Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters

Vacuolar compartmentation of Na(+) is an essential mechanism for salinity tolerance since it lowers cytosolic Na(+) levels while contributing to osmotic adjustment for cell turgor and expansion. The AtNHX1 protein of Arabidopsis thaliana substituted functionally for ScNHX1, the endosomal Na(+)/H(+) antiporter of yeast. Ion tolerance conferred by AtNHX1 and ScNHX1 correlated with ion uptake into an intracellular pool that was energetically dependent on the vacuolar (H(+))ATPase. AtNHX1 localized to vacuolar membrane fractions of yeast. Hence, both transporters share an evolutionarily conserved function in Na(+) compartmentation. AtNHX1 mRNA levels were upregulated by ABA and NaCl treatment in leaf but not in root tissue.     

Chimeric Na(+)/H(+) antiporters constructed from NhaA of Helicobacter pylori and Escherichia coli: implications for domains of NhaA for pH sensing

In order to delineate regions which play a role in the regulation of Na(+)/H(+) antiporter NhaA activity by pH, we analyzed the antiporter activities of various chimeric mutants constructed from specific portions of NhaA from Escherichia coli and Helicobacter pylori (EC and HP NhaA). HP NhaA contains 10 residues at the amino-terminus, and 38 residues in a loop region between the eighth and ninth transmembrane spans (loop 8), which are absent in EC NhaA. Deletion from HP NhaA or insertion into EC NhaA of the sequences caused almost no change in pH-dependent antiport activities relative to in the case of the wild-type parent molecules. Chimeras consisting of various combinations of the amino-terminal (amino terminus to sixth or eighth transmembrane span) and carboxy-terminal (seventh or ninth transmembrane span to the carboxy-terminus) regions of EC and HP NhaA showed antiporter activity profiles intermediate between those of the parent molecules. These results show that the two HP-specific sequences are not directly involved in the mechanism of pH sensing by HP NhaA and that the pH sensitivity of NhaA activity is not determined by the amino- or carboxy-terminal regions of NhaA alone, but may be due to interaction between unconserved residues in the two domains. In addition, it was suggested that loop 8 functions primarily as a hinge in both NhaA molecules.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC(50) of 0.9 muM) inhibit the proton motive force dependent Na(+)(Li(+))/H(+) exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of (22)Na(+)/Na(+) exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.     

How do MgATP analogues differentially modify high-affinity and low-affinity ATP binding sites of Na+/K(+)-ATPase?

The exchange-inert tetra-ammino-chromium complex of ATP [Cr(NH3)4ATP], unlike the analogous cobalt complex Co(NH3)4ATP, inactivated Na+/K(+)-ATPase slowly by interacting with the high-affinity ATP binding site. The inactivation proceeded at 37 degrees C with an inactivation rate constant of 1.34 x 10(-3) min-1 and with a dissociation constant of 0.62 microM. To assess the potential role of the water ligands of metal in binding and inactivation, a kinetic analysis of the inactivation of Na+/K(+)-ATPase by Cr(NH3)4ATP, and its H2O-substituted derivatives Cr(NH3)3(H2O)ATP, Cr(NH3)2(H2O)2ATP and Cr(H2O)4ATP was carried out. The substitution of the H2O ligands with NH3 ligands increased the apparent binding affinity and decreased the inactivation rate constants of the enzyme by these complexes. Inactivation by Cr(H2O)4ATP was 29-fold faster than the inactivation by Cr(NH3)4ATP. These results suggested that substitution to Cr(III) occurs during the inactivation of the enzyme. Additionally hydrogen bonding between water ligands of metal and the enzyme's active-site residues does not seem to play a significant role in the inactivation of Na+/K(+)-ATPase by Cr(III)-ATP complexes. Inactivation of the enzyme by Rh(H2O)nATP occurred by binding of this analogue to the high-affinity ATP site with an apparent dissociation constant of 1.8 microM. The observed inactivation rate constant of 2.11 x 10(-3) min-1 became higher when Na+ or Mg2+ or both were present. The presence of K+ however, increased the dissociation constant without altering the inactivation rate constant. High concentrations of Na+ reactivated the Rh(H2O)nATP-inactivated enzyme. Co(NH3)4ATP inactivates Na+/K(+)-ATPase by binding to the low-affinity ATP binding site only at high concentrations. However, inactivation of the enzyme by Cr(III)-ATP or Rh(III)-ATP complexes was prevented when low concentrations of Co(NH3)4ATP were present. This indicates that, although Co(NH3)4ATP interacts with both ATP sites, inactivation occurs only through the low-affinity ATP site. Inactivation of Na+/K(+)-ATPase was faster by the delta isomer of Co(NH3)4ATP than by the delta isomer. Co(NH3)4ATP, but not Cr(H2O)4ATP or adenosine 5'-[beta,gamma-methylene]triphosphate competitively inhibited K(+)-activated p-nitrophenylphosphatase activity of Na+/K(+)-ATPase, which is assumed to be a partial reaction of the enzyme catalyzed by the low-affinity ATP binding site.     

Heterologous expression of mammalian Na/H antiporters in Saccharomyces cerevisiae

Na⁺/H⁺ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1–3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.     

Functional and structural dynamics of NhaA,a prototype for Na and H antiporters,which are responsible for Na and H homeostasis in cells

The crystal structure of down-regulated NhaA crystallized at acidic pH 4 [21] has provided the first structural insights into the antiport mechanism and pH regulation of a Na⁺/H⁺ antiporter [22]. On the basis of the NhaA crystal structure [21] and experimental data (reviewed in [2,22,38] we have suggested that NhaA is organized into two functional regions: (i) a cluster of amino acids responsible for pH regulation (ii) a catalytic region at the middle of the TM IV/XI assembly, with its unique antiparallel unfolded regions that cross each other forming a delicate electrostatic balance in the middle of the membrane. This unique structure contributes to the cation binding site and allows the rapid conformational changes expected for NhaA. Extended chains interrupting helices appear now a common feature for ion binding in transporters. However the NhaA fold is unique and shared by ASBTNM [30] and NapA [29]. Computation [13], electrophysiology [69] combined with biochemistry [33,47] have provided intriguing models for the mechanism of NhaA. However, the conformational changes and the residues involved have not yet been fully identified. Another issue which is still enigma is how energy is transduced “in this ‘nano-machine.’” We expect that an integrative approach will reveal the residues that are crucial for NhaA activity and regulation, as well as elucidate the pHand ligand-induced conformational changes and their dynamics. Ultimately, integrative results will shed light on the mechanism of activity and pH regulation of NhaA, a prototype of the CPA2 family of transporters. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Requirements for conversion of the Na(+)-driven flagellar motor of Vibrio cholerae to the H(+)-driven motor of Escherichia coli

Bacterial flagella are powered by a motor that converts a transmembrane electrochemical potential of either H(+) or Na(+) into mechanical work. In Escherichia coli, the MotA and MotB proteins form the stator and function in proton translocation, whereas the FliG protein is located on the rotor and is involved in flagellar assembly and torque generation. The sodium-driven polar flagella of Vibrio species contain homologs of MotA and MotB, called PomA and PomB, and also contain two other membrane proteins called MotX and MotY, which are essential for motor rotation and that might also function in ion conduction. Deletions in pomA, pomB, motX, or motY in Vibrio cholerae resulted in a nonmotile phenotype, whereas deletion of fliG gave a nonflagellate phenotype. fliG genes on plasmids complemented fliG-null strains of the parent species but not fliG-null strains of the other species. FliG-null strains were complemented by chimeric FliG proteins in which the C-terminal domain came from the other species, however, implying that the C-terminal part of FliG can function in conjunction with the ion-translocating components of either species. A V. cholerae strain deleted of pomA, pomB, motX, and motY became weakly motile when the E. coli motA and motB genes were introduced on a plasmid. Like E. coli, but unlike wild-type V. cholerae, motility of some V. cholerae strains containing the hybrid motor was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone under neutral as well as alkaline conditions but not by the sodium motor-specific inhibitor phenamil. We conclude that the E. coli proton motor components MotA and MotB can function in place of the motor proteins of V. cholerae and that the hybrid motors are driven by the proton motive force.     

Physiological consequences of expression of the Na+/H+ antiporter sod2 in Escherichia coli

Sod2 is the sodium-proton antiporter on the plasma membrane of the fission yeast Schizosaccharomyces pombe. It is vitally important for sodium export and pH homeostasis in this organism. Recently, the sod2 gene has been cloned and sequenced. However, initial attempts to express sod2 in Escherichia coli using the T7 promoter failed. In the present work we examined physiological consequences of expression of sod2 in E. coli. To alleviate problems caused by expression of sod2 we: (i) used sodium-free media at all steps; (ii) used the moderate tac promoter for expression and; (iii) used E. coli strain MH1 which has impaired sodium exchange. The effect of sod2 expression on E. coli varied depending on the E. coli genotype. When sod2 was expressed in BL21 cells which have normal N a⁺/H⁺ antiporters, the result was a Li⁺ sensitive phenotype. LiCl completely arrested or prevented growth of BL21 E. coli transformed with the sod2 gene. The effect on growth was pronounced in media of low external pH. Sod2 was then expressed in E. coli MH1 which is devoid of endogenous Na⁺/H⁺ antiporters. These cells became more resistant to external LiCl, but only in Na⁺ containing media. In the absence of external Na⁺, the presence of sod2 reduced growth. The results are explained in a model which demonstrates the physiological consequences of interference by expression of a foreign electroneutral Na⁺/H⁺ antiporter in conjunction with different housekeeping systems of E. coli host cells.     

细菌Na+/H+逆向转运蛋白的研究进展

Na+/H+逆向转运蛋白在维持细胞内pH稳态、Na+离子动态平衡和调控细胞体积方面发挥着重要作用。目前,细菌中许多参与高盐或高碱性环境压力应答的Na+/H+逆向转运蛋白得到了鉴定和功能阐释。继续挖掘高效的Na+/H+逆向转运蛋白,深入探究Na+/H+逆向转运蛋白的分子机理,将为工业菌株或农作物的改良提供新的研究思路。本文以4种模式菌株为例,简要概述细菌Na+/H+逆向转运蛋白的种类和特征,同时对其结构和功能等方面也进行探讨。     

Na(+)-dependent high-affinity uptake of L-glutamate in cultured fibroblasts.

Uptake of 1 microM [3H]L-glutamate by cultured 3T3 fibroblasts was strongly dependent on extracellular Na+; it was reduced by elevated concentrations of K+ (60 mM) but it was not influenced by variations in the concentration of Ca2+ (0-9.6 mM). D- and L-Asparate, D- and L-threo-3-hydroxyaspartate DL-threo-3-methylaspartate and a few other glutamate derivatives and analogues inhibited the uptake but several close analogues of L-glutamate (including D-glutamate) had no effect, implying that the uptake system is highly structurally selective. The recently identified inhibitor of glutamate uptake in synaptosomal preparations, L-trans-pyrrolidine-2,4-dicarboxylate, was also among the inhibitors. Apparent Km of the uptake was found to be less than 10 microM. The present observations indicate that Na(+)-dependent 'high-affinity' uptake of L-glutamate may appear in structures which are apparently unrelated to glutamatergic synaptic transmission in the CNS.     

Na+/H+ antiporters,molecular devices that couple the Na+ and H+ circulation in cells

Na⁺/H⁺ antiporters are universal devices involved in the Na⁺ and H⁺ circulation of both eukaroyotes and prokaryotes, thus playing an essential role in the pH and Na⁺ homeostasis of cells. This review focuses on the major impact of the application of molecular biology tools in the study of the antiporters. These tools permit the verification of the role of the antiporters and provide insights into their unique biology. A novel signal transduction to Na⁺ involvingnhaR, a positive regulator, controls the expression ofnhaA inE. coli. A pH sensor regulates the activity of Na⁺/H⁺ antiporters, both in eukaryotes and prokaryotes. A most intricate signal transduction to pH involving phosphorylation steps controls the activity ofnhel in higher mammals. The identification of Histidine 226 in the pH sensor of NhaA is a step forward towards the understanding of the pH regulation of these proteins.     

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and S?ll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.     

Inhibition studies on Rhodospirillum rubrum H(+)-pyrophosphatase expressed in Escherichia coli

The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N,N'-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5'-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg(2)PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg(2+), possibly reflecting a change in substrate preference from Mg(2)PPi to MgPPi. BPB is a H(+)-pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.     

Functional expression of a Drosophila antifungal peptide in Escherichia coli

Drosomycin is a key effector molecule involved in Drosophila innate immunity against fungal infection. This peptide is composed of 44 residues stabilized by four disulfide bridges. As the first step towards the understanding of the molecular basis for its specific antifungal activity, rapid and efficient production of the wild-type peptide and its mutants is needed. Here, we report a pGEX system for high-level expression of recombinant Drosomycin. The fusion Drosomycin protein with a carrier of Glutathione S-transferase (GST) was initially purified by affinity chromatography followed by Enterokinase cleavage. The digested product was separated by gel filtration and reverse phase HPLC. Mass spectrometry and circular dichroism spectroscopy analysis revealed that the recombinant peptide has identical molecular weight and correct structural conformation to native Drosomycin. Classical inhibition assay showed clear antifungal activity against Neurospora crassa with the IC(50) of 1.0muM. Successful expression of the CSalphabeta-type antifungal peptide in E. coli offers a basis for further studying its functional surface by alanine scanning mutagenesis strategy. Also, our work should be helpful in developing this peptide to an antifungal drug.     

Functional expression of His-tagged sensory rhodopsin I in Escherichia coli

Sensory rhodopsin I (SRI) from Halobacterium salinarum was functionally expressed in Escherichia coli and subsequently purified to homogeneity using a C-terminal His-tag anchor. Yields of 3-4 mg SRI/l cell culture can be obtained. The absorption and photocycle properties of SRI were similar if not indistinguishable from those of the homologously expressed SRI. A global fit analysis of the photocycle data and the calculation of the spectra of states provided strong evidence for the existence of an N-like intermediate.