Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA.
J Cell Biol. 2013 Jan 21;200(2):163-72. doi: 10.1083/jcb.201210111. Epub 2013 Jan 14.
Genetic studies indicate that the mitochondrial kinase PINK1 and the RING-between-RING E3 ubiquitin ligase Parkin function in the same pathway. In concurrence, mechanistic studies show that PINK1 can recruit Parkin from the cytosol to the mitochondria, increase the ubiquitination activity of Parkin, and induce Parkin-mediated mitophagy. Here, we used a cell-free assay to recapitulate PINK1-dependent activation of Parkin ubiquitination of a validated mitochondrial substrate, mitofusin 1. We show that PINK1 activated the formation of a Parkin-ubiquitin thioester intermediate, a hallmark of HECT E3 ligases, both in vitro and in vivo. Parkin HECT-like ubiquitin ligase activity was essential for PINK1-mediated Parkin translocation to mitochondria and mitophagy. Using an inactive Parkin mutant, we found that PINK1 stimulated Parkin self-association and complex formation upstream of mitochondrial translocation. Self-association occurred independent of ubiquitination activity through the RING-between-RING domain, providing mechanistic insight into how PINK1 activates Parkin.
遗传研究表明，线粒体激酶 PINK1 和 RING 之间环 E3 泛素连接酶 Parkin 作用于同一路径。同时，机制研究表明 PINK1 可以将 Parkin 从细胞质招募到线粒体，增加 Parkin 的泛素化活性，并诱导 Parkin 介导的线粒体自噬。在这里，我们使用无细胞测定法重新构建了依赖 PINK1 的 Parkin 泛素化已验证的线粒体底物，即线粒体融合蛋白 1。我们表明 PINK1 在体外和体内都激活了 Parkin-泛素硫酯中间产物的形成，这是 HECT E3 连接酶的标志。Parkin HECT 样泛素连接酶活性对于 PINK1 介导的 Parkin 易位到线粒体和线粒体自噬至关重要。使用无活性的 Parkin 突变体，我们发现 PINK1 刺激 Parkin 自我缔合和线粒体易位之前的复合物形成。自我缔合不依赖于泛素化活性通过 RING 之间环结构域发生，为 PINK1 如何激活 Parkin 提供了机制见解。
