Heme allocation in eukaryotic cells relies on mitochondrial heme export through FLVCR1b to cytosolic GAPDH.

Heme is an iron-containing cofactor generated in mitochondria that must leave this organelle to reach protein targets in other cell compartments. Because mitochondrial heme binding by cytosolic GAPDH enables its distribution in cells, we sought to uncover how heme reaches GAPDH. Experiments utilizing two human cell lines and a GAPDH reporter protein whose heme binding can be followed by fluorescence reveal that the mitochondrial protein FLVCR1b provides heme to GAPDH in concert with a rise and fall in their association. An absence of FLVCR1b diminishes GAPDH association with mitochondria and prevents GAPDH and cell hemeproteins from receiving heme. GAPDH heme procurement also requires the TANGO2 protein, which interacts with FLVCR1b to presumably support heme export. In isolated mitochondria, GAPDH associates with FLVCR1b to trigger heme release and delivery to client hemeproteins. Identifying FLVCR1b as the source of mitochondrial heme for GAPDH reveals a path by which this essential cofactor can reach multiple protein targets within eukaryotic cells.