Abstract
Pink1, a mitochondrial kinase, and Parkin, an E3 ubiquitin ligase, function in mitochondrial maintenance. Pink1 accumulates on depolarized mitochondria, where it recruits Parkin to mainly induce K63-linked chain ubiquitination of outer membrane proteins and eventually mitophagy. Parkin belongs to the RBR E3 ligase family. Recently, it has been proposed that the RBR domain transfers ubiquitin to targets via a cysteine∼ubiquitin enzyme intermediate, in a manner similar to HECT domain E3 ligases. However, direct evidence for a ubiquitin transfer mechanism and its importance for Parkin's in vivo function is still missing. Here, we report that Parkin E3 activity relies on cysteine-mediated ubiquitin transfer during mitophagy. Mutating the putative catalytic cysteine to serine (Parkin C431S) traps ubiquitin, and surprisingly, also abrogates Parkin mitochondrial translocation, indicating that E3 activity is essential for Parkin translocation. We found that Parkin can bind to K63-linked ubiquitin chains, and that targeting K63-mimicking ubiquitin chains to mitochondria restores Parkin C431S localization. We propose that Parkin translocation is achieved through a novel catalytic activity coupled mechanism.
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Acknowledgements
We thank Dr Zhuohua Zhang (Sanford-Burnham Medical Research Institute, USA) for Pink1-null and WT MEFs, and Dr Richard Youle (NINDS, NIH, USA) for helpful discussions. We thank other members of the Hunter lab for helpful discussions, Suzy Simon for assistance in picking MGC cDNA clones, and Jill Meisenhelder and Justin Zimmerman for laboratory support. This study was supported by NIH grants (CA14195, CA80100 and CA82683) to TH. TH is a Frank and Else Schilling American Cancer Society Professor, and the Renato Dulbecco Chair in Cancer Biology. XZ is supported by fellowship from California Institute of Regenerative Medicine.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
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(A) Mutation of the putative catalytic cysteine 431 to alanine in the Parkin RBR domain abrogated mitochondrial translocation and clearance. (PDF 158 kb)
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Zheng, X., Hunter, T. Parkin mitochondrial translocation is achieved through a novel catalytic activity coupled mechanism. Cell Res 23, 886–897 (2013). https://doi.org/10.1038/cr.2013.66
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DOI: https://doi.org/10.1038/cr.2013.66
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