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

Abstract

Ammonium* is an important nitrogen (N) source for living organisms, and a key metabolite for pH control but also a potent cytotoxic compound. Ammonium is transported by the widespread AMT-Mep-Rh membrane proteins and despite their significance in patho-physiological processes, the nature of the substrate translocate (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 15N relative to 14N when compared to the external ammonium source. 15N depletion was observed over a wide range of external pH indicating its independence of NH3 formation in solution. Based on inhibitor studies, ammonium transport by non-specific 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 14N over 15N, due to dissociation of NH4+ into NH3+H+ in the protein leading to 15N depletion in the cell and allowing the NH3 passage or NH3/H+ cotransport. This deprotonation mechanism explains these proteins essential functions in environments under low NH4+/K+ ratio, allowing organisms to specifically scavenge NH4+. We show that 15N isotope fractionation may be used in vivo to determine the molecular species being transported by ammonium transport proteins but also to track ammonium toxicity and associated amino acids excretion.