Conformational changes in NhaA Na+/H+ antiporter

Abstract

Na⁺/H⁺ antiporters play a primary role in Na⁺/H⁺ homeostasis in cells and many organelles and have long been drug targets. The X-ray structure of NhaA, the main antiporter of Escherichia coli, provided structural insights into the antiport mechanism and its pH regulation and revealed a novel fold; six of the 12 TMs (Trans membrane segments) are organized in two topologically inverted repeats, each with one TM interrupted by an extended chain creating a unique electrostatic environment in the middle of the membrane at the cation binding site. Remarkably, inverted repeats containing interrupted helices with similar functional implications have since been observed in structures of other bacterial secondary transporters with almost no sequence homology. Finally, the structure reveals that NhaA is organized into two functional regions: a ‘pH sensor' – a cluster of amino acyl side chains that are involved in pH regulation; and a catalytic region that is 9 Å removed from the pH sensor. Alternative accessibility of the binding site to either side of the membrane, i.e., functional-dynamics, is the essence of secondary transport mechanism. Because NhaA is tightly pH regulated, structures of the pH-activated and ligand-activated NhaA conformations are needed to identify its functional-dynamics. However, as these are static snapshots of a dynamic protein, the dynamics of the protein both in vitro and in situ in the membrane are also required as reviewed here in detail. The results reveal two different conformational changes characterizing NhaA: One is pH-induced for NhaA activation; the other is ligand-induced for antiport activity.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

细菌钠离子输出系统的类型及其可能机制

在高盐条件下,绝大多数微生物通过拒盐策略适应其生存环境,其中,钠离子输出系统在维持细胞正常的盐浓度和pH稳态等生命活动过程中扮演十分重要的角色。细菌的钠离子输出系统包括初级钠泵和次级钠泵两种类型,前者所介导的钠离子输出与呼吸相偶联,后者又分为单亚基和多亚基两种类型。到目前为止,有关初级钠泵和次级钠泵转运钠离子的分子机制还停留在推测阶段。对细菌Na 输出系统的类型和Na 外排的可能机制进行综述,并对有待深入研究的问题进行探讨。     

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.)

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca^2 +/H⁺ antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca^2 + ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca^2 + ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet.     

Ectopic Expression of a Bacterium NhaD-type Na~+/H~+ Antiporter Leads to Increased Tolerance to Combined Salt/Alkali Stresses

AaNhaD,a gene isolated from the soda lake alkaliphile Alkalimonas amylolytica,encodes a Na+/H+ antiporter crucial for the bacterium’s resistance to salt/alkali stresses.However,it remains unknown whether this type of bacterial gene may be able to increase the tolerance of flowering plants to salt/alkali stresses.To investigate the use of extremophile genetic resources in higher plants,transgenic tobacco BY-2 cells and plants harboring AaNhaD were generated and their stress tolerance was evaluated.Ectopic expression of AaNhaD enhanced the salt tolerance of the transgenic BY-2 cells in a pH-dependent manner.Compared to wild-type controls,the transgenic cells exhibited increased Na+concentrations and pH levels in the vacuoles.Subcellular localization analysis indicated that AaNhaD-GFP fusion proteins were primarily localized in the tonoplasts.Similar to the transgenic BY-2 cells,AaNhaD-overexpressing tobacco plants displayed enhanced stress tolerance when grown in saline-alkali soil.These results indicate that AaNhaD functions as a pH-dependent tonoplast Na+/H+antiporter in plant cells,thus presenting a new avenue for the genetic improvement of salinity/alkalinity tolerance.     

Purification of two putative type II NADH dehydrogenases with different substrate specificities from alkaliphilic Bacillus pseudofirmus OF4

A putative Type II NADH dehydrogenase from Halobacillus dabanensis was recently reported to have Na⁺/H⁺ antiport activity (and called Nap), raising the possibility of direct coupling of respiration to antiport-dependent pH homeostasis. This study characterized a homologous type II NADH dehydrogenase of genetically tractable alkaliphilic Bacillus pseudofirmus OF4, in which evidence supports antiport-based pH homeostasis that is mediated entirely by secondary antiport. Two candidate type II NADH dehydrogenase genes with canonical GXGXXG motifs were identified in a draft genome sequence of B. pseudofirmus OF4. The gene product designated NDH-2A exhibited homology to enzymes from Bacillus subtilis and Escherichia coli whereas NDH-2B exhibited homology to the H. dabanensis Nap protein and its alkaliphilic Bacillus halodurans C-125 homologue. The ndh-2A, but not the ndh-2B, gene complemented the growth defect of an NADH dehydrogenase-deficient E. coli mutant. Neither gene conferred Na⁺-resistance on an antiporter-deficient E. coli strain, nor did they confer Na⁺/H⁺ antiport activity in vesicle assays. The purified hexa-histidine-tagged gene products were approximately 50 kDa, contained noncovalently bound FAD and oxidized NADH. They were predominantly cytoplasmic in E. coli, consonant with the absence of antiport activity. The catalytic properties of NDH-2A were more consistent with a major respiratory role than those of NDH-2B.     

Functional characterization of a wheat plasma membrane Na+/H+ antiporter in yeast

The functional analysis of the sodium exchanger SOS1 from wheat, TaSOS1, was undertaken using Saccharomyces cerevisiae as a heterologous expression system. The TaSOS1 protein, with significant sequence homology to SOS1 sodium exchangers from Arabidopsis and rice, is abundant in roots and leaves, and is induced by salt treatment. TaSOS1 suppressed the salt sensitivity of a yeast strain lacking the major Na⁺ efflux systems by decreasing the cellular Na⁺ content while increasing K⁺ content. Na⁺/H⁺ exchange activity of purified plasma membrane from yeast cells expressing TaSOS1 was higher than controls transformed with empty vector. These results demonstrate that TaSOS1 contributes to plasma membrane Na⁺/H⁺ exchange.     

Uncoupling of a CLC Cl-/H+ exchange transporter by polyatomic anions

CLC-ec1 is a bacterial archetype of CLC transporters, a ubiquitous class of proteins that catalyze transmembrane exchange of Cl- and H+ necessary for pH regulation of numerous physiological processes. Despite a profusion of high-resolution structures, the molecular mechanism of exchange remains unknown. Here, we rigorously demonstrate strict exchange stoichiometry of 2 Cl-/1 H+. In addition to Cl- and Br-, two non-halide ions, NO3- and SCN-, are shown to be transported by CLC-ec1, but with reduced H+ counter-transport. The loss of proton coupling to these anions is accompanied by an absence of bound anions in the central and external Cl- binding sites in the protein's anion selectivity region, as revealed by crystallographic comparison of Br- and SeCN- bound to this region.     

Identification of Three Distinct Phylogenetic Groups of CAX Cation/Proton Antiporters

Ca²⁺/cation antiporter (CaCA) proteins are integral membrane proteins that transport Ca²⁺ or other cations using the H⁺ or Na⁺ gradient generated by primary transporters. The CAX (for CAtion eXchanger) family is one of the five families that make up the CaCA superfamily. CAX genes have been found in bacteria, Dictyostelium, fungi, plants, and lower vertebrates, but only a small number of CAXs have been functionally characterized. In this study, we explored the diversity of CAXs and their phylogenetic relationships. The results demonstrate that there are three major types of CAXs: type I (CAXs similar to Arabidopsis thaliana CAX1, found in plants, fungi, and bacteria), type II (CAXs with a long N-terminus hydrophilic region, found in fungi, Dictyostelium, and lower vertebrates), and type III (CAXs similar to Escherichia coli ChaA, found in bacteria). Some CAXs were found to have secondary structures that are different from the canonical six transmembrane (TM) domains–acidic motif-five TM domain structure. Our phylogenetic tree indicated no evidence to support the cyanobacterial origin of plant CAXs or the classification of Arabidopsis exchangers CAX7 to CAX11. For the first time, these results clearly define the CAX exchanger family and its subtypes in phylogenetic terms. The surprising diversity of CAXs demonstrates their potential range of biochemical properties and physiologic relevance. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: David Guttman]