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

Hexokinase (HK) catalyzes the synthesis of glucose-6-phosphate, marking the first committed step of glucose metabolism. Most cancer cells express two homologous isoforms (HK1 and HK2) that can each bind to the outer mitochondrial membrane (OMM). CRISPR screens across hundreds of cancer cell lines indicate that both are dispensable for cell growth in traditional culture media. By contrast, deletion impairs cell growth in Human Plasma-Like Medium (HPLM). Here, we find that HK2 is required to maintain sufficient cytosolic (OMM-detached) HK activity under conditions that enhance HK1 binding to the OMM. Notably, OMM-detached rather than OMM-docked HK promotes "aerobic glycolysis" (Warburg effect), an enigmatic phenotype displayed by most proliferating cells. We show that several proposed theories for this phenotype cannot explain the dependence and instead find that deletion severely impairs glycolytic ATP production with little impact on total ATP yield for cells in HPLM. Our results reveal a basis for conditional essentiality and suggest that demand for compartmentalized ATP synthesis underlies the Warburg effect.