RESUMEN
Mitochondrial porins or voltage-dependent anion channels (VDAC) are the main route for solute transport through outer mitochondrial membranes (OMM). In mammals, hexokinase (HK) binds to VDAC, which allows the channeling of ATP synthesized by oxidative phosphorylation toward HK. In plants, although HK has been found associated with OMM, evidence for an interaction with VDAC is scarce. Thus, in this work, we studied the physical and functional interaction between these proteins in beetroot mitochondria. To observe a physical interaction between HK and VDAC, OMM presenting HK activity were prepared from purified mitochondria. Protein complexes were solubilized from OMM with mild detergents and separated by centrifugation in glycerol gradients. Both HK activity and immunodetected VDAC were found in small (9S-13S) and large (>40S) complexes. OMM proteins were also separated according to their hydropathy by serial phase partitioning with Triton X-114. Most of HK activity was found in hydrophobic fractions where VDAC was also present. These results indicated that HK could be bound to VDAC in beetroot mitochondria. The functional interaction of HK with VDAC was demonstrated by observing the effect of apyrase on HK-catalyzed glucose phosphorylation in intact mitochondria. Apyrase, which hydrolyzes freely soluble ATP, competed efficiently with hexokinase for ATP when it was produced outside mitochondria (with PEP and pyruvate kinase), but not when it was produced inside mitochondria by oxidative phosphorylation. These results suggest that HK closely interacts with VDAC in beetroot mitochondria, and that this interaction allows the channeling of respiratory ATP toward HK through VDAC.