A widened substrate selectivity filter of eukaryotic formate-nitrite transporters enables high-level lactate conductance.

Bacterial formate-nitrite transporters (FNT) regulate the metabolic flow of small weak mono-acids derived from anaerobic mixed-acid fermentation, such as formate, and further transport nitrite and hydrosulfide. The eukaryotic Plasmodium falciparumFNT is vital for the malaria parasite by its ability to release the larger l-lactate substrate as the metabolic end product of anaerobic glycolysis in symport with protons preventing cytosolic acidification. However, the molecular basis for substrate discrimination by FNTs has remained unclear. Here, we identified a size-selective FNT substrate filter region around an invariant lysine at the bottom of the periplasmic/extracellular vestibule. The selectivity filter is reminiscent of the aromatic/arginine constriction of aquaporin water and solute channels regarding composition, location in the protein, and the size-selection principle. Bioinformatics support an adaptation of the eukaryotic FNT selectivity filter to accommodate larger physiologically relevant substrates. Mutations that affect the diameter at the filter site predictably modulated substrate selectivity. The shape of the vestibule immediately above the filter region further affects selectivity. This study indicates that eukaryotic FNTs evolved to transport larger mono-acid substrates, especially l-lactic acid as a product of energy metabolism.