功能失调的线粒体的聚集，其方式类似于其多泛素化蛋白的聚集。令人惊讶的是，与最近报道的泛素诱导的pexophagy 和 xenophagy 相反，p62 似乎对于线粒体自噬是可有可无的。同样，锚定在线粒体上的泛素足以募集 p62 并促进线粒体聚集，但不促进线粒体自噬。虽然 VDAC1（而非 VDAC2）在线粒体去极化后被泛素化，但我们发现 VDAC1 不能完全解释去极化后观察到的线粒体 K63 连接泛素免疫反应性，因为在 VDAC1/3-/- 小鼠胚胎成纤维细胞中也观察到了这种现象。此外，我们发现 VDAC1 和 VDAC3 对于 p62 的募集、线粒体聚集和线粒体自噬是可有可无的。这些结果表明，p62 在线粒体去极化后通过 Parkin 以不依赖于 VDAC1 的方式募集，线粒体被聚集。它们还表明，Parkin 底物除了 VDAC1 之外，其他蛋白质可能是线粒体自噬所必需的。
