Suchergebnisse

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 N-15 relative to N-14 when compared to the external ammonium source. We observed N-15 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 N-14 over N-15, owing to the dissociation of NH4+ into NH3+ H+ in the protein, leading to N-15 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 N-15 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. ; I. A. was supported by a postdoctoral fellowship from the Government of Navarra, Spain (Anabasid outgoing Programme, 2011) and by a postdoctoral fellowship from the Portuguese Fundaçao para a Ciencia e a Tecnologia (SFRH/BPD/90436/2012). A.M.M. is a senior research associate of the Belgian Fonds de la Recherche Scientifique Fonds de la Recherche Scientifique-FNRS (grants CDR J017617F, PDR T011515F, and ARC) and a WELBIO investigator, and M.B. is a scientific research worker supported by WELBIO. This work was also developed in the context of the following projects: PTDC/BIA-BEC/099323/2008 and PTDC/AGR-PRO/115888/2009 to cE3c and FCUL, UID/DTP/04138/2013 to iMed. ULisboa, and AGL2015-64582-C3-1-R and AGL2012-37815-C05-05 to UPNa.