Fig. 2: SUMOylation controls HK2 binding to the mitochondria.
a SUMO-defective HK2 preferentially interacted with VDAC1. 293T cells were transfected with HA-tag HK2 WT, HK2-K315R, HK2-K492R, HK2-DKR, or MTD (mitochondrial binding deficient). Cell lysates were precipitated with HA antibody and blotted by VDAC1 antibody. b SUMOylation of HK2 prevented its interaction to VDAC1. Endogenous HK2 and VDAC1 interaction was accessed by immunoprecipitation with IgG or anti-HK2 antibody and then western blotting with anti-VDAC1. c, d Representative fluorescent images showed colocalization of ectopic expression HK2 and mitochondria in PC3 cells. Wild-type or DKR mutant HK2 with HA tag was expressed in PC3 cells and detected by HA antibody (green), co-staining of mitochondria with MitoTrackerTM Red CMXRos (Red). Scale bar, 5 μm. Quantification is calculated with Pearson’s correlation using ImageJ. Data are presented as mean ± SEM of five biologically independent samples. Statistical significance was determined by a two-tailed Student’s t test. e, f Representative fluorescent images showed colocalization of endogenous HK2 and mitochondria in PC3 cells. Scramble control or UBC9/ SENP1 knockdown PC3 cells were stained with HK2 antibody (green) and MitoTrackerTM Red CMXRos (Red). Scale bar, 25 μm. Quantification is calculated with Pearson’s correlation using ImageJ. Statistical significance was determined by a two-tailed Student’s t test. g Endogenous SUMOylation of HK2 was detected in the mitochondria and mitochondria-free cytosolic fractions in PC3 cells. Immunoprecipitation was performed with IgG or HK2 antibody, and then western blotting with SUMO1 antibody. h Western blotting showed endogenous HK2 expression in mitochondrial and cytoplasmic extraction from UBC9 shRNA, SENP1 shRNA or scramble PC3 cells. i PC3 cells expressing different mutant forms of HK2 were prepared for mitochondrial and cytoplasmic extraction and probed by HA and VDAC1 antibody in western blotting. Source data are provided as a Source Data file.
