RESUMEN

Na+/H+ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na+ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a 'CPA motif', we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li+-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li+ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li+ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Mutación , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Concentración de Iones de Hidrógeno , Litio/metabolismo , Modelos Moleculares , Unión Proteica , Pliegue de Proteína , Intercambiadores de Sodio-Hidrógeno/genética

RESUMEN

Transient or partial formation of complexes between biomacromolecules is a general mechanism used to control cellular functions. Several of these complexes escape structure determination by crystallographic means. We developed a new approach for determining the structure of protein dimers in the native environment (e.g., in the membrane) with high resolution in cases where the structure of the two monomers is known. The approach is based on measurements of distance distributions between spin labels in the range between 2 and 6 nanometers by a pulsed electron paramagnetic resonance technique and explicit modeling of spin label conformations. By applying this method to the membrane protein homodimer of the Na(+)/H(+) antiporter NhaA of Escherichia coli, the structure of the presumably physiological dimer was determined. It reveals two points of contact between the two monomers, with one of them confirming results of earlier cross-linking experiments.

Asunto(s)

Intercambiadores de Sodio-Hidrógeno/química , Alelos , Sitios de Unión , Microscopía por Crioelectrón , Cisteína/química , Bases de Datos de Proteínas , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Modelos Estadísticos , Conformación Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/metabolismo

RESUMEN

The Na+/H+ antiporter NhaA is the main Na+ extrusion system in E. coli. Using direct current measurements combined with a solid supported membrane (SSM), we obtained electrical data of the function of NhaA purified and reconstituted in liposomes. These measurements demonstrate NhaA's electrogenicity, its specificity for Li+ and Na+ and its pronounced pH dependence in the range pH 6.5-8.5. The mutant G338S, in contrast, presents a pH independent profile, as reported previously. A complete right-side-out orientation of the NhaA antiporter within the proteoliposomal membrane was determined using a NhaA-specific antibody based ELISA assay. This allowed for the first time the investigation of NhaA in the passive downhill uptake mode corresponding to the transport of Na+ from the periplasmic to the cytoplasmic side of the membrane. In this mode, the transporter has kinetic properties differing significantly from those of the previously investigated efflux mode. The apparent Km values were 11 mM for Na+ and 7.3 mM for Li+ at basic pH and 180 mM for Na+ and 50 mM for Li+ at neutral pH. The data demonstrate that in the passive downhill uptake mode pH regulation of the carrier affects both apparent Km as well as turnover (Vmax).

Asunto(s)

Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Anticuerpos Monoclonales/metabolismo , Transporte Biológico Activo/fisiología , Electrofisiología , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Proteínas de Escherichia coli/genética , Concentración de Iones de Hidrógeno , Transporte Iónico/fisiología , Cinética , Liposomas , Mutación/genética , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Espectrometría de Fluorescencia

RESUMEN

Na(+)/H(+) antiporters are ubiquitous membrane proteins that are involved in homeostasis of H(+) and Na(+) throughout the biological kingdom. Corroborating their role in pH homeostasis, many of the Na(+)/H(+) antiporter proteins are regulated directly by pH. The pH regulation of NhaA, the Escherichia coli Na(+)/H(+) antiporter (EcNhaA), as of other, both eukaryotic and prokaryotic Na(+)/H(+) antiporters, involves a pH sensor and conformational changes in different parts of the protein that transduce the pH signal into a change in activity. Thus, residues that affect the pH response, the translocation or both activities cluster in separate domains along the antiporter molecules. Importantly, in the NhaA family, these domains are conserved. Helix-packing model of EcNhaA based on cross-linking data suggests, that in the three dimensional structure of NhaA, residues that affect the pH response may be in close proximity, forming a single pH sensitive domain. Therefore, it is suggested that, despite considerable differences in the primary structure of the antiporters from the bacterial NhaA to the mammalian NHEs, their three-dimensional architectures are conserved. Test of this possibility awaits the atomic resolution of the 3D structure of the antiporters.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencia de Aminoácidos , Escherichia coli , Proteínas de Escherichia coli/genética , Células Eucariotas/metabolismo , Homeostasis , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Células Procariotas/metabolismo , Conformación Proteica , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio cholerae

RESUMEN

The unique trypsin cleavable site of NhaA, the Na(+)/H(+) antiporter of Escherichia coli, was exploited to detect a change in mobility of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin cleavage site (Lys 249). The proximity of paired Cys residues was assessed by disulfide cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis(maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomly. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H225C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C, N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C; (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformation with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helices at both the cytoplasmic and the periplasmic face of NhaA.

Asunto(s)

Citoplasma/química , Proteínas de Escherichia coli/química , Periplasma/química , Intercambiadores de Sodio-Hidrógeno/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Cricetinae , Reactivos de Enlaces Cruzados/metabolismo , Cisteína/genética , Citoplasma/genética , Citoplasma/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Periplasma/genética , Periplasma/metabolismo , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Células Tumorales Cultivadas

RESUMEN

Recently, a two-dimensional crystal structure of NhaA, the Na+/H+ antiporter of Escherichia coli has been obtained [Williams, K. A., Kaufer, U. G., Padan, E., Schuldiner, S. and Kühlbrandt, W. (1999) EMBO J., 18, 3558-3563]. In these crystals NhaA exists as a dimer. Using biochemical and genetic approaches here we show that NhaA exists in the native membrane as a homooligomer. Functional complementation between the polypeptides of NhaA was demonstrated by coexpression of pairs of conditional lethal (at high pH in the presence of Na+) mutant alleles of nhaA in EP432, a strain lacking antiporters. Physical interaction in the membrane was shown between the His-tagged NhaA polypeptide which is readily affinity purified from DM-solubilized membranes with a Ni2+-NTA column and another which is not; only when coexpressed did both copurify on the column. The organization of the oligomer in the membrane was studied in situ by site-directed cross-linking experiments. Cysteine residues were introduced--one per NhaA--into certain loops of Cys-less NhaA, so that only intermolecular cross-linking could take place. Different linker-size cross-linkers were applied to the membranes, and the amount of the cross-linked protein was analyzed by mobility shift on SDS-PAGE. The results are consistent with homooligomeric NhaA and the location of residue 254 in the interface between monomers. Intermolecular cross-linking of V254C caused an acidic shift in the pH profile of NhaA.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Alelos , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Estructuras de la Membrana Celular/química , Estructuras de la Membrana Celular/genética , Estructuras de la Membrana Celular/metabolismo , Estructuras de la Membrana Celular/ultraestructura , Reactivos de Enlaces Cruzados/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Genes Letales , Prueba de Complementación Genética , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/ultraestructura , Relación Estructura-Actividad , Transformación Genética

RESUMEN

Na(+)/H(+) antiporters are membrane proteins that play a major role in pH and Na(+) homeostasis of cells throughout the biological kingdom, from bacteria to humans and higher plants. The emerging genomic sequence projects already have started to reveal that the Na(+)/H(+) antiporters cluster in several families. Structure and function studies of a purified antiporter protein have as yet been conducted mainly with NhaA, the key Na(+)/H(+) antiporter of Escherichia coli. This antiporter has been overexpressed, purified and reconstituted in a functional form in proteoliposomes. It has recently been crystallized in both 3D as well as 2D crystals. The NhaA 2D crystals were analyzed by cryoelectron microscopy and a density map at 4 A resolution was obtained and a 3D map was reconstructed. NhaA is shown to exist in the 2D crystals as a dimer of monomers each composed of 12 transmembrane segments with an asymmetric helix packing. This is the first insight into the structure of a polytopic membrane protein. Many Na(+)/H(+) antiporters are characterized by very dramatic sensitivity to pH, a property that corroborates their role in pH homeostasis. The molecular mechanism underlying this pH sensitivity has been studied in NhaA. Amino acid residues involved in the pH response have been identified. Conformational changes transducing the pH change into a change in activity were found in loop VIII-IX and at the N-terminus by probing trypsin digestion or binding of a specific monoclonal antibody respectively. Regulation by pH of the eukaryotic Na(+)/H(+) antiporters involves an intricate signal transduction pathway (recently reviewed by Yun et al., Am. J. Physiol. 269 (1995) G1-G11). The transcription of NhaA has been shown to be regulated by a novel Na(+)-specific regulatory network. It is envisaged that interdisciplinary approaches combining structure, molecular and cell biology as well as genomics should be applied in the future to the study of this important group of transporters.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Animales , Cationes Monovalentes , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Modelos Genéticos , Datos de Secuencia Molecular , Filogenia , Plantas , Conformación Proteica , Transducción de Señal , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Transcripción Genética

RESUMEN

The transcription of nhaA, encoding the main Na(+)/H(+) antiporter of Escherichia coli, is induced by Na(+), regulated by NhaR, and affected by H-NS. In this work the roles of the two nhaA promoters (P1 and P2) were studied by analysis of transcription both in vivo and in vitro and promoter mutations. We found that P1 is an NhaR-dependent, Na(+)-induced, and H-NS-affected promoter both in the exponential and stationary phases. An in vitro transcription assay demonstrated that P1 is activated by sigma(70)-RNA polymerase and both NhaR and H-NS increase the specificity of P1. Remarkably, in marked contrast to P1, P2 exhibits very low activity during the exponential phase but is induced in the stationary phase to become the major promoter. Furthermore, P2 is activated by sigma(S) and is neither induced by Na(+) nor dependent on NhaR or affected by H-NS. Hence, this work establishes that nhaA has a dual mode of regulation, each involving a different promoter, and reveals that P2 and sigma(S) together are responsible for the survival of stationary-phase cells in the presence of high Na(+), alkaline pH, and the combination of high Na(+) and alkaline pH, the most stressful condition.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/genética , Intercambiadores de Sodio-Hidrógeno/genética , Sodio/farmacología , Proteínas Bacterianas/metabolismo , Secuencia de Bases , División Celular , Proteínas de Unión al ADN/metabolismo , Escherichia coli/citología , Regulación Bacteriana de la Expresión Génica , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.

Asunto(s)

Clonación Molecular , Cianobacterias/enzimología , Cianobacterias/genética , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , Secuencia de Aminoácidos , Membrana Celular/enzimología , Cianobacterias/crecimiento & desarrollo , ADN Bacteriano/análisis , ADN Bacteriano/genética , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Filogenia , Quinona Reductasas/química , Alineación de Secuencia , Análisis de Secuencia de ADN

RESUMEN

One of the most interesting properties of the NhaA Na(+)/H(+) antiporter of Escherichia coli is the strong regulation of its activity by pH. This regulation is accompanied by a conformational change that can be probed by digestion with trypsin and involves the hydrophilic loop connecting the transmembrane helices VIII-IX. In the present work we show that a monoclonal antibody (mAb), 1F6, recognizes yet another domain of NhaA in a pH-dependent manner. This antibody binds NhaA at pH 8.5 but not at pH 4.5, whereas two other mAbs bind to NhaA independently of pH. The epitope of mAb 1F6 was located at the NH(2) terminus of NhaA by probing proteolytic fragments in Western blot analysis and amino acid sequencing. The antibody bound to the peptide HLHRFFSS, starting at the third amino acid of NhaA. A synthetic peptide with this sequence was shown to bind mAb 1F6 both at acidic and alkaline pH suggesting that this peptide is accessible to mAb 1F6 in the native protein only at alkaline pH. Although slightly shifted to acidic pH, the pH profile of the binding of mAb 1F6 to the antiporter is similar to that of both the Na(+)/H(+) antiporter activity as well as to its sensitivity to trypsin. We thus suggest that these pH profiles reflect a pH-dependent conformational change, which leads to activation of the antiporter. Indeed, a replacement of Gly-338 by Ser (G338S), which alleviates the pH dependence of both the NhaA activity as well as its sensitivity to trypsin, affects in a similar pattern the binding of mAb 1F6 to NhaA. Furthermore, the binding site of mAb 1F6 is involved in the functioning of the antiporter as follows: a double Cys replacement H3C/H5C causes an acidic shift by half a pH unit in the pH dependence of the antiporter; N-ethylmaleimide, which does not inhibit the wild-type protein, inhibits H3C/H5C antiporter to an extent similar to that exerted by mAb 1F6.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Proteínas de Escherichia coli , Escherichia coli/química , Concentración de Iones de Hidrógeno , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/química , Secuencia de Aminoácidos , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Mutagénesis Sitio-Dirigida , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/inmunología

RESUMEN

Digestion with trypsin of purified His-tagged NhaA in a solution of dodecyl maltoside yields two fragments at alkaline pH but only one fragment at acidic pH. Determination of the amino acid sequence of the N terminus of the cleavage products show that the pH-sensitive cleavage site of NhaA, both in isolated everted membrane vesicles as well as in the pure protein in detergent, is Lys-249 in loop VIII-IX, which connects transmembrane segment VIII to IX. Interestingly, the two polypeptide products of the split antiporter remain complexed and co-purify on Ni(2+)-NTA column. Loop VIII-IX has also been found to play a role in the pH regulation of NhaA; three mutations introduced into the loop shift the pH profile of the Na(+)/H(+) antiporter activity as measured in everted membrane vesicles. An insertion mutation introducing Ile-Glu-Gly between residues Lys-249 and Arg-250 (K249-IEG-R250) and Cys replacement of either Val-254 (V254C) or Glu-241 (E241C) cause acidic shift of the pH profile of the antiporter by 0.5, 1, and 0.3 pH units, respectively. Interestingly, the double mutant E241C/V254C introduces a basic shift of more than 1 pH unit with respect to the single mutation V254C. Taken together these results imply the involvement of loop VIII-IX in the pH-induced conformational change, which leads to activation of NhaA at alkaline pH.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/química , Glucósidos/farmacología , Conformación Proteica , Intercambiadores de Sodio-Hidrógeno/química , Secuencia de Aminoácidos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Análisis de Secuencia , Intercambiadores de Sodio-Hidrógeno/genética , Tripsina

RESUMEN

Electron cryomicroscopy of frozen-hydrated two-dimensional crystals of NhaA, a Na+/H+ antiporter from Escherichia coli predicted to have 12 transmembrane alpha-helices, has facilitated the calculation of a projection map of NhaA at 4.0 A resolution. NhaA was homologously expressed in E.coli with a His6 tag, solubilized in dodecyl maltoside and purified in a single step using Ni2+ affinity chromatography. Two-dimensional crystals were obtained after reconstitution of purified protein with E.coli lipids. The projection map reveals that this secondary transporter has a highly asymmetric structure in projection. NhaA exhibits overall dimensions of approximately 38x48 A with a ring-shaped density feature probably corresponding to a bundle of tilted helices, adjacent to an elongated region of density containing several peaks indicative of transmembrane helices. Two crystal forms with p22121 symmetry show tightly packed dimers of NhaA which differ in the interactions between adjacent dimers. This work provides the first direct glimpse into the structure of a secondary transporter.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/química , Intercambiadores de Sodio-Hidrógeno/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/ultraestructura , Cromatografía de Afinidad , Microscopía por Crioelectrón , Cristalización , Dimerización , Electrones , Escherichia coli/genética , Análisis de Fourier , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/ultraestructura , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/ultraestructura , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Intercambiadores de Sodio-Hidrógeno/aislamiento & purificación

RESUMEN

The NhaA Na+/H+ antiporter is the main system responsible for adaptation to Na+ and alkaline pH (in the presence of Na+) in Escherichia coli and many other enteric bacteria. It is under intricate control. At the protein level it is regulated directly by pH, one of its regulatory signals. A pH shift from 7 to 8.5 activates the antiporter and, in a fashion correlated with the activity change, confers a conformation change that, in isolated membrane vesicles, is reflected in the exposure of trypsin-cleavable sites. H225 and G338 are essential for the pH response of NhaA. nhaA transcription is dependent on NhaR, a positive regulator of the LysR family, and is regulated by Na+, the other environmental signal. Na+ affects the NhaR/nhaA interaction directly by changing the footprint of NhaR on nhaA in a pH-dependent fashion. The expression of nhaA is also under global regulation of H-NS. We suggest that the pattern of regulation of nhaA found in E. coli is a paradigm for the response of proteins and genes to H+ and Na+, the most common ions that challenge every cell.

Asunto(s)

Adaptación Fisiológica/fisiología , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/fisiología , Intercambiadores de Sodio-Hidrógeno/metabolismo , Secuencia de Aminoácidos , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Protones , Sodio , Factores de Transcripción/metabolismo

RESUMEN

pH controls the activity of the NhaA Na+/H+ antiporter of Escherichia coli. In the present work we show that replacement of glycine 338 of NhaA with serine (G338S) alleviates the pH control of the antiporter. Monitoring Na+-dependent collapse of DeltapH, to assess antiporter activity in isolated membrane vesicles, shows that the mutant protein is practically independent of pH, between pH 7 and 9, and even at pH 6 is 70% active. Similarly the purified reconstituted mutant protein catalyzes pH-independent passive efflux of 22Na from proteoliposomes as well as DeltapH-driven influx. Whereas the native NhaA in isolated membrane vesicles is exposed to digestion by trypsin only above pH 7, the mutated protein is degraded already at pH 6.5. DeltanhaA DeltanhaB cells transformed with a plasmid encoding the pH-independent antiporter are sensitive to Na+ but not to K+ at alkaline pH, while growing in the presence of both ions at neutral pH. Several possibilities that could explain the Na+ sensitivity of the mutant at alkaline pH were excluded; Western analysis and measurement of Na+/H+ antiporter activity in membrane vesicles, isolated from cells shifted to the non-permissive growth conditions, showed neither reduced expression of G338S-NhaA nor defective activity. The finding that the mutated protein is electrogenic led to the retraction of the idea that the protein is active in vitro but not in vivo at alkaline pH, when only Deltapsi exists in the cells. The Na+ concentration needed for half-maximal activity of G338S in isolated everted membrane vesicles is similar to that of the wild type. Therefore an increase in intracellular Na+ due to a reduced antiporter affinity could not explain the results. It is suggested that the loss of growth at alkaline pH in the presence of Na+ is due to the loss of the pH control of the mutated NhaA. Indeed, in the four mutations suppressing G338S phenotype, growth at alkaline pH was restored together with the pH regulation of NhaA. Three of the four suppressor mutations cluster in helix IV, whereas the original mutation is in helix XI, suggesting that the two helixes interact.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/crecimiento & desarrollo , Mutación Puntual , Intercambiadores de Sodio-Hidrógeno/metabolismo , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Fenotipo , Tripsina/metabolismo

RESUMEN

The bacterial Na+ (Li+)/H+ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na+, Li+) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H+ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na+, Li+) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H+ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionally of plasma membrane (Na+, Li+) ATPase and vacuolar H+ ATPase.

Asunto(s)

Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Litio/metabolismo , Saccharomyces cerevisiae/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Sodio/metabolismo , ATPasas de Translocación de Protón Vacuolares , Proteínas Bacterianas/genética , Secuencia de Bases , Membrana Celular/metabolismo , Cartilla de ADN/genética , Homeostasis , Membranas Intracelulares/metabolismo , Transporte Iónico , Mutación , Fenotipo , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Intercambiadores de Sodio-Hidrógeno/genética

RESUMEN

NhaB, an Na+/H+ antiporter, of Vibrio alginolyticus is a 528-amino-acid protein. Hydropathy profile-based computer analysis predicted that the NhaB might contain up to 13 membrane-spanning domains. To examine this hypothesis, we applied the phoA fusion method to the cloned nhaB gene. Eighteen plasmid-borne nhaB-phoA fusion genes were constructed in Escherichia coli cells and the alkaline phosphatase activity and expression level of the fusion proteins analyzed. These results and the results obtained with additional constructs indicated that V. alginolyticus NhaB has a unique topology consisting of nine transmembrane segments with the N-terminus in the cytoplasm and the C-terminus in the periplasm.

Asunto(s)

Fusión Artificial Génica , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/genética , Vibrio/genética , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Membrana Celular/química , Activación Enzimática/genética , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/síntesis química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Vibrio/química

RESUMEN

NtcA is a transcriptional activator involved in global nitrogen control in cyanobacteria. In the absence of ammonium it regulates the transcription of a series of genes encoding proteins required for the uptake and assimilation of alternative nitrogen sources (I. Luque, E. Flores, and A. Herrero, EMBO J. 13:2862-2869, 1994). ntcA, present in a single copy in the marine Synechococcus sp. strain WH 7803, was cloned and sequenced. The putative amino acid sequence shows a high degree of identity to NtcA from freshwater cyanobacteria in two functional domains. The expression of ntcA was negatively regulated by ammonium from a putative transcription start point located downstream of an NtcA consensus recognition sequence. Addition of either rifampin or ammonium led to a rapid decline in ntcA transcript levels with half-lives of less than 2 min in both cases. Nitrate-grown cells showed high ntcA transcript levels, as well as the capacity for active nitrite uptake. However, ammonium-grown cells showed low levels of the ntcA transcript and did not utilize nitrite. The addition of ammonium to nitrite uptake-active cells resulted in a gradual decline in the rate of uptake over a 24-h period. Active nitrite uptake was not induced in cells transferred to medium lacking a nitrogen source despite evidence of elevated expression of ntcA, indicating that ntcA expression is not sufficient for uptake capacity to develop. Nitrate and nitrite addition led to the development of nitrite uptake, whereas the addition of leucine did not. Furthermore, nitrite addition triggered the de novo protein synthesis required for uptake capacity to develop. These data suggest that nitrite and nitrate act as specific inducers for the synthesis of proteins required for nitrite uptake.

Asunto(s)

Proteínas Bacterianas , Cianobacterias/metabolismo , Proteínas de Unión al ADN/genética , Regulación Bacteriana de la Expresión Génica , Nitritos/metabolismo , Factores de Transcripción/genética , Secuencia de Aminoácidos , Cianobacterias/crecimiento & desarrollo , Datos de Secuencia Molecular , Compuestos de Amonio Cuaternario/farmacología , Transcripción Genética

RESUMEN

Monoclonal antibodies (mAbs) recognizing native epitopes are an important tool for functional and structural studies of proteins, yet they have rarely been used with transport proteins. In an attempt to raise monoclonal antibodies against the NhaA Na+/H+ antiporter of Escherichia coli we encountered difficulties in the screening procedure, which is based on the standard enzyme-linked immunosorbent assay (ELISA). Here we report a rapid and efficient method of screening for anti-NhaA mAbs which recognize native epitopes of the antiporter. The method is based on the use of His-tagged protein, Ni2+-nitrilotriacetic acid coated plates and non-denaturing conditions in the assay. With this procedure four mAbs were obtained, three of which recognize the NhaA in its native conformation and one preferentially recognizes the denatured form. The latter mAb is Western blot positive, the others are Western blot negative and bind the detergent solubilized NhaA as assayed by gel filtration chromatography. Competition experiments show that the native epitopes are common to both the His-tagged and the wild-type protein. We suggest that in the standard ELISA the NhaA protein is not presented to the antibody in the native conformation whereas the His tag based protocol favors this presentation. Indeed, we could remarkably improve the low reactivity of the standard ELISA by coating the plates with anti-NhaA mAb and use it to present NhaA ('sandwich' ELISA or two antibodies assay). Remarkably, two of the mAbs (5H4, 2C5) which bind native NhaA inhibit drastically the deltapH driven 22Na uptake mediated by His-tagged NhaA reconstituted in proteoliposomes. Hence, these mAbs afford a new tool to study the structure/function relationship of the antiporter.

Asunto(s)

Anticuerpos Monoclonales , Epítopos/análisis , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/análisis , Intercambiadores de Sodio-Hidrógeno/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Proteínas Bacterianas/análisis , Proteínas Bacterianas/inmunología , Western Blotting , Cromatografía en Gel , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunoglobulina G , Cadenas kappa de Inmunoglobulina , Liposomas , Ratones , Ratones Endogámicos BALB C , Proteolípidos

RESUMEN

One of the most striking features of NhaA, an Escherichia coli Na+/H+ antiporter, is its extreme sensitivity to pH. The activity of NhaA increases 2000-fold between pH 6.5 and 8.5. In this work, we investigated whether the activation of NhaA by pH is accompanied by conformational changes which can be detected using trypsin as a probe. We have found that NhaA is susceptible to proteolytic digestion at the pH range where it is activated, suggesting that these two events may be related; at alkaline pH, the protein becomes active and adopts an "open" conformational state in which more domains are exposed to the enzyme. This idea was further supported by results from two mutants of NhaA in which His-225, a residue involved in pH sensing, has been replaced by either Arg or Asp. The mutant H225R is activated at more acidic pH values, while H225D at more alkaline pH. In accordance with the results described for the wild-type protein, H225R was susceptible to digestion by trypsin at the pH at which it undergoes main activation. NhaA has many potential tryptic cleavage sites. However, analysis of the tryptic digestion fragments suggests that at alkaline pH, the protein is exposed to cleavage mainly at hydrophilic loops 6, 7, and 8. Thus, upon activation, NhaA appears to undergo a change in conformation that is reflected in specific regions of the protein.

Asunto(s)

Proteínas de Escherichia coli , Escherichia coli/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

We used partially purified NhaR and a highly purified His-tagged NhaR derivative to identify the cis-regulatory sequences of nhaA recognized by NhaR and to study the specific effect of Na+ on this interaction. Gel retardation assay with DNase I footprinting analysis showed that NhaR binds a region of nhaA which spans 92 bp and contains three copies of the conserved LysR-binding motif. Na+, up to 100 mM, had no effect on the binding of NhaR to nhaA. The dimethylsulfate methylation protection assay in vivo and in vitro, showed that bases G-92, G-60, G-29 and A-24 form direct contacts with NhaR; in the absence of added Na+ in vivo, these bases were protected but became exposed to methylation in a DeltanhaR strain; accordingly, these bases were protected in vitro by the purified His-tagged NhaR. 100 mM Na+, but not K+, removed the protection of G-60 conferred by His-tagged NhaR in vitro. Exposure of intact cells to 100 mM Na+, but not K+, exposed G-60. The maximal effect of Na+ in vitro was observed at 20 mM and was pH dependent, vanishing below pH 7.5. In contrast to G-60, G-92 was exposed to methylation by the ion only in vivo, suggesting a requirement for another factor existing only in vivo for this interaction. We suggest that NhaR is both sensor and transducer of the Na+ signal and that it regulates nhaA expression by undergoing a conformational change upon Na+ binding which modifies the NhaR-nhaA contact points.

Asunto(s)

Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Intercambiadores de Sodio-Hidrógeno/genética , Sodio/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Sitios de Unión , Huella de ADN , Metilación de ADN , ADN Bacteriano/química , Escherichia coli/genética , Eliminación de Gen , Histidina/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Intercambiadores de Sodio-Hidrógeno/metabolismo , Factores de Transcripción/química