Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination

doi: 10.1073/pnas.1909814117

Figure Lengend Snippet: Poly- and monoubiquitination on VDAC1 determine the fate of mitochondria to mitophagy or apoptosis, respectively. (A) Confocal images of the translocation of p62/SQSTM1 to mitochondria. VDAC1 KO MEF cells were transfected with HA-tagged VDAC1 WT, K274R, Poly-KR, or All-KR and treated with 30 µM CCCP for 8 h. Subcellular localizations of YFP-Parkin (green), VDAC1 (red), and p62/SQSTM1 (gray) were observed. (Scale bars, 20 µm.) (B) Percentage of the cells with p62/SQSTM1 localized to the mitochondria in A (n > 500 cells from three independent experiments). Data were analyzed by ANOVA Tukey test. **P < 0.01; ***P < 0.001. (C) Concentrations of mitochondrial proteins from the outer membrane (OM) and inner membrane (IM) in VDAC1 KO MEF cells with indicated VDAC1 proteins transfected for 48 h and treated with or without 30 µM CCCP for 16 h. (D) Confocal images of Bax translocated to the mitochondria. Subcellular localizations of GFP-Parkin (green), Bax (red), and VDAC1 proteins (blue) were observed. (Scale bars, 20 µm.) (E) Percentage of the cells with Bax localized to mitochondria in D (n > 500 cells from three independent experiments). Data were analyzed by one-way ANOVA with Tukey multiple-comparison test and are presented as means ± SD. ***P < 0.001. (F) HeLa cells stably expressing GFP-Parkin were transfected with indicated VDAC1 proteins in a dose-dependent manner and analyzed by immunoblotting to examine indicated proteins.

Article Snippet: VDAC1 WT or KO MEF cells were obtained from Geert Bultynck (KU Leuven University) and William Craigen (Baylor College of Medicine).

Techniques: Translocation Assay, Transfection, Membrane, Comparison, Stable Transfection, Expressing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination

doi: 10.1073/pnas.1909814117

Figure Lengend Snippet: Monoubiquitination-deficient VDAC1 induces apoptosis by increasing mitochondrial calcium uptake. (A and B) Mitochondrial calcium dynamics in VDAC1 KO MEF cells. VDAC1 KO MEF cells were transfected with HA-tagged VDAC1 WT or K274R and cotransfected with 4mitD3, a mitochondrial calcium indicator, for 48 h. Cells were then treated with 100 µM ATP for 3 min and were monitored for the changes in 4mitD3 fluorescence. (B) Maximum mitochondrial calcium uptake for VDAC1 KO MEF cells expressing VDAC1 WT or K274R in A. All of the values were normalized to the basal level of VDAC1 KO MEF cells transfected with empty vector (−) (n = 50 to ∼70 cells). Data were analyzed by ANOVA Tukey test. **P < 0.05. (C and D) Confocal images of Bax translocated to the mitochondria. (C) Flag-tagged Bax (red) and HA-tagged VDAC1 WT or K274R (blue) were transfected and immune-stained with Flag and HA antibodies in HeLa cells stably expressing GFP-Parkin (green). Cells were treated with or without 0.5 mM Ru360 for 8 h or transfected with MCU siRNA. (Scale bars, 20 µm.) (D) Percentage of cells with Bax localized in the cytosol or the mitochondria shown in C (n > 500 cells from three independent experiments). Data were analyzed by one-way ANOVA with Tukey multiple-comparison test and are presented as means ± SD. ***P < 0.001. (E and F) HA-tagged VDAC1 WT or K274R was transfected in HeLa cells stably expressing GFP-Parkin. Cells were treated with or without 0.5 mM Ru360 for 8 h in E or transfected with MCU siRNA in F. Cell lysates were analyzed by immunoblotting to examine indicated proteins.

Article Snippet: VDAC1 WT or KO MEF cells were obtained from Geert Bultynck (KU Leuven University) and William Craigen (Baylor College of Medicine).

Techniques: Transfection, Fluorescence, Expressing, Plasmid Preparation, Staining, Stable Transfection, Comparison, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Decision between mitophagy and apoptosis by Parkin via VDAC1 ubiquitination

doi: 10.1073/pnas.1909814117

Figure Lengend Snippet: Impaired monoubiquitination of VDAC1 by Parkin T415N leads to apoptosis. (A) Parkin PD patient mutations in the domains of Parkin protein. Listing from the N-terminal to the C-terminal region, Parkin consists of UBL, really-interesting-new-gene (RING) 0, RING1, in-between RING (IBR), and RING2 domains. (B) Summarized results of the ubiquitination assays for VDAC1 by 63 different Parkin PD patient mutants. Mono- and polyubiquitination activities of these Parkin mutants were normalized to the mono- and polyubiquitination of VDAC1 by Parkin WT (red). The Parkin T415N mutant (green) showed significant reduction in the monoubiquitination of VDAC1. We quantitated band intensities of ubiquitination on VDAC1 using Image J program. (C) Ubiquitination assays on the Parkin T415N mutant. Monoubiquitination of VDAC1 is indicated by arrows, and the bracket represents the polyubiquitination. IP, immunoprecipitated; WCL, whole-cell lysates. (D) Mitochondrial proteins analyzed by immunoblotting. HeLa cells were expressed with Myc-tagged Parkin WT or T415N with or without 20 µM CCCP for 12 h. A quantitation graph shows the protein band intensity of MFN2 (blue), MFN1 (green), NDUFS3 (yellow), and TIM23 (red) normalized to tubulin from CCCP-treated cells. Data were obtained from two independent experiments and are shown as means ± SD. (E) Parkin KO MEF cells expressing Myc-tagged Parkin WT or T415N were compared with Parkin WT MEF cells. Whole-cell lysates were analyzed for immunoblotting to detect indicated proteins. (F) Confocal images of Bax translocated to the mitochondria. HeLa cells were transfected with Myc-tagged Parkin WT or T415N and treated with 1 mM H2O2 for 6 h. Subcellular localizations of Bax (green), COXIV (blue), and Parkin (red) were examined by confocal microscopy. (Scale bars, 20 µm.) (G) Percentage of cells with Bax in mitochondria in F (n > 450 cells from three independent experiments). Data were analyzed by ANOVA Tukey test. ***P < 0.001

Article Snippet: VDAC1 WT or KO MEF cells were obtained from Geert Bultynck (KU Leuven University) and William Craigen (Baylor College of Medicine).

Techniques: Ubiquitin Proteomics, Mutagenesis, Immunoprecipitation, Western Blot, Quantitation Assay, Expressing, Transfection, Confocal Microscopy