Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production.

Hexokinase (HK) catalyses the phosphorylation of glucose to glucose 6-phosphate, marking the first step of glucose metabolism. Most cancer cells co-express two homologous HK isoforms, HK1 and HK2, which can each bind the outer mitochondrial membrane (OMM). CRISPR screens performed across hundreds of cancer cell lines indicate that both isoforms are dispensable for growth in conventional culture media. By contrast, HK2 deletion impaired cell growth in human plasma-like medium. Here we show that this conditional HK2 dependence can be traced to the subcellular distribution of HK1. Notably, OMM-detached (cytosolic) rather than OMM-docked HK supports cell growth and aerobic glycolysis (the Warburg effect), an enigmatic phenotype of most proliferating cells. We show that under conditions promoting increased translocation of HK1 to the OMM, HK2 is required for cytosolic HK activity to sustain this phenotype, thereby driving sufficient glycolytic ATP production. Our results reveal a basis for conditional HK2 essentiality and suggest that demand for compartmentalized ATP synthesis explains why cells engage in aerobic glycolysis.