Fig. 1: Mitochondrial localization of HKI relies on its N-terminal α-helix and a membrane-buried Glu in VDACs.
From: Hexokinase-I directly binds to a charged membrane-buried glutamate of mitochondrial VDAC1 and VDAC2
a Fluorescence images of wild-type (WT) and VDAC1/2-DKO HeLa cells expressing EGFP-tagged HKI (green) alone or in combination with HA-tagged VDAC1, VDAC1E73Q, VDAC1E73D, VDAC2, VDAC2E84Q or VDAC2E84D, fixed and then stained with DAPI (blue) and an antibody against Tom20 (magenta). Line scans showing degree of overlap between HKI and Tom20 signals along the path of the arrow shown in the zoom-in. Scale bar, 10 μm. b Pearson’s correlation co-efficient analysis between HKI and Tom20 signals in cells as in (a). For each violin plot, the middle line denotes the median, and the top and bottom lines indicate the 75th and 25th percentile. From left to right, n = 20 (WT), 20 (VDAC1/2-DKO), 20 (VDAC1/2-DKO + VDAC1), 20 (VDAC1/2-DKO + VDAC1E73Q), 20 (VDAC1/2-DKO + VDAC2), 20 (VDAC1/2-DKO + VDAC2E84Q), 46 (WT), 20 (VDAC1/2-DKO), 20 (VDAC1/2-DKO + VDAC1), 23 (VDAC1/2-DKO + VDAC1E73D), 20 (VDAC1/2-DKO + VDAC2) and 20 cells (VDAC1/2-DKO + VDAC2E84D) over at least 2 independent experiments. p values were calculated by unpaired two-tailed t test. c Fluorescence images of WT HeLa cells expressing EGFP-tagged HKI or N-terminal truncation mutant HKI∆2-14, fixed and then stained with DAPI (blue) and an antibody against Tom20 (magenta). Line scans showing degree of overlap between HKI and Tom20 signals along the path of the arrow shown in the zoom-in. Scale bar, 10 μm. d Fluorescence images of live WT and VDAC1/2-DKO HeLa cells co-expressing EGFP-tagged Tom20 (magenta) and Halo-tagged HKI-N (N-terminal HKI residues 1-17, green). Line scans showing degree of overlap between HKI-N and Tom20 signals along the path of the arrow shown in the zoom-in. Scale bar, 10 μm. e Models of complexes formed between HKI, Halo-tagged HKI-N and VDAC1/2. The membrane-buried Glu is marked in red.
