KRAS4A promotes oligomerization of hexokinase 1 on mitochondria.

Among the ways by which oncogenic KRAS upregulates glycolysis in cancer is direct interaction of KRAS4A with hexokinase 1 (HK1), but the mechanism is unknown. HK1 associates with the outer mitochondrial membrane (OMM) where its allosteric regulation depends on homodimerization. Using affinity capture, FRET, and blue native gels, we show that KRAS4A enhances oligomerization of HK1 on the OMM. Modeling the HK1/KRAS4A complex with AlphaFold3 predicts that the membrane association sequences of both HK1 and KRAS4A are oriented toward the OMM. Super-resolution microscopy showed colocalization of HK1 and KRAS4A on the OMM with HK1 enriched at discrete locations. Single-molecule tracking reveals HK1 diffusing freely along the OMM and dwelling at discrete regions where two molecules can be seen to colocalize transiently. KRAS4A expression decreased the diffusion coefficient of HK1 on the organelle. Thus, KRAS4A alters the dynamics of HK1 on the OMM and promotes oligomerization.