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. 2015 Aug 3;290(38):23226–23239. doi: 10.1074/jbc.M115.663492

FIGURE 5.

FIGURE 5.

Import of TbTim17 in the wild type control and TbTim62-depleted mitochondria. A, radiolabeled TbTim17 were incubated with isolated mitochondria from the Wt and TbTim62 KD T. brucei procyclic form for different time periods (1–10 min). Post-import, mitochondria were washed and treated with proteinase K (20 μg/ml) for 30 min at 4 °C, and proteins were analyzed by SDS-PAGE and autoradiography. Import reactions for COIV were also run in parallel. The precursor (p) and the mature (m) proteins are shown. Input lanes represent 10% of radiolabeled substrate proteins used for each reaction. For some reactions mitochondrial membrane potential was disrupted by pretreatment of mitochondria with CCCP. Intensity of the Tim17 (B) and matured COIV (C) imported into Wt and TbTim17 KD mitochondria were quantitated using densitometry. The intensity of the matured protein imported into the Wt mitochondria at the longest time point was set as maximum (100%), and the calculated percentage of the maximum import at other time points into mitochondria from the Wt and TbTim17 KD was plotted versus time. S.E. are calculated from three independent experiments. p values: ***, <0.001; **, <0.05. D, expression and targeting of TbTim17-myc into mitochondria in T. brucei Wt and TbTim62 KD. TbTim17–2X-myc was expressed in the wild type and in T. brucei where TbTim62 levels were reduced simultaneously by RNAi. At different time points (24–96 h) after induction mitochondrial fractions were isolated, and proteins were analyzed by immunoblot using anti-myc, anti-TbTim17, anti-TbTim62, and anti-VDAC antibodies. The ectopically expressed TbTim17–2X-myc and the endogenous TbTim17 were both detected by anti-TbTim17 antibody. E, intensities of the Tim17-myc and Tim62 protein bands were quantitated and normalized with that for the corresponding VDAC band, and values were plotted as relative intensity (arbitrary unit) in Wt and TbTim62 KD mitochondria. The number of experiment was three. Tim62 levels were significantly reduced in Tim17-myc/Tim62 KD relative to TbTim17-myc/Wt mitochondria. *, p values <0.05.