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 translocatio...

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Autores: Ariz Arnedo, Idoia, Boeckstaens, Mélanie, Gouveia, Catarina, Martins, Ana Paula, Sanz-Luque, Emanuel, Fernández, Emilio, Soveral, Graça, Wiren, Nicolaus von, Marini, Anna M., Aparicio Tejo, Pedro María, Cruz, Cristina
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2018
País:España
Institución:Universidad Pública de Navarra
Repositorio:Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
OAI Identifier:oai:academica-e.unavarra.es:2454/33508
Acceso en línea:https://hdl.handle.net/2454/33508
Access Level:acceso abierto
Palabra clave:AMT-Mep-Rh proteins
Nitrogen
Ammonium
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spelling Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamilyAriz Arnedo, IdoiaBoeckstaens, MélanieGouveia, CatarinaMartins, Ana PaulaSanz-Luque, EmanuelFernández, EmilioSoveral, GraçaWiren, Nicolaus vonMarini, Anna M.Aparicio Tejo, Pedro MaríaCruz, CristinaAMT-Mep-Rh proteinsNitrogenAmmoniumAmmonium 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.American Association for the Advancement of ScienceCienciasZientziak2018info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://hdl.handle.net/2454/33508reponame:Academica-e. Repositorio Institucional de la Universidad Pública de Navarrainstname:Universidad Pública de NavarraInglésinfo:eu-repo/grantAgreement/MINECO//AGL2015-64582-C3-1-Rinfo:eu-repo/grantAgreement/MINECO//AGL2012-37815-C05-05© 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).https://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccessoai:academica-e.unavarra.es:2454/335082026-06-17T12:41:47Z
dc.title.none.fl_str_mv Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
title Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
spellingShingle Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
Ariz Arnedo, Idoia
AMT-Mep-Rh proteins
Nitrogen
Ammonium
title_short Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
title_full Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
title_fullStr Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
title_full_unstemmed Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
title_sort Nitrogen isotope signature evidences ammonium deprotonation as a common transport mechanism for the AMT-Mep-Rh protein superfamily
dc.creator.none.fl_str_mv Ariz Arnedo, Idoia
Boeckstaens, Mélanie
Gouveia, Catarina
Martins, Ana Paula
Sanz-Luque, Emanuel
Fernández, Emilio
Soveral, Graça
Wiren, Nicolaus von
Marini, Anna M.
Aparicio Tejo, Pedro María
Cruz, Cristina
author Ariz Arnedo, Idoia
author_facet Ariz Arnedo, Idoia
Boeckstaens, Mélanie
Gouveia, Catarina
Martins, Ana Paula
Sanz-Luque, Emanuel
Fernández, Emilio
Soveral, Graça
Wiren, Nicolaus von
Marini, Anna M.
Aparicio Tejo, Pedro María
Cruz, Cristina
author_role author
author2 Boeckstaens, Mélanie
Gouveia, Catarina
Martins, Ana Paula
Sanz-Luque, Emanuel
Fernández, Emilio
Soveral, Graça
Wiren, Nicolaus von
Marini, Anna M.
Aparicio Tejo, Pedro María
Cruz, Cristina
author2_role author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Ciencias
Zientziak
dc.subject.none.fl_str_mv AMT-Mep-Rh proteins
Nitrogen
Ammonium
topic AMT-Mep-Rh proteins
Nitrogen
Ammonium
description 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.
publishDate 2018
dc.date.none.fl_str_mv 2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://hdl.handle.net/2454/33508
url https://hdl.handle.net/2454/33508
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv info:eu-repo/grantAgreement/MINECO//AGL2015-64582-C3-1-R
info:eu-repo/grantAgreement/MINECO//AGL2012-37815-C05-05
dc.rights.none.fl_str_mv https://creativecommons.org/licenses/by-nc/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv American Association for the Advancement of Science
publisher.none.fl_str_mv American Association for the Advancement of Science
dc.source.none.fl_str_mv reponame:Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
instname:Universidad Pública de Navarra
instname_str Universidad Pública de Navarra
reponame_str Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
collection Academica-e. Repositorio Institucional de la Universidad Pública de Navarra
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repository.mail.fl_str_mv
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