Compartmentalization of folate-dependent one-carbon metabolism. In mammalian mitochondria, monofunctional MTHFD1L catalyzes the 10-formyl-THF (CHO-THF) synthetase reaction. This enzyme is missing in Mthfd1l knockout mice. Bifunctional MTHFD2 or MTHFD2L enzymes catalyse 5,10-methenyl-THF (CH+-THF) cyclohydrolase and 5,10-methylene-THF (CH2-THF) dehydrogenase activities. MTHFD1 is the cytoplasmic trifunctional C1-THF synthase that catalyzes 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase activities. SHMT1 and SHMT2 represent cytoplasmic and mitochondrial serine hydroxymethyltransferase isozymes, respectively. Gray ovals represent putative metabolite transporters. CH3-THF, 5-methyl-THF; dTMP, thymidylate