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. 2019 Aug 14;9:11829. doi: 10.1038/s41598-019-47352-9

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Parkin modulates mitochondrial import through its E3 ubiquitin-protein ligase activity induced by PINK1. Effect of relief of Parkin autoinhibition by (A) the activating W403A substitution, or by mimicking the PINK1-mediated phosphorylation of Parkin UBL (S65E) or (B) Ub (S65D), showing exacerbation of the Parkin-dependent enhancement of the bioluminescent signal of Probe 2 in HEK293T cells. By contrast, non-phosphorylatable S65A Parkin or Ub variants abolish the effect of Parkin. (C) In these cells, Ub S65D mimics the effect of Parkin overproduction in a manner dependent on endogenous Parkin (Parkin siRNA). (D) Effect of the lysine-less Ub K0 variant on the intensity of the bioluminescent signal of Probe 2 in HEK293T, indicating that the effect of Parkin is mediated by polyubiquitylation. (E) Quantification by UB-AQUA proteomics of individual Ub chain linkage types associated with mitochondria in HeLa Flp-In T-REx Parkin^WT or Parkin^C431S cells depleted or not of PINK1 and expressing Probe 2 in the presence of Shield1 (P + S), or treated with S or AO. (F) Immunoblot analysis of ubiquitylated proteins pulled down with TUBEs from mitochondrion-enriched fractions analyzed in (E) shows ubiquitylation of the TOM subunits TOM20, TOM22 and TOM70 in cells expressing Probe 2 (P + S). (G) Analysis of the impact of the mitochondrial ubiquitin-specific protease USP30 or its inactive C77A variant on the signal of Probe in the presence or absence of overexpressed Parkin. n = 4 (A,B,G), 5 (D) or 6 (C) independent experiments. Results are expressed as means ± SEM. Data were analyzed by one-way (A) or two-way ANOVA (B,C,G) with Dunnett’s or Holm-Sidak’s posthoc tests. *p < 0.05, **p < 0.01, ***p < 0.001. ns: non-significant.