Fig. 4: Cytosolic pH controls mitochondrial association of HKI-N.
From: Hexokinase-I directly binds to a charged membrane-buried glutamate of mitochondrial VDAC1 and VDAC2
a Schematic outline of experimental strategy to determine the impact of cytosolic acidification on mitochondrial association of HKI-N. b Fluorescence images of live HeLa cells co-expressing EGFP-tagged Tom20 (green), OMM-mCherry (cyan) and Halo-tagged HKI-N (magenta) grown in Optimem (top), treated with 10 μM nigericin in pH 6.0 buffer for 5 min (middle) and then with 10 μM nigericin in pH 7.4 buffer for 5 min (bottom). Line scans showing degree of overlap between OMM and HKI-N signals along the path of the arrow shown in the zoom-in. Scale bar, 10 μm. c Fluorescence images of live HeLa cells co-expressing EGFP-tagged Tom20 (green), OMM-anchored mCherry (cyan) and Halo-tagged Tom20 (magenta) treated as in (b). Line scans showing degree of overlap between OMM and Tom20-Halo signals along the path of the arrow shown in the zoom-in. Scale bar, 10 μm. d Quantitative assessment of mitochondria-associated levels of OMM-mCherry, Tom20-EGFP, Tom20-Halo and HKI-N-Halo in live HeLa cells after treatment with nigericin in buffer at indicated pH for 5 min. Fluorescence values in corresponding pH buffer were set relative to values of same cell in Opti-MEM. Data are means ± SD, n = 6 cells per condition over four independent experiments. p values were calculated by unpaired two-tailed t test.
