RESUMEN
Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.
Asunto(s)
Microscopía por Crioelectrón , Proteínas de Escherichia coli , Escherichia coli , Potasio , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Glutatión/metabolismo , Simulación de Dinámica Molecular , Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Dominios ProteicosRESUMEN
bis-(3',5')-cyclic diadenosine monophosphate (c-di-AMP) is a second messenger with roles in virulence, cell wall and biofilm formation, and surveillance of DNA integrity in many bacterial species, including pathogens. Strikingly, it has also been proposed to coordinate the activity of the components of K+ homeostasis machinery, inhibiting K+ import, and activating K+ export. However, there is a lack of quantitative evidence supporting the direct functional impact of c-di-AMP on K+ transporters. To gain a detailed understanding of the role of c-di-AMP on the activity of a component of the K+ homeostasis machinery in B. subtilis, we have characterized the impact of c-di-AMP on the functional, biochemical, and physiological properties of KhtTU, a K+/H+ antiporter composed of the membrane protein KhtU and the cytosolic protein KhtT. We have confirmed c-di-AMP binding to KhtT and determined the crystal structure of this complex. We have characterized in vitro the functional properties of KhtTU and KhtU alone and quantified the impact of c-di-AMP and of pH on their activity, demonstrating that c-di-AMP activates KhtTU and that pH increases its sensitivity to this nucleotide. Based on our functional and structural data, we were able to propose a mechanism for the activation of KhtTU by c-di-AMP. In addition, we have analyzed the impact of KhtTU in its native bacterium, providing a physiological context for the regulatory function of c-di-AMP and pH. Overall, we provide unique information that supports the proposal that c-di-AMP is a master regulator of K+ homeostasis machinery.
Asunto(s)
Proteínas Bacterianas/metabolismo , AMP Cíclico/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Potasio/metabolismo , Bacillus subtilis , Sitios de Unión , AMP Cíclico/química , Homeostasis , Antiportadores de Potasio-Hidrógeno/química , Unión ProteicaRESUMEN
Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na+ ) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na+ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na+ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na+ from the root xylem leading to a high shoot Na+ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.
Asunto(s)
Proteínas de Plantas/genética , Brotes de la Planta/metabolismo , Sodio/metabolismo , Triticum/genética , Triticum/metabolismo , Animales , Femenino , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Oocitos/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Brotes de la Planta/genética , Polimorfismo de Nucleótido Simple , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Antiportadores de Potasio-Hidrógeno/metabolismo , Tolerancia a la Sal/genética , Xenopus laevis , Xilema/genética , Xilema/metabolismoRESUMEN
NhaP2 is a K+/H+ antiporter from Vibrio cholerae which consists of a transmembrane domain and a cytoplasmic domain of approximately 200 amino acids, both of which are required for cholera infectivity. Here we present the solution structure for a 165 amino acid minimal cytoplasmic domain (P2MIN) form of the protein. The structure reveals a compact N-terminal domain which resembles a Regulator of Conductance of K+ channels (RCK) domain connected to a more open C-terminal domain via a flexible 20 amino acid linker. NMR titration experiments showed that the protein binds ATP through its N-terminal domain, which was further supported by waterLOGSY and Saturation Transfer Difference NMR experiments. The two-domain organisation of the protein was confirmed by BIOSAXS, which also revealed that there are no detectable-ATP-induced conformational changes in the protein structure. Finally, in contrast to all known RCK domain structures solved to date, the current work shows that the protein is a monomer.
Asunto(s)
Proteínas Bacterianas/química , Antiportadores de Potasio-Hidrógeno/química , Dominios Proteicos , Vibrio cholerae/química , Adenosina Trifosfato/metabolismo , Antiportadores/química , Antiportadores/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Citoplasma/química , Resonancia Magnética Nuclear Biomolecular , Antiportadores de Potasio-Hidrógeno/metabolismo , Conformación ProteicaRESUMEN
The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Vibrio cholerae/metabolismo , Proteínas Bacterianas/química , Cólera/microbiología , Humanos , Simulación de Dinámica Molecular , Antiportadores de Potasio-Hidrógeno/química , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Vibrio cholerae/químicaRESUMEN
Plants remodel their cells through the dynamic endomembrane system. Intracellular pH is important for membrane trafficking, but the determinants of pH homeostasis are poorly defined in plants. Electrogenic proton (H+) pumps depend on counter-ion fluxes to establish transmembrane pH gradients at the plasma membrane and endomembranes. Vacuolar-type H+-ATPase-mediated acidification of the trans-Golgi network is crucial for secretion and membrane recycling. Pump and counter-ion fluxes are unlikely to fine-tune pH; rather, alkali cation/H+ antiporters, which can alter pH and/or cation homeostasis locally and transiently, are prime candidates. Plants have a large family of predicted cation/H+ exchangers (CHX) of obscure function, in addition to the well-studied K+(Na+)/H+ exchangers (NHX). Here, we review the regulation of cytosolic and vacuolar pH, highlighting the similarities and distinctions of NHX and CHX members. In planta, alkalinization of the trans-Golgi network or vacuole by NHXs promotes membrane trafficking, endocytosis, cell expansion, and growth. CHXs localize to endomembranes and/or the plasma membrane and contribute to male fertility, pollen tube guidance, pollen wall construction, stomatal opening, and, in soybean (Glycine max), tolerance to salt stress. Three-dimensional structural models and mutagenesis of Arabidopsis (Arabidopsis thaliana) genes have allowed us to infer that AtCHX17 and AtNHX1 share a global architecture and a translocation core like bacterial Na+/H+ antiporters. Yet, the presence of distinct residues suggests that some CHXs differ from NHXs in pH sensing and electrogenicity. How H+ pumps, counter-ion fluxes, and cation/H+ antiporters are linked with signaling and membrane trafficking to remodel membranes and cell walls awaits further investigation.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Pared Celular/metabolismo , Homeostasis , Concentración de Iones de Hidrógeno , Proteínas de Plantas/química , Proteínas de Plantas/genética , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.
Asunto(s)
Adenosina Monofosfato/química , Antiportadores de Potasio-Hidrógeno/química , Pliegue de Proteína , Multimerización de Proteína , Shewanella/química , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Antiportadores de Potasio-Hidrógeno/genética , Antiportadores de Potasio-Hidrógeno/metabolismo , Unión Proteica , Dominios Proteicos , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Shewanella/genética , Shewanella/metabolismoRESUMEN
Halomonas sp. Y2 is a halotolerant alkaliphilic strain from Na+-rich pulp mill wastewater with high alkalinity (pH >11.0). Transcriptome analysis of this isolate revealed this strain may use various transport systems for pH homeostasis. In particular, the genes encoding four putative Na+/H+ antiporters were differentially expressed upon acidic or alkaline conditions. Further evidence, from heterologous expression and mutant studies, suggested that Halomonas sp. Y2 employs its Na+/H+ antiporters in a labor division way to deal with saline and alkaline environments. Ha-NhaD2 displayed robust Na+(Li+) resistance and high transport activities in Escherichia coli; a ΔHa-nhaD2 mutant exhibited growth inhibition at high Na+(Li+) concentrations at pH values of 6.2, 8.0, and 10.0, suggesting its physiological role in osmotic homeostasis. In contrast, Ha-NhaD1 showed much weaker activities in ion exporting and pH homeostasis. Ha-Mrp displayed a combination of properties similar to those of Mrp transporters from some Bacillus alkaliphiles and neutrophiles. This conferred obvious Na+(Li+, K+) resistance in E. coli-deficient strains, as those ion transport spectra of some neutrophil Mrp antiporters. Conversely, similar to the Bacillus alkaliphiles, Ha-Mrp showed central roles in the pH homeostasis of Halomonas sp. Y2. An Ha-mrp-disrupted mutant was seriously inhibited by high concentrations of Na+(Li+, K+) but only under alkaline conditions. Ha-NhaP was determined to be a K+/H+ antiporter and shown to confer strong K+ resistance both at acidic and alkaline stresses.
Asunto(s)
Proteínas Bacterianas/metabolismo , Halomonas/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Halomonas/química , Halomonas/genética , Concentración de Iones de Hidrógeno , Mutación , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
Crop canopies create environments of highly fluctuating light intensities. In such environments, photoprotective mechanisms and their relaxation kinetics have been hypothesized to limit photosynthetic efficiency and therefore crop yield potential. Here, we show that overexpression of the Arabidopsis thylakoid K+/H+ antiporter KEA3 accelerates the relaxation of photoprotective energy-dependent quenching after transitions from high to low light in Arabidopsis and tobacco. This, in turn, enhances PSII quantum efficiency in both organisms, supporting that in wild-type plants, residual light energy quenching following a high to low light transition represents a limitation to photosynthetic efficiency in fluctuating light. This finding underscores the potential of accelerating quenching relaxation as a building block for improving photosynthetic efficiency in the field. Additionally, by overexpressing natural KEA3 variants with modification to the C-terminus, we show that KEA3 activity is regulated by a mechanism involving its lumen-localized C-terminus, which lowers KEA3 activity in high light. This regulatory mechanism fine-tunes the balance between photoprotective energy dissipation in high light and maximum quantum yield in low light, likely to be critical for efficient photosynthesis in fluctuating light conditions.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Luz , Fotosíntesis/efectos de la radiación , Antiportadores de Potasio-Hidrógeno/metabolismo , Tilacoides/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Secuencia de Bases , Complejo de Proteína del Fotosistema II/metabolismo , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Tilacoides/efectos de la radiación , Nicotiana/fisiología , Nicotiana/efectos de la radiaciónRESUMEN
The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of â¼10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.
Asunto(s)
Proteínas de Escherichia coli/química , Glutatión/análogos & derivados , Antiportadores de Potasio-Hidrógeno/química , Potasio/química , Succinimidas/química , Transporte Biológico Activo/fisiología , Proteínas de Escherichia coli/metabolismo , Glutatión/química , Glutatión/metabolismo , Activación del Canal Iónico/fisiología , Ligandos , Potasio/metabolismo , Antiportadores de Potasio-Hidrógeno/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Shewanella/química , Shewanella/metabolismo , Succinimidas/metabolismoRESUMEN
AtKEAs, homologs of bacterial KefB/KefC, are predicted to encode K(+)/H(+) antiporters in Arabidopsis. The AtKEA family contains six genes forming two subgroups in the cladogram: AtKEA1-3 and AtKEA4-6. AtKEA1 and AtKEA2 have a long N-terminal domain; the full-length AtKEA1 was inactive in yeast. The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K(+) and hygromycin B but not to salt and Li(+) stress. AtKEAs expressed in both the shoot and root of Arabidopsis. The expression of AtKEA1, -3 and -4 was enhanced under low K(+) stress, whereas AtKEA2 and AtKEA5 were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2 and AtKEA5 was not observed in aba2-3 mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs' expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants. The GFP tagging analysis showed that AtKEAs distributed diversely in yeast. The Golgi localization of AtKEA3 was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts. Overall, AtKEAs expressed and localized diversely, and may play roles in K(+) homeostasis and osmotic adjustment in Arabidopsis.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Homeostasis , Ósmosis , Antiportadores de Potasio-Hidrógeno/metabolismo , Potasio/metabolismo , Secuencia de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Familia de Multigenes , Filogenia , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Antiportadores de Potasio-Hidrógeno/química , Antiportadores de Potasio-Hidrógeno/genética , Transporte de Proteínas , Alineación de Secuencia , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , Estrés Fisiológico , Levaduras/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismoRESUMEN
The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/química , Modelos Moleculares , Antiportadores de Potasio-Hidrógeno/química , Multimerización de Proteína/fisiología , Sustitución de Aminoácidos , Sitios de Unión , Cloruros/química , Cloruros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transporte Iónico , Mutación Missense , Antiportadores de Potasio-Hidrógeno/genética , Antiportadores de Potasio-Hidrógeno/metabolismo , ProtonesRESUMEN
The Saccharomyces cerevisiae genome contains three genes encoding alkali metal cation/H+ antiporters (Nha1p, Nhx1p, Kha1p) that differ in cell localization, substrate specificity and physiological function. Systematic genome sequencing of other yeast species revealed highly conserved homologous ORFs in all of them. We compared the yeast sequences both at DNA and protein levels. The subfamily of yeast endosomal/prevacuolar Nhx1 antiporters is closely related to mammalian plasma membrane NHE proteins and to both plasma membrane and vacuolar plant antiporters. The high sequence conservation within this subfamily of yeast antiporters suggests that Nhx1p is of great importance in cell physiology. Yeast Kha1 proteins probably belong to the same subfamily as bacterial antiporters, whereas Nhal proteins form a distinct subfamily.
Asunto(s)
Proteínas de Transporte de Catión/química , Proteínas de la Membrana/química , Antiportadores de Potasio-Hidrógeno/química , Proteínas de Saccharomyces cerevisiae/química , Intercambiadores de Sodio-Hidrógeno/química , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/clasificación , Proteínas de Transporte de Catión/genética , ADN de Hongos/análisis , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Filogenia , Antiportadores de Potasio-Hidrógeno/clasificación , Antiportadores de Potasio-Hidrógeno/genética , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Intercambiadores de Sodio-Hidrógeno/clasificación , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers.