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. 2016 Dec 30;26(4):702–716. doi: 10.1093/hmg/ddw431

Figure 7.

Figure 7

NDUFB10C107S is not efficiently oxidized and accumulates in the cytosol. (A) NDUFB10C107S accumulates in the cytosol. HeLa cells were transfected with plasmids coding either for NDUFB10-HA or NDUFB10C107S-HA. 24 h after transfection cells were fixed and analyzed by immunofluorescence. HEK293 cells stably expressing the respective NDUFB10 variants were induced with doxycycline for 24 h, then fixed and analyzed by immunofluorescence. The colocalization of NDUFB10-HA variants with Mitotracker was quantified and the fraction of cells exhibiting cytosolic or mitochondrial localization was plotted for HeLa and HEK293 cells. Bars contain the number of cells assessed for quantification. (B) Oxidation kinetics of NDUFB10 variants with CHCHD4 variants show that C107S is oxidized slower and presumably to a misoxidized product. Purified oxidized CHCHD4 wild type or a redox-inactive variants (C4S, C53S, C55S) were incubated with radioactive lysate of reduced NDUFB10 variants. At the indicated times the reaction was stopped by addition of mmPEG12-containing buffer. Samples were analyzed by SDS-PAGE and autoradiography. The arrowhead indicates the correctly oxidized NDUFB10, while the hashtag indicates the presumable mis-oxidized NDUFB10C107S.