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      Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

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          Abstract

          Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased mitochondrial delivery to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1-light chain-3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modeling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an “eat-me” signal for the elimination of damaged mitochondria from neuronal cells.

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          Most cited references35

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          Nix is a selective autophagy receptor for mitochondrial clearance.

          Autophagy is the cellular homeostatic pathway that delivers large cytosolic materials for degradation in the lysosome. Recent evidence indicates that autophagy mediates selective removal of protein aggregates, organelles and microbes in cells. Yet, the specificity in targeting a particular substrate to the autophagy pathway remains poorly understood. Here, we show that the mitochondrial protein Nix is a selective autophagy receptor by binding to LC3/GABARAP proteins, ubiquitin-like modifiers that are required for the growth of autophagosomal membranes. In cultured cells, Nix recruits GABARAP-L1 to damaged mitochondria through its amino-terminal LC3-interacting region. Furthermore, ablation of the Nix:LC3/GABARAP interaction retards mitochondrial clearance in maturing murine reticulocytes. Thus, Nix functions as an autophagy receptor, which mediates mitochondrial clearance after mitochondrial damage and during erythrocyte differentiation.
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            Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts.

            Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.
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              Is Open Access

              PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

              Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nat. Cell Biol.
                Nature cell biology
                1465-7392
                1476-4679
                12 September 2013
                15 September 2013
                October 2013
                01 April 2014
                : 15
                : 10
                : 1197-1205
                Affiliations
                [1 ]Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213.
                [2 ]Department of Environmental and Occupational Health and Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, PA 15213.
                [3 ]Department of Critical Care Medicine and Safar Center for Resuscitation Research, University of Pittsburgh, Pittsburgh, PA 15213.
                [4 ]Department of Cell Biology and Physiology and Center for Biologic Imaging, University of Pittsburgh, Pittsburgh, PA 15213.
                [5 ]Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15213.
                [6 ]Department of Computational and Systems Biology, University of Pittsburgh, PA 15213.
                Author notes
                [* ]Correspondence to: Charleen T. Chu, ctc4@ 123456pitt.edu ; or Hülya Bayir, bayihx@ 123456ccm.upmc.edu ; or Valerian Kagan, kagan+@ 123456pitt.edu .
                Article
                NIHMS513865
                10.1038/ncb2837
                3806088
                24036476
                3674fa1f-a270-4b9e-86c3-0711b359a575

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                History
                Funding
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: U19 AI068021 || AI
                Funded by: National Institute of Allergy and Infectious Diseases Extramural Activities : NIAID
                Award ID: U19 AI068021 || AI
                Funded by: National Institute of Child Health & Human Development : NICHD
                Award ID: R21 HD057587 || HD
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Award ID: R21 ES021068 || ES
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS076511 || NS
                Funded by: National Institute of Neurological Disorders and Stroke : NINDS
                Award ID: R01 NS061817 || NS
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Award ID: R01 HL094488 || HL
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Award ID: R01 HL070755 || HL
                Funded by: National Institute of Environmental Health Sciences : NIEHS
                Award ID: R01 ES020693 || ES
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                Cell biology
                Cell biology

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