The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells

M Jacoupy,M J Millan,E Hamon-Keromen,D Guedin,J A Boutin,C A Gautier,J.-C Corvol,J W Harper,F Coge,O Nosjean,O Corti,C Mannoury La Cour,Z Erpapazoglou,A Ordureau,A Brice

doi:10.1038/s41598-019-47352-9

Abstract

Most of over a thousand mitochondrial proteins are encoded by nuclear genes and must be imported from the cytosol. Little is known about the cytosolic events regulating mitochondrial protein import, partly due to the lack of appropriate tools for its assessment in living cells. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway with a quantitative, luminescence-based readout. This tool was used to explore the regulation of mitochondrial import by the PINK1 kinase-driven Parkin ubiquitin ligase, which is dysfunctional in autosomal recessive Parkinson’s disease. We show that mitochondrial import was stimulated by Parkin, but not by disease-causing Parkin variants. This effect was dependent on Parkin activation by PINK1 and accompanied by an increase in the abundance of K11 ubiquitin chains on mitochondria and by ubiquitylation of subunits of the translocase of outer mitochondrial membrane. Mitochondrial import efficiency was abnormally low in cells from patients with PINK1- and PARK2-linked Parkinson’s disease and was restored by phosphomimetic ubiquitin in cells with residual Parkin activity. Altogether, these findings uncover a role of ubiquitylation in mitochondrial import regulation and suggest that loss of this regulatory loop may underlie the pathophysiology of Parkinson’s disease, providing novel opportunities for therapeutic intervention.

Highlights

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We found that mitochondrial protein import efficiency was abnormally low in cells derived from Parkinson’s disease (PD) patients with PARK2 or PINK1 mutations, suggesting that the loss of this regulatory function of PINK1 and Parkin contributes to the pathophysiology of PD
Probe 2 import was not modified by a UBL domain-deficient Parkin variant, or by any of the nine PD-related variants that we previously found to be unable to associate with the the outer mitochondrial membrane (TOM) complex in Förster resonance energy transfer (FRET) experiments in cells treated with CCCP31 (Fig. 3C and Supplementary Fig. 2D)

Summary

Introduction

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The mitochondrial serine/threonine kinase PINK1 and the cytosolic ubiquitin (Ub)-protein ligase Parkin, which are dysfunctional in autosomal recessive PD, function together as a molecular quality control system, coupling the loss of mitochondrial import efficiency via the TOM machinery to the degradation of dysfunctional mitochondria (mitophagy) under conditions of severe mitochondrial stress[9]. This process involves the stabilization of PINK1 in close proximity to the TOM complex, the recruitment, www.nature.com/scientificreports/. Whether PINK1 and Parkin can modulate mitochondrial import has not been reported so far

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