Molecular Cell
Volume 77, Issue 5, 5 March 2020, Pages 1124-1142.e10
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Global Landscape and Dynamics of Parkin and USP30-Dependent Ubiquitylomes in iNeurons during Mitophagic Signaling

https://doi.org/10.1016/j.molcel.2019.11.013Get rights and content
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Highlights

  • Global phospho and ubiquitylome analysis of PINK1-Parkin pathway in iNeurons

  • Dynamics and specificity of Parkin-mediated ubiquitylation revealed in iNeurons

  • p97-mediated MFN turnover not required for Parkin substrate “gating” in iNeurons

  • USP30 acts primarily on translocon and supports import quality control in iNeurons

Summary

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.

Keywords

Parkin
ubiquitin
ubiquitin ligase
mitophagyĆ
USP30
deubiquitylating enzyme
p97
quantitative proteomics
Ub-clipping

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Present address: Merck, West Point, PA 19486, USA

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