Mutations in PINK1 and Parkin impair ubiquitination of Mitofusins in human fibroblasts

Aleksandar Rakovic; Anne Grünewald; Jan Kottwitz; Norbert Brüggemann; Peter P Pramstaller; Katja Lohmann; Christine Klein

doi:10.1371/journal.pone.0016746

Mutations in PINK1 and Parkin impair ubiquitination of Mitofusins in human fibroblasts

PLoS One. 2011 Mar 8;6(3):e16746. doi: 10.1371/journal.pone.0016746.

Authors

Aleksandar Rakovic¹, Anne Grünewald, Jan Kottwitz, Norbert Brüggemann, Peter P Pramstaller, Katja Lohmann, Christine Klein

Affiliation

¹ Section of Clinical and Molecular Neurogenetics, Department of Neurology, University of Lübeck, Lübeck, Germany.

Abstract

PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H(2)O(2) resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
Fibroblasts / drug effects
Fibroblasts / metabolism*
GTP Phosphohydrolases / metabolism*
Humans
Hydrogen Peroxide / pharmacology
Leupeptins / pharmacology
Macrolides / pharmacology
Membrane Proteins / metabolism*
Membrane Transport Proteins / metabolism*
Mitochondria / drug effects
Mitochondria / metabolism
Mitochondrial Membrane Transport Proteins
Mitochondrial Proteins / metabolism*
Models, Biological
Mutation / genetics*
Oligopeptides / pharmacology
Proteasome Endopeptidase Complex / metabolism
Protein Kinases / genetics*
Protein Transport / drug effects
Ubiquitin / metabolism
Ubiquitin-Protein Ligases / genetics*
Ubiquitination* / drug effects
Valinomycin / pharmacology

Substances

Leupeptins
Macrolides
Membrane Proteins
Membrane Transport Proteins
Mitochondrial Membrane Transport Proteins
Mitochondrial Proteins
Oligopeptides
Ubiquitin
bafilomycin A
Valinomycin
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Hydrogen Peroxide
Ubiquitin-Protein Ligases
parkin protein
Protein Kinases
PTEN-induced putative kinase
Proteasome Endopeptidase Complex
GTP Phosphohydrolases
MFN2 protein, human
Mfn1 protein, human
benzyloxycarbonylleucyl-leucyl-leucine aldehyde
epoxomicin