Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

One-carbon (1C) units for purine and thymidine synthesis can be generated from serine by cytosolic or mitochondrial folate metabolism. The mitochondrial 1C pathway is consistently overexpressed in cancer. Here, we show that most but not all proliferating mammalian cell lines use the mitochondrial pathway as the default for making 1C units. Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mitochondrial pathway knockout activates cytosolic 1C-unit production. This reversal in cytosolic flux is triggered by depletion of a single metabolite, 10-formyl-tetrahydrofolate (10-formyl-THF), and enables rapid cell growth in nutrient-replete conditions. Loss of the mitochondrial pathway, however, renders cells dependent on extracellular serine to make 1C units and on extracellular glycine to make glutathione. HCT-116 colon cancer xenografts lacking mitochondrial 1C pathway activity generate the 1C units required for growth by cytosolic serine catabolism. Loss of both pathways precludes xenograft formation. Thus, either mitochondrial or cytosolic 1C metabolism can support tumorigenesis, with the mitochondrial pathway required in nutrient-poor conditions. [Display omitted] •In most growing cells, mitochondrial 1C metabolism maintains cytosolic formyl-THF•Mitochondrial 1C metabolism also supports redox homeostasis by making glycine and NADPH•Depletion of formyl-THF induces cytosolic flux reversal to make 1C units from serine•Cytosolic folate metabolism is sufficient to support tumor growth Using genetic and metabolomic approaches, Ducker et al. dissect the roles of cytosolic and mitochondrial folate metabolism in cell proliferation, revealing that most cells default to the mitochondria for making 1C units, simultaneously generating glycine, NADH, and NADPH. Upon loss of the mitochondrial pathway, however, cytosolic metabolism supports tumor growth.