title: Lysine 27 ubiquitination of the mitochondrial transport protein Miro is dependent on serine 65 of the Parkin ubiquitin ligase.
abstract: Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2-3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.
authors.affiliation: From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Nicol
authors.initials: N
authors.lastname: Birsa
authors.affiliation: From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom.
authors.forename: Rosalind
authors.initials: R
authors.lastname: Norkett
authors.affiliation: the Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Tobias
authors.initials: T
authors.lastname: Wauer
authors.affiliation: the Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: Tycho E T
authors.initials: TE
authors.lastname: Mevissen
authors.affiliation: the University College London Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom, and.
authors.forename: Hsiu-Chuan
authors.initials: HC
authors.lastname: Wu
authors.affiliation: the University College London Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom, and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Foltynie
authors.affiliation: the University College London Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom, and.
authors.forename: Kailash
authors.initials: K
authors.lastname: Bhatia
authors.affiliation: the Neuroscience Research Unit, Pfizer, Cambridge, Massachusetts 02139.
authors.forename: Warren D
authors.initials: WD
authors.lastname: Hirst
authors.affiliation: the Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Komander
authors.affiliation: the University College London Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom, and.
authors.forename: Helene
authors.initials: H
authors.lastname: Plun-Favreau
authors.affiliation: From the Department of Neuroscience, Physiology and Pharmacology, University College London, Gower Street, London WC1E 6BT, United Kingdom, j.kittler@ucl.ac.uk.
authors.forename: Josef T
authors.initials: JT
authors.lastname: Kittler
mesh_terms: D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal; D051336:Mitochondrial Membranes / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D008239:Lysine; D024101:Mitochondrial Proteins; D046988:Proteasome Endopeptidase Complex; D011494:Protein Kinases; D012694:Serine; D044767:Ubiquitin-Protein Ligases; D020741:rho GTP-Binding Proteins; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein; C473124:RHOT1 protein, human
keywords: Mitochondrial Transport; Mitophagy; Parkin; Parkinson Disease; Pink1; Ubiquitination