Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Department of Molecular Machines and Signaling, Max Planck Institute of Biochemistry, Martinsried 82152, Germany; Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Mol Cell. 2020 Mar 5;77(5):1124-1142.e10. doi: 10.1016/j.molcel.2019.11.013.
The ubiquitin ligase Parkin, protein kinase PINK1, USP30 deubiquitylase, and p97 segregase function together to regulate turnover of damaged mitochondria via mitophagy, but our mechanistic understanding in neurons is limited. Here, we combine induced neurons (iNeurons) derived from embryonic stem cells with quantitative proteomics to reveal the dynamics and specificity of Parkin-dependent ubiquitylation under endogenous expression conditions. Targets showing elevated ubiquitylation in USP30 iNeurons are concentrated in components of the mitochondrial translocon, and the ubiquitylation kinetics of the vast majority of Parkin targets are unaffected, correlating with a modest kinetic acceleration in accumulation of pS65-Ub and mitophagic flux upon mitochondrial depolarization without USP30. Basally, ubiquitylated translocon import substrates accumulate, suggesting a quality control function for USP30. p97 was dispensable for Parkin ligase activity in iNeurons. This work provides an unprecedented quantitative landscape of the Parkin-modified ubiquitylome in iNeurons and reveals the underlying specificity of central regulatory elements in the pathway.
泛素连接酶 Parkin、蛋白激酶 PINK1、USP30 去泛素化酶和 p97 分离酶共同作用，通过线粒体自噬来调节受损线粒体的周转，但我们对神经元中的机制理解有限。在这里，我们将胚胎干细胞衍生的诱导神经元（iNeurons）与定量蛋白质组学相结合，揭示了内源性表达条件下 Parkin 依赖性泛素化的动态和特异性。在 USP30 iNeurons 中显示出升高的泛素化的靶标集中在线粒体转位复合物的成分中，并且绝大多数 Parkin 靶标的泛素化动力学不受影响，这与线粒体去极化时 pS65-Ub 的积累和线粒体自噬通量的适度动力学加速相关，而没有 USP30。基础上，泛素化的转位复合物导入底物积累，表明 USP30 具有质量控制功能。p97 在 iNeurons 中对 Parkin 连接酶活性不是必需的。这项工作提供了 iNeurons 中 Parkin 修饰的泛素组的前所未有的定量图谱，并揭示了该途径中中央调控元件的基础特异性。
