RESUMO
The plant plasma membrane Na+/H+ antiporter SOS1 (Salt Overlay Sensitive 1) of Arabidopsis thaliana is the major transporter extruding Na+ out of cells in exchange for an intracellular H+. The sodium extrusion process maintains a low intracellular Na+ concentration and thereby facilitates salt tolerance. A. thaliana SOS1 consists of 1146 amino acids, with the first 450 in a N-terminal membrane transport domain and the balance forming a cytosolic regulatory domain. For studies on characterization of the protein, two different constructs of SOS1 comprising of the residues 28 to 460 and 28 to 990 were cloned and overexpressed in methylotropic yeast strain of Pichia pastoris with a C-terminal histidine tag using the expression vector pPICZA. Styrene malic acid copolymers (SMA) were used as a cost-effective alternative to detergent for solubilization and isolation of this membrane protein. Immobilized Ni2+-ion affinity chromatography was used to purify the expressed protein resulting in a yield of ~0.6-2â¯mg of SOS1 per liter of Pichia pastoris culture. The SMA purified protein containing amino acids 28 to 990 was directly reconstituted into liposomes for determination of Na+ transport activity and was functionally active. However, similar reconstitution with amino acids 28-460 did not yield a functional protein. Other results have shown that the truncated SOS1 protein at amino acid 481 is active, which infers the presence of an element between residues 461-481 which is necessary for SOS1 activity. This region contains several conserved segments that may be important in SOS1 structure and function.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Clonagem Molecular/métodos , Citoplasma/metabolismo , Detergentes/metabolismo , Proteínas de Membrana/metabolismo , Pichia/metabolismo , Tolerância ao Sal/genética , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Living cells are critically dependent on processes that regulate intracellular pH, Na(+) content, and volume. Na(+)/H(+) antiporters play a primary role in these homeostatic mechanisms. They are found in the cytoplasmic and intracellular membranes of most organisms from bacteria to humans and have long been human drug targets. NhaA, the principal Na(+)/H(+) antiporter in Escherichia coli, plays an essential role in homeostasis of Na(+) and H(+). It constitutes a paradigm for the study of its numerous prokaryotic homologs and of several human Na(+)/H(+) antiporters. The crystal structure of NhaA, determined at pH4, has provided the first structural and functional insights into the antiport mechanism and pH regulation of an Na(+)/H(+) antiporter. Remarkably, the NhaA structure revealed a new and unique fold (the "NhaA fold") that has since been observed in four additional bacterial secondary transporters. The NhaA structure has facilitated the rational interpretation of mutational data obtained in NhaA, revealing the antiporter's functional organization. Nevertheless, the crystal structure is a single snapshot, determined at acidic pH, when NhaA is downregulated; NhaA is activated at pH6.5 and reaches maximal activity at pH8.5. Therefore, it is crucial to crystallize the active conformations of NhaA. Herein, we present a procedure for determining the structure of NhaA.
Assuntos
Cristalização/métodos , Proteínas de Escherichia coli/química , Escherichia coli/química , Trocadores de Sódio-Hidrogênio/química , Cromatografia de Afinidade , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Hidrogênio/metabolismo , Modelos Moleculares , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Trocadores de Sódio-Hidrogênio/metabolismo , Regulação para CimaRESUMO
Mrp antiporters and their homologues in the cation/proton antiporter 3 family of the Membrane Transporter Database are widely distributed in bacteria. They have major roles in supporting cation and cytoplasmic pH homeostasis in many environmental, extremophilic, and pathogenic bacteria. These antiporters require six or seven hydrophobic proteins that form hetero-oligomeric complexes, while most other cation/proton antiporters require only one membrane protein for their activity. The resemblance of three Mrp subunits to membrane-embedded subunits of the NADH:quinone oxidoreductase of respiratory chains and to subunits of several hydrogenases has raised interest in the evolutionary path and commonalities of their proton-translocating domains. In order to move toward a greater mechanistic understanding of these unusual antiporters and to rigorously demonstrate that they function as secondary antiporters, powered by an imposed proton motive force, we established a method for purification and functional reconstitution of the seven-subunit Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4. Na(+)/H(+) antiporter activity was demonstrated by a fluorescence-based assay with proteoliposomes in which the Mrp complex was coreconstituted with a bacterial FoF1-ATPase. Proton pumping by the ATPase upon addition of ATP generated a proton motive force across the membranes that powered antiporter activity upon subsequent addition of Na(+).
Assuntos
Bacillus/enzimologia , Multimerização Proteica , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Trocadores de Sódio-Hidrogênio/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Força Próton-Motriz , Sódio/metabolismoRESUMO
Na(+)/H(+) exchanger (NHE) 1 is a member of the solute carrier superfamily, which regulates intracellular ionic homeostasis. NHE1 is known to require cellular ATP for its activity, despite there being no requirement for energy input from ATP hydrolysis. In this study, we investigated whether NHE1 is an ATP-binding protein. We designed a baculovirus vector carrying both epitope-tagged NHE1 and its cytosolic subunit CHP1, and expressed the functional NHE1-CHP1 complex on the surface of Sf9 insect cells. Using the purified complex protein consisting of NHE1 and CHP1 from Sf9 cells, we examined a photoaffinity labeling reaction with 8-azido-ATP-biotin. UV irradiation promoted the incorporation of 8-azido-ATP into NHE1, but not into CHP1, with an apparent Kd of 29.1 µM in the presence of Mg(2+). The nonlabeled nucleotides ATP, GTP, TTP and CTP all inhibited this crosslinking. However, ATP had the strongest inhibitory effect, with an apparent inhibition constant (IC50) for ATP of 2.2 mM, close to the ATP concentration giving the half-maximal activation of NHE1 activity. Importantly, crosslinking was more strongly inhibited by ATP than by ADP, suggesting that ATP is dissociated from NHE1 upon ATP hydrolysis. Limited proteolysis with thrombin and deletion mutant analysis revealed that the 8-azido-ATP-binding site is within the C-terminal cytoplasmic domain of NHE1. Equilibrium dialysis with NHE1-derived peptides provided evidence that ATP directly binds to the proximal cytoplasmic region (Gly542-Pro598), which is critical for ATP-dependent regulation of NHE1. These findings suggest that NHE1 is an ATP-binding transporter. Thus, ATP may serve as a direct activator of NHE1.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Animais , Azidas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Transporte de Cátions/isolamento & purificação , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Magnésio/metabolismo , Marcadores de Fotoafinidade/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteólise , Células Sf9 , Radioisótopos de Sódio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Transfecção , Raios UltravioletaRESUMO
A metagenomic library containing 8,000 clones was constructed by using genomic DNA obtained from Chaerhan Salt Lake in northwest China. Three Na(+)/H(+) antiporters, C4-NhaG, C47-NhaG and C49-NhaG that grouped to the NhaG family, were screened and cloned from this metagenome by complementing Escherichia coli strain KNabc (ΔnhaA ΔnhaB ΔchaA) in medium containing 0.2 M NaCl. The three putative Na(+)/H(+) antiporters were membrane proteins with 10, 11 and 11 transmembrane segments, respectively. They enabled E. coli KNabc to grow in medium containing 0.2-0.6 M Na(+) or 7-14 mM Li(+). Everted membrane vesicles prepared from E. coli KNabc cells carrying C49-NhaG exhibited Na(+)/H(+) and Li(+)/H(+) antiport activities.
Assuntos
Lagos/microbiologia , Metagenoma , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , China , Clonagem Molecular , Meios de Cultura/química , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Teste de Complementação Genética , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
A full-length cDNA homologue of SOS1 (salt overly sensitive 1) was isolated from the salinity-tolerant species Chrysanthemum crassum and found to encode a Na(+) /H(+) antiporter, using degenerate PCR and RACE-PCR. The 3752-bp sequence comprised a 3438 bp open reading frame, encoding a 127-kDa protein with 12 transmembrane domains within its N terminal portion, and a hydrophilic cytoplasmic tail in its C-terminal portion. CcSOS1 appears to be a plasma membrane protein, and shares â¼62% identity at the peptide level with its Arabidopsis thaliana homologue. Expression of CcSOS1 in the roots of C. crassum was sensitive to salinity stress, while in the leaves CcSOS1 was down-regulated in the presence of abscisic acid. CcSOS1 transcript abundance was reduced in both roots and leaves of plants exposed to low temperature, while it was increased in leaves (but not in roots) after drought stress. CcSOS1 expression was not regulated in the presence of CaCl2 . A heterologous complementation assay in yeast suggested that CcSOS1 directs Na(+) efflux, mimicking the function of the endogenous NHA1 protein. Thus CcSOS1 appears to encode a salinity-inducible plasma membrane Na(+) /H(+) antiporter. This gene may be useful in transgenic approaches to improving the salinity tolerance of related ornamental species.
Assuntos
Membrana Celular/metabolismo , Chrysanthemum/metabolismo , Proteínas de Plantas/isolamento & purificação , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Ácido Abscísico/farmacologia , Sequência de Aminoácidos , Cálcio/farmacologia , Membrana Celular/efeitos dos fármacos , Chrysanthemum/efeitos dos fármacos , Chrysanthemum/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Proteínas de Membrana/química , Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutação/genética , Cebolas/citologia , Filogenia , Epiderme Vegetal/citologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.
Assuntos
Proteínas de Bactérias/metabolismo , Hidrogênio/metabolismo , Potássio/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Vibrio cholerae/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Biocatálise , Clonagem Molecular , Concentração de Íons de Hidrogênio , Homologia de Sequência de Aminoácidos , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Especificidade por SubstratoRESUMO
We isolated cDNAs for Na(+)/H(+) antiporter genes (PhaNHA1s) from salt-sensitive and salt-tolerant reed plants. A phylogenetic analysis and localization analysis using yeast strains expressing PhaNHA1-GFP protein showed that PhaNHA1s were plasma membrane Na(+)/H(+) antiporters. Yeast strains expressing PhaNHA1 from salt-tolerant reed plants (PhaNHA1-n) grew well than yeast strains expressing PhaNHA1 from salt-sensitive reed plants (PhaNHA1-u) in the presence of 100mM NaCl. Furthermore, Na(+) contents of yeast cells expressing PhaNHA1-n were less than half of those of yeast cells expressing PhaNHA1-u. These results suggest that PhaNHA1-n is more efficient at excluding Na(+) from the cells than PhaNHA1-u.
Assuntos
Membrana Celular/genética , Poaceae/efeitos dos fármacos , Poaceae/genética , Plantas Tolerantes a Sal/genética , Cloreto de Sódio/farmacologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Sequência de Aminoácidos , Membrana Celular/efeitos dos fármacos , DNA Complementar/isolamento & purificação , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Filogenia , Poaceae/citologia , Poaceae/crescimento & desenvolvimento , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Plantas Tolerantes a Sal/citologia , Plantas Tolerantes a Sal/efeitos dos fármacos , Plantas Tolerantes a Sal/crescimento & desenvolvimento , Alinhamento de Sequência , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Transformação Genética/efeitos dos fármacosRESUMO
Sod2, is a Na(+)/H(+) exchanger present on the cytoplasmic membrane of the fission yeast Schizosaccharomyces pombe. It expels toxic Na(+) from the cytosol. Sod2 was expressed in Saccharomyces cerevisiae with a C-terminal histidine tag under control of the GAL1 promoter. Western blots using anti-V5 antibodies identified the tagged protein. Solubilization of the protein was by n-dodecyl beta-D: -maltoside. Immobilized Ni-ion column affinity chromatography partially purified the protein at a yield of ~240 microg per liter of culture. Sod2 was present as a 40-kDa and an 80-kDa protein, however, it co-purified with a number of other proteins. Cross linking of sod2 with N,N'-(o-phenylene)dimaleimide showed that sod2 was present in association with a number of other proteins as a larger molecular weight complex. Partially purified sod2 protein was reconstituted in proteoliposomes and functionally active. Our results suggest that the sod2 protein associates with a number of other proteins and can be expressed in S. cerevisiae in active form.
Assuntos
Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/biossíntese , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Schizosaccharomyces/enzimologia , Trocadores de Sódio-Hidrogênio/biossíntese , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Expressão Gênica , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Muscle contraction is normally mediated by the release of neurotransmitters from motor neurons. Here we demonstrate that protons can act as a direct transmitter from intestinal cells to stimulate muscle contraction. During the C. elegans defecation motor program the posterior body muscles contract even in the absence of neuronal inputs or vesicular neurotransmission. In this study, we demonstrate that the space between the intestine and the muscle is acidified just prior to muscle contraction and that the release of caged protons is sufficient to induce muscle contraction. PBO-4 is a putative Na+/H+ ion exchanger expressed on the basolateral membrane of the intestine, juxtaposed to the posterior body muscles. In pbo-4 mutants the extracellular space is not acidified and the muscles fail to contract. The pbo-5 and pbo-6 genes encode subunits of a "cys-loop" proton-gated cation channel required for muscles to respond to acidification. In heterologous expression assays the PBO receptor is half-maximally activated at a pH of 6.8. The identification of the mechanisms for release and reception of proton signals establishes a highly unusual mechanism for intercellular communication.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Mucosa Intestinal/metabolismo , Contração Muscular/fisiologia , Prótons , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/isolamento & purificação , Comunicação Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Defecação/fisiologia , Intestinos/citologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Canais Iônicos/metabolismo , Músculos/metabolismo , Músculos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Transdução de Sinais/fisiologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
The Na(+)/H(+) exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH by extruding an intracellular H(+) in exchange for one extracellular Na(+). The human NHE1 isoform is involved in heart disease and cell growth and proliferation. Although details of NHE1 regulation and transport are being revealed, there is little information available on the structure of the intact protein. In this report, we demonstrate overexpression, purification, and characterization of the human NHE1 (hNHE1) protein in Saccharomyces cerevisiae. Overproduction of the His-tagged protein followed by purification via nickel-nitrilotriacetic acid-agarose chromatography yielded 0.2 mg of pure protein/liter of cell culture. Reconstitution of hNHE1 in proteoliposomes demonstrated that the protein was active and responsive to an NHE1-specific inhibitor. Circular dichroism spectroscopy of purified hNHE1 revealed that the protein contains 41% alpha-helix, 23% beta-sheet, and 36% random coil. Size exclusion chromatography indicated that the protein-detergent micelle was in excess of 200 kDa, consistent with an hNHE1 dimer. Electron microscopy and single particle reconstruction of negatively stained hNHE1 confirmed that the protein was a dimer, with a compact globular domain assigned to the transmembrane region and an apical ridge assigned to the cytoplasmic domain. The transmembrane domain of the hNHE1 reconstruction was clearly dimeric, where each monomer had a size and shape consistent with the predicted 12 membrane-spanning segments for hNHE1.
Assuntos
Proteínas de Transporte de Cátions/química , Saccharomyces cerevisiae/genética , Trocadores de Sódio-Hidrogênio/química , Sequência de Bases , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/isolamento & purificação , Cromatografia Líquida , Dicroísmo Circular , Primers do DNA , Dimerização , Humanos , Microscopia Eletrônica , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
Sodium proton antiporters are ubiquitous membrane proteins. Their importance for cell viability is the result of their role in homeostasis of intracellular pH, cellular Na+ content and cell volume. Recently, the first structure of this family of secondary transporters, namely of NhaA from Escherichia coli, revealed a novel fold and elucidated the molecular basis for the mechanism of transport and its regulation by pH. Here, we describe the key steps for the structure determination of NhaA, an iterative process of improving protein quality as well as crystallization conditions. Protein quality was optimized by shortening the purification to a single step and by changing the expression host. The major steps for crystal improvement were the exchange of the detergent during protein purification from the beta- to the alpha-anomer of DDM, the addition of OG to the crystallization set ups, and the growth of the crystals under conditions suitable for cryo-temperatures. Unexpectedly, the dimeric association of the transporter in the 3D crystal lattice is non-physiological. A comparison of the X-ray structure with the electron density map from cryo-electron microscopy of 2D crystals demonstrates that the NhaA helix packing in the 3D crystal is identical with the one in the lipid environment. Thus, the antiporter is in a native conformation in the 3D crystals.
Assuntos
Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Estrutura Terciária de Proteína , Trocadores de Sódio-Hidrogênio/química , Sequência de Aminoácidos , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
According to the sequences of the gene nhaA coding for Na+ / H+ antiporter,a structural gene was cloned from Pseudomonas sp. cn4902 by PCR reaction with a set of primers. It was 1089 bp in length and codes for 362 amino acids sharing homology with the gene nhaA of E. coli K12 as high as 97.0%. It was inserted into plasmid pBV220 to form a high level expression reconstruction plasmid pBVA. So an overexpression 41 kD protein band could be found in the lane of transformant harbored with pBVA after SDS-PAGE electrophoresis. The detection of growth curve showed that the biomass of the transformant was 2.3 times over that of the control in the medium containing 1.0 mol/L NaCl. It was found that Na+ concentration in cytoplasm of the transformant was low to 60.4% of the control by the detection of atomic absorption spectrum. Evidence of SDS-PAGE electrophoresis of membrane proteins also showed that the NhaA was located in membrane. Purified NhaA was harvested and digested by FXa proteinase. The sequence of eight amino acids in N termination of NhaA protein was entirely identical with the polypeptide deduced from the nhaA gene. Then ten strains of transformant were continuously cultivated for 18 generations under 42 degrees C hot shock condition,all of their reconstructed plasmids were lost with the result that salt-tolerant-level went back to the original standard. In summary, all the experiments proved that the cloned gene is nhaA gene. The gene has been accepted in GenBank by the accession number AY643494.
Assuntos
Proteínas de Bactérias/genética , Pseudomonas/genética , Trocadores de Sódio-Hidrogênio/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Pseudomonas/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.
Assuntos
Gossypium/genética , Gossypium/metabolismo , Proteínas de Plantas/genética , Cloreto de Sódio/toxicidade , Trocadores de Sódio-Hidrogênio/genética , Ácido Abscísico/farmacologia , Sequência de Aminoácidos/genética , Sequência de Bases/genética , DNA Complementar/análise , DNA Complementar/genética , Gossypium/efeitos dos fármacos , Dados de Sequência Molecular , Mutação/efeitos dos fármacos , Mutação/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Homologia de Sequência de Aminoácidos , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
A NHE1 variant that exhibits very high resistance to (3-methyl sulfonyl-4-piperidinobenzoyl) guanidine methane sulfonate (HOE694), a potent inhibitor of Na(+)-H(+) exchangers, was selected and characterized. Sequencing of the coding region corresponding to the N-terminal domain of this variant revealed the presence of only one mutation located within membrane-spanning segment 9 (M9). This base pair change replaces a glutamate (Glu) with an aspartate (Asp). We reproduced this amino acid change in wild-type NHE1 and found that this mutation alone is responsible for the huge decrease in sensitivity to the HOE694 compound and to ethylisopropylamiloride (EIPA). We found that the NHE1-Glu(346)Asp mutant was more than 2000-fold more resistant to HOE694 and up to 300-fold more resistant to EIPA than wild-type NHE1, with the size, rather than the charge, of the amino acid in position 346 having the greatest effect. Interestingly, its affinity for Na(+) was at least 4-fold lower than that of wild-type NHE1. Mutation of amino acids in the vicinity of Glu(346) did not change the sensitivity of mutated NHE1 proteins to inhibitors. We suggest there is a direct interaction of Glu(346) or involvement of Glu(346) in a coordination site with NHE inhibitors and with Na(+).
Assuntos
Amilorida/análogos & derivados , Amilorida/química , Ácido Glutâmico/química , Trocadores de Sódio-Hidrogênio/química , Sódio/química , Amilorida/farmacologia , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular , Cricetinae , Resistência a Medicamentos/genética , Variação Genética , Ácido Glutâmico/genética , Guanidinas/farmacologia , Humanos , Mutagênese Sítio-Dirigida , Potássio/química , Prótons , RNA Mensageiro/análise , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Sulfonas/farmacologiaRESUMO
It has long been speculated that intracellular pH is a critical regulator of both invertebrate and vertebrate sperm motility, and sodium-hydrogen exchange has been suggested as a mediator of such pH(i) regulation in various instances. Two sodium-hydrogen exchangers (NHE1 and NHE5) are expressed in spermatozoa. However, elimination of the NHE1 gene fails to cause infertility, suggesting that normal sperm function is maintained in NHE1-null animals. Here, we used a functionally unbiased signal peptide trap screen to identify a novel sperm-specific NHE. The NHE contains 14 predicted transmembrane segments, including a potential voltage sensor and a consensus cyclic nucleotide-binding motif. Testis histology, sperm numbers and morphology were normal, but NHE-null males were completely infertile with severely diminished sperm motility. The addition of ammonium chloride, which elevates intracellular pH, partially rescued the motility and fertility defects. Surprisingly, cyclic AMP analogues almost completely rescued the motility and infertility phenotypes. The existence of this new sperm NHE provides an attractive contraceptive target, given its cell-specific expression and absolute requirement for fertility.
Assuntos
Fertilidade/fisiologia , Líquido Intracelular/metabolismo , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Cloreto de Amônio/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , DNA Complementar/análise , DNA Complementar/genética , Feminino , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Líquido Intracelular/efeitos dos fármacos , Masculino , Camundongos , Dados de Sequência Molecular , Mutação/genética , Estrutura Terciária de Proteína/fisiologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
Since their advent some 25 years ago, monoclonal antibodies have developed into powerful tools for structural and functional analysis of their cognate antigens. Together with the respective antigen binding fragments, antibodies offer exclusive capacities in detection, characterization, purification and functional assays for every given ligand. Antibody-fragment mediated crystallization represents a major advance in determining the three-dimensional structure of membrane-bound protein complexes. In this review, we focus on the methods used to generate monoclonal antibodies against the NhaA antiporter from Escherichia coli as a paradigm of secondary transporters. We describe examples on how antibodies are helpful in understanding structure and function relationships for this important class of integral membrane proteins. The generated conformation-specific antibody fragments are highly valuable reagents for co-crystallization attempts and structure determination of the antiporter.
Assuntos
Anticorpos Monoclonais/química , Proteínas de Escherichia coli/biossíntese , Proteínas de Membrana/biossíntese , Trocadores de Sódio-Hidrogênio/biossíntese , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Clonagem Molecular , Cristalização , Epitopos/química , Proteínas de Escherichia coli/isolamento & purificação , Fragmentos Fab das Imunoglobulinas/química , Fragmentos de Imunoglobulinas/química , Cinética , Proteínas de Membrana/isolamento & purificação , Conformação Proteica , Trocadores de Sódio-Hidrogênio/imunologia , Trocadores de Sódio-Hidrogênio/isolamento & purificaçãoRESUMO
The Na,K-ATPase generates electrochemical gradients that are used to drive the coupled transport of many ions and nutrients across the plasma membrane. The functional enzyme is comprised of an alpha and beta subunit and families of isoforms for both subunits exist. Recent studies in this laboratory have identified a biological role for the Na,K-ATPase alpha4 isoform in sperm motility. Here we further investigate the role of the Na,K-ATPase carrying the alpha4 isoform, showing again that ouabain eliminates sperm motility, and in addition, that nigericin, a H+/K+ ionophore, and monensin, a H+/Na+ ionophore, reinitiate motility. These data, along with the observation that the K+ ionophore valinomycin has no effect on the motility of ouabain-inhibited sperm, suggest that ouabain may change intracellular H+ levels in a manner that is incompatible with sperm motility. We have also localized NHE1 and NHE5, known regulators of intracellular H+ content, to the same region of the sperm as the Na,K-ATPase alpha4 isoform. These data highlight the important role of the Na,K-ATPase alpha4 isoform in regulating intracellular H(+) levels, and provide evidence suggesting the involvement of the Na+/H+ exchanger, which is critical for maintaining normal sperm motility.
Assuntos
Trocadores de Sódio-Hidrogênio/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Imunofluorescência , Ionóforos/farmacologia , Isoenzimas/fisiologia , Masculino , Proteínas de Membrana , Monensin/farmacologia , Nigericina/farmacologia , Ouabaína/farmacologia , Ratos , Ratos Sprague-Dawley , Trocadores de Sódio-Hidrogênio/imunologia , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Valinomicina/farmacologiaRESUMO
On the basis of intracellular acidifications in the presence of 30 microM cariporide, we selected a fibroblast cell line termed CR5, expressing a mutated Na(+)/H(+) exchanger NHE-1 with a low affinity for cariporide (87 +/- 11.6 microM) and extracellular sodium (248 +/- 63.7 mM). This mutated exchanger displays a Phe162Ser substitution in its fourth transmembrane segment. Using intracellular acidifications in the presence of 3 mM external sodium on the CR5 fibroblasts, we isolated two revertants which exhibited a complete recovery for sodium affinity but were still resistant to cariporide. Sequencing the cDNAs encoding these revertants revealed the presence of two mutations situated at a distant location from Phe162 in the same fourth transmembrane segment (Ile169Ser and Ile170Thr). Interestingly, introducing these two mutations in the wild-type cDNA did not result in a significant increase in affinity for sodium. Furthermore, all the mutants characterized in this study display an unchanged affinity for lithium, another transported cation. These data suggest that the mutation resulting in the low sodium affinity and the two mutations responsible for the reversion of this phenotype affect the binding of sodium itself instead of the conformational changes triggering substrate translocation. Taken together, these results allow us to propose that optimal sodium binding by the Na(+)/H(+) exchangers requires the geometrical integrity of a highly constrained sodium coordination site.
Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutagênese , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Sódio/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Linhagem Celular , Células Clonais , Cricetinae , Resistência a Medicamentos , Metanossulfonato de Etila , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Guanidina/farmacologia , Guanidinas/farmacologia , Lítio/antagonistas & inibidores , Lítio/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/isolamento & purificação , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Fenótipo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/isolamento & purificação , Sulfonas/farmacologia , TransfecçãoRESUMO
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.