Abstract
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 (NH(3)/NH(4)(+)) 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 NH(4)(+)/NH(3) 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 NH(3) 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 NH(4)(+) into NH(3) + H(+) in the protein, leading to (15)N depletion in the cell and allowing NH(3) passage or NH(3)/H(+) cotransport. This deprotonation mechanism explains these proteins' essential functions in environments under a low NH(4)(+)/K(+) ratio, allowing organisms to specifically scavenge NH(4)(+). 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.