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

Heme is an iron-containing cofactor essential for life. In eukaryotes heme is generated in the mitochondria and must leave this organelle to reach protein targets in other cell compartments. Mitochondrial heme binding by cytosolic GAPDH was recently found essential for heme distribution in eukaryotic cells. Here, we sought to uncover how mitochondrial heme reaches GAPDH. Experiments involving a human cell line and a novel GAPDH reporter construct whose heme binding in live cells can be followed by fluorescence revealed that the mitochondrial transmembrane protein FLVCR1b exclusively transfers mitochondrial heme to GAPDH through a direct protein-protein interaction that rises and falls as heme transfers. In the absence of FLVCR1b, neither GAPDH nor downstream hemeproteins received any mitochondrial heme. Cell expression of TANGO2 was also required, and we found it interacts with FLVCR1b to likely support its heme exporting function. Finally, we show that purified GAPDH interacts with FLVCR1b in isolated mitochondria and triggers heme transfer to GAPDH and its downstream delivery to two client proteins. Identifying FLVCR1b as the sole heme source for GAPDH completes the path by which heme is exported from mitochondria, transported, and delivered into protein targets within eukaryotic cells.