title: The ubiquitin-conjugating enzymes UBE2N, UBE2L3 and UBE2D2/3 are essential for Parkin-dependent mitophagy.
abstract: Depolarized mitochondria are degraded by mitophagy in a process that depends on the Parkinson's disease gene products PINK1 and Parkin. This is accompanied by ubiquitylation of several mitochondrial substrates. The roles of E2 ubiquitin-conjugating enzymes (UBE2) in mitophagy are poorly understood. Here, we investigate a set of UBE2 enzymes that might regulate Parkin-mediated mitophagy. Knockdown of the E2 enzymes UBE2N, UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly reduced autophagic clearance of depolarized mitochondria. However, this did not interfere with mitochondrial PINK1 stabilization and Parkin translocation. UBE2N knockdown prevented specifically K63-linked ubiquitylation at mitochondrial sites. Nevertheless, polyubiquitin and p62 (officially known as SQSTM1) were still found on mitochondria after individual UBE2 knockdown. Knockdown of all of these UBE2s together significantly reduced mitochondrial polyubiquitylation and p62 recruitment. Moreover, reduced ubiquitylation of mitofusins, the mitochondrial import receptor subunits TOM20 and TOM70, the voltage-dependent anion channel protein 1 and Parkin was observed in cells silenced for all of these UBE2s. A version of Parkin with a mutation in the active site (C431S) failed to ubiquitylate these mitochondrial substrates even in the presence of UBE2s. We conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically contribute to Parkin-mediated mitophagy.
authors.affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, 72076 Tübingen, Germany sven.geisler@uni-tuebingen.de philipp.kahle@uni-tuebingen.de.
authors.forename: Sven
authors.initials: S
authors.lastname: Geisler
authors.affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, 72076 Tübingen, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Vollmer
authors.affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, 72076 Tübingen, Germany.
authors.forename: Sonia
authors.initials: S
authors.lastname: Golombek
authors.affiliation: Laboratory of Functional Neurogenetics, Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, 72076 Tübingen, Germany German Center for Neurodegenerative Diseases, 72076 Tübingen, Germany sven.geisler@uni-tuebingen.de philipp.kahle@uni-tuebingen.de.
authors.forename: Philipp J
authors.initials: PJ
authors.lastname: Kahle
mesh_terms: D006367:HeLa Cells; D006706:Homeostasis; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D026901:Membrane Transport Proteins / Q000378:metabolism; D008928:Mitochondria / Q000502:physiology*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D063306:Mitophagy / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010300:Parkinson Disease / Q000201:enzymology* / Q000235:genetics; D011494:Protein Kinases / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics; D050995:Voltage-Dependent Anion Channel 1 / Q000378:metabolism; D000099202:PTEN-Induced Putative Kinase
chemical_list: D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011494:Protein Kinases; D034741:RNA, Small Interfering; D011956:Receptors, Cell Surface; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D050995:Voltage-Dependent Anion Channel 1; D000099202:PTEN-Induced Putative Kinase; C084008:TOMM20 protein, human; C402118:TOMM70 protein, human; C097309:UBE2D2 protein, human; C079437:UBE2L3 protein, human; C471607:UBE2N protein, human; C111567:parkin protein
keywords: Mitophagy; Parkin; Proteasome; UBE2; Ubiquitin