Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily.

Ammonium is an important nitrogen (N) source for living organisms, a key metabolite for pH control, and a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins, and despite their significance in physiological processes, the nature of substrate translocation (NH3 /NH4 + ) by the distinct members of this family is still a matter of controversy. Using Saccharomyces cerevisiae cells expressing representative AMT-Mep-Rh ammonium carriers and taking advantage of the natural chemical-physical property of the N isotopic signature linked to NH4 + /NH3 conversion, this study shows that only cells expressing AMT-Mep-Rh proteins were depleted in 15 N relative to 14 N when compared to the external ammonium source. We observed 15 N depletion over a wide range of external pH, indicating its independence of NH3 formation in solution. On the basis of inhibitor studies, ammonium transport by nonspecific cation channels did not show isotope fractionation but competition with K+ . We propose that kinetic N isotope fractionation is a common feature of AMT-Mep-Rh-type proteins, which favor 14 N over 15 N, owing to the dissociation of NH4 + into NH3 + H+ in the protein, leading to 15 N depletion in the cell and allowing NH3 passage or NH3 /H+ cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH4 + /K+ ratio, allowing organisms to specifically scavenge NH4 + . We show that 15 N isotope fractionation may be used in vivo not only to determine the molecular species being transported by ammonium transport proteins, but also to track ammonium toxicity and associated amino acids excretion.