title: Parkin interacts with Ambra1 to induce mitophagy.
abstract: Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy.
authors.affiliation: Department of Experimental Neurology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
authors.forename: Cindy
authors.initials: C
authors.lastname: Van Humbeeck
authors.forename: Tom
authors.initials: T
authors.lastname: Cornelissen
authors.forename: Hilde
authors.initials: H
authors.lastname: Hofkens
authors.forename: Wim
authors.initials: W
authors.lastname: Mandemakers
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.forename: Bart
authors.initials: B
authors.lastname: De Strooper
authors.forename: Wim
authors.initials: W
authors.lastname: Vandenberghe
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001343:Autophagy / Q000235:genetics / Q000502:physiology*; D001921:Brain / Q000378:metabolism*; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C521754:Ambra1 protein, mouse; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein