Heme is a vital cofactor required for many cellular functions, with dysregulation of heme being detrimental to the cell. Heme biosynthesis involves eight enzymatic steps, ending in the mitochondrial matrix. Heme must then be exported out of mitochondria and is distributed to the rest of the cell. The molecular mechanism of how this occurs, as well as the identity of the mitochondrial heme transporters, has been controversial and elusive.
To investigate the proteins involved in mitochondrial heme export, we have combined the genetically encoded, fluorescent heme biosensor, HS1, with a whole-genome CRISPR knockout screen. When cells have heme available, the GFP fluorescence is quenched, while in the absence of heme, the cells fluoresce green. We hypothesised that knockout of the mitochondrial heme transporter would lead to increased GFP fluorescence of cytosolic HS1 due to impaired heme transport across the mitochondrial membranes.
The screen identified several targets that are required for the production and cytosolic transport of heme. The known heme biosynthetic enzymes were strongly enriched. Additionally, several membrane proteins belonging to the solute carrier (SLC) family of metabolite transporters were also enriched. Current work to functionalize these proteins to determine their involvement in heme export from mitochondria is ongoing.