Figure 6. Identification of... | Rockefeller University Press

Figure 6.

$Identification of mitochondrial proteins ubiquitinated in cells with reduced MTF.(A) Summary of mass spectrometry analyzes of ubiquitinated proteins enriched in membrane fractions of Acn-treated versus control (DMSO-treated) cells. For the complete dataset, see Data S1. (B–D) Mitochondria-enriched HM and LM fractions from DMSO (control)– or Acn-treated WT (B) or ATG5−/− HCT116 (C) cells were subjected to Ub pull-down (PD) under denaturing conditions and analyzed by Western blot as indicated in the figure. Inputs (3% of samples used for the Ub pull-down) are shown in D. High-molecular-weight (apparently ubiquitinated) subsets of CPOX, TRAP1, and Mfn2 are marked with a red “Ub.” Upon stress, Mfn2 is degraded by the UPS (McLelland et al., 2018; Tanaka et al., 2010), and ubiquitination of this protein in Acn-treated cells was used as a reference. (E and F) WT HCT116 cells treated with DMSO (control; E) or Acn (F) were labeled to detect cytochrome c (Cyt. c; green), CPOX (red), and Tom20 (blue). Arrowheads in F indicate examples of high–cytochrome c/high-CPOX mitochondria. Detail images are from areas outlined with light green rectangles. Scale bars represent 20 µm (2 µm in detail images). (G) Overlap among cytochrome c, CPOX, and Tom20 in control and Acn-treated WT and Drp1−/− and Acn plus CP–treated WTHCT116 cells was estimated from maximum intensity projection images (exemplified in E and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 40 cells per condition. **, P < 0.01 versus control in WT HCT116 cells (all other samples were not significant); one-way ANOVA with Bonferroni correction (α = 0.05).$

Identification of mitochondrial proteins ubiquitinated in cells with reduced MTF. (A) Summary of mass spectrometry analyzes of ubiquitinated proteins enriched in membrane fractions of Acn-treated versus control (DMSO-treated) cells. For the complete dataset, see Data S1. (B–D) Mitochondria-enriched HM and LM fractions from DMSO (control)– or Acn-treated WT (B) or ATG5^−/− HCT116 (C) cells were subjected to Ub pull-down (PD) under denaturing conditions and analyzed by Western blot as indicated in the figure. Inputs (3% of samples used for the Ub pull-down) are shown in D. High-molecular-weight (apparently ubiquitinated) subsets of CPOX, TRAP1, and Mfn2 are marked with a red “Ub.” Upon stress, Mfn2 is degraded by the UPS (McLelland et al., 2018; Tanaka et al., 2010), and ubiquitination of this protein in Acn-treated cells was used as a reference. (E and F) WT HCT116 cells treated with DMSO (control; E) or Acn (F) were labeled to detect cytochrome c (Cyt. c; green), CPOX (red), and Tom20 (blue). Arrowheads in F indicate examples of high–cytochrome c/high-CPOX mitochondria. Detail images are from areas outlined with light green rectangles. Scale bars represent 20 µm (2 µm in detail images). (G) Overlap among cytochrome c, CPOX, and Tom20 in control and Acn-treated WT and Drp1^−/− and Acn plus CP–treated WTHCT116 cells was estimated from maximum intensity projection images (exemplified in E and F). The values are shown as Pearson’s correlation coefficient. Data are represented as mean ± SD; n = 40 cells per condition. **, P < 0.01 versus control in WT HCT116 cells (all other samples were not significant); one-way ANOVA with Bonferroni correction (α = 0.05).