Figure 3. Protein blocked in the... | Rockefeller University Press

Figure 3.

$Protein blocked in the translocase becomes destabilized upon dissipation of mitochondrial membrane potential. (A–D) Cellular levels of ATP5MG-tFT protein in HEK-293 cells following indicated treatments: (A) cells were treated with CHX (70 µM) to inhibit protein synthesis for the specified time; (B) cells were treated for 4 h with CCCP (40 µM) to dissipate mitochondrial membrane potential; (C) cells were treated with indicated concentrations of protonophores CCCP or BAM15 for 2 h; (D) cells were treated for 2 h with CCCP, valinomycin, oligomycin, antimycin A, or rotenone at the indicated concentrations. (E) Confocal imaging of HEK-293 cells expressing ATP5MG-tFT (GFP signal) treated with 40 µM CCCP for the indicated time. Mitochondria were visualized by TOMM20 immunostaining; cell nuclei were stained with DAPI. Scale bar: 5 μm. (F) Quantification of GFP fluorescence intensity in different cellular compartments. Graphs present means ± SD of n = 3 independent experiments. *P < 0.05, **P < 0.01 in two-sided Student’s t test. (G) Impact of 2 h of CCCP treatment on the presence of ATP5MG-tFT in cytosolic, mitochondrial (Mito.), and nuclear fractions of HEK-293 cells. Fractions were analyzed by western blotting using anti-GFP antibody to detect ATP5MG-tFT. For validation, cytosolic proteins GAPDH and Hsp105, mitochondrial marker SDHA, and nuclear marker Histone H3 were detected. (H) HEK-293 (after tetracycline induction) and HeLa (plasmid transfected) cells expressing ATP5MG-tFT were treated or not treated with 40 µM CCCP for the final 2 h of the culture. Total protein extracts were analyzed by SDS-PAGE and western blotting. Expression of the fusion proteins was tested with an anti-GFP antibody. (I) HEK-293 cells were transfected with mitoT (Viana et al., 2021) or ATP5MG-tFT expression vectors. CCCP treatment was applied for 2 h. Total protein extracts were analyzed by SDS-PAGE and western blotting. Expression of the fusion proteins was tested with an anti-GFP antibody; schematic depiction of the mitoT membrane tether construct (Viana et al., 2021). All panels: the expression of fusion proteins was induced for 24 h by adding tetracycline or by plasmid transfection 24 h before indicated treatments. Source data are available for this figure: SourceData F3.$

Protein blocked in the translocase becomes destabilized upon dissipation of mitochondrial membrane potential. (A–D) Cellular levels of ATP5MG-tFT protein in HEK-293 cells following indicated treatments: (A) cells were treated with CHX (70 µM) to inhibit protein synthesis for the specified time; (B) cells were treated for 4 h with CCCP (40 µM) to dissipate mitochondrial membrane potential; (C) cells were treated with indicated concentrations of protonophores CCCP or BAM15 for 2 h; (D) cells were treated for 2 h with CCCP, valinomycin, oligomycin, antimycin A, or rotenone at the indicated concentrations. (E) Confocal imaging of HEK-293 cells expressing ATP5MG-tFT (GFP signal) treated with 40 µM CCCP for the indicated time. Mitochondria were visualized by TOMM20 immunostaining; cell nuclei were stained with DAPI. Scale bar: 5 μm. (F) Quantification of GFP fluorescence intensity in different cellular compartments. Graphs present means ± SD of n = 3 independent experiments. *P < 0.05, **P < 0.01 in two-sided Student’s t test. (G) Impact of 2 h of CCCP treatment on the presence of ATP5MG-tFT in cytosolic, mitochondrial (Mito.), and nuclear fractions of HEK-293 cells. Fractions were analyzed by western blotting using anti-GFP antibody to detect ATP5MG-tFT. For validation, cytosolic proteins GAPDH and Hsp105, mitochondrial marker SDHA, and nuclear marker Histone H3 were detected. (H) HEK-293 (after tetracycline induction) and HeLa (plasmid transfected) cells expressing ATP5MG-tFT were treated or not treated with 40 µM CCCP for the final 2 h of the culture. Total protein extracts were analyzed by SDS-PAGE and western blotting. Expression of the fusion proteins was tested with an anti-GFP antibody. (I) HEK-293 cells were transfected with mitoT (Viana et al., 2021) or ATP5MG-tFT expression vectors. CCCP treatment was applied for 2 h. Total protein extracts were analyzed by SDS-PAGE and western blotting. Expression of the fusion proteins was tested with an anti-GFP antibody; schematic depiction of the mitoT membrane tether construct (Viana et al., 2021). All panels: the expression of fusion proteins was induced for 24 h by adding tetracycline or by plasmid transfection 24 h before indicated treatments. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only