Physiological roles of three Na+/H+ antiporters in the halophilic bacterium Vibrio parahaemolyticus

Vibrio parahaemolyticus mutants lacking three Na+/H+ antiporters (NhaA, NhaB, NhaD) were constructed. The DeltanhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The DeltanhaA and DeltanhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na+/H+ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na+/H+ and Li+/H+ antiport activities were observed with everted membrane vesicles of DeltanhaB strains. However, Li+/H+ antiport activities of DeltanhaB strains were two times higher than those of DeltanhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2-heptyl-4-hydroxyquinoline N-oxide), which is a potent inhibitor of the respiratory Na+ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na+/H+ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na+/H+ antiporters cooperate with the respiratory Na+ pump in ionic homeostasis in V. parahaemolyticus.     

Plastid KEA-type cation/H+ antiporters are required for vacuolar protein trafficking in Arabidopsis

Arabidopsis plastid antiporters KEA1 and KEA2 are critical for plastid development, photosynthetic efficiency, and plant development. Here, we show that KEA1 and KEA2 are involved in vacuolar protein trafficking. Genetic analyses found that the kea1 kea2 mutants had short siliques, small seeds, and short seedlings. Molecular and biochemical assays showed that seed storage proteins were missorted out of the cell and the precursor proteins were accumulated in kea1 kea2. Protein storage vacuoles (PSVs) were smaller in kea1 kea2. Further analyses showed that endosomal trafficking in kea1 kea2 was compromised. Vacuolar sorting receptor 1 (VSR1) subcellular localizations, VSR–cargo interactions, and p24 distribution on the endoplasmic reticulum (ER) and Golgi apparatus were affected in kea1 kea2. Moreover, plastid stromule growth was reduced and plastid association with the endomembrane compartments was disrupted in kea1 kea2. Stromule growth was regulated by the cellular pH and K⁺ homeostasis maintained by KEA1 and KEA2. The organellar pH along the trafficking pathway was altered in kea1 kea2. Overall, KEA1 and KEA2 regulate vacuolar trafficking by controlling the function of plastid stromules via adjusting pH and K⁺ homeostasis.     

K+/H+ antiporter in alkaliphilic Bacillus sp. no. 66 (JCM 9763)

A K⁺/H⁺ antiport system was detected for the first time in right-side-out membrane vesicles prepared from alkaliphilic Bacillus sp. no. 66 (JCM 9763). An outwardly directed K⁺ gradient (intravesicular K⁺ concentration, K_in, 100 mM; extravesicular K⁺ concentration, K_out, 0.25 mM) stimulated uphill H⁺ influx into right-side-out vesicles and created the inside-acidic pH gradient (ΔpH). This H⁺ influx was pH-dependent and increased as the pH increased from 6.8 to 8.4. Addition of 100 μM quinine inhibited the H⁺ influx by 75%. This exchange process was electroneutral, and the H⁺ influx was not stimulated by the imposition of the membrane potential (interior negative). Addition of K⁺ at the point of maximum ΔpH caused a rapid K⁺-dependent H⁺ eflux consistent with the inward exchange of external K⁺ for internal H⁺ by a K⁺/H⁺ antiporter. Rb⁺ and Cs⁺ could replace K⁺ but Na⁺ and Li⁺ could not. The H⁺ efflux rate was a hyperbolic function of K⁺ and increased with increasing extravesicular pH (pH_out) from 7.5 to 8.5. These findings were consistent with the presence of K⁺/H⁺ antiport activity in these membrane vesicles. Received: March 20, 1997 / Accepted: May 22, 1997     

Na+/H+ 逆向转运蛋白与植物耐盐性关系

Na+/H+ 逆向转运蛋白与植物的耐盐性有密切的关系。在高等植物体内,主要存在两种Na+/H+ 逆向转运蛋白,分别为位于细胞质膜上的逆向转运蛋白SOS1,以及存在于液泡膜上的AtNHX1。质膜Na+/H+ 逆向转运蛋白主要负责Na+ 的外排,液泡膜Na+/H+ 逆向转运蛋白主要负责把Na+ 区隔化入液泡。过量表达质膜Na+/H+ 逆向转运蛋白SOS1或液泡膜Na+/H+ 逆向转运蛋白AtNHX1能够明显提高植物的耐盐性。本文对植物中Na+/H+ 逆向转运蛋白及其与植物耐盐性之间的关系研究最新进展作一概述。     

Na+/H+逆向转运蛋白和植物耐盐性

Na^ /H^ 逆向转运蛋白对植物耐盐起着重要作用,它利用质膜H^ -ATPase或液泡膜H^ -ATPase及PPiase泵H^ 产生的驱动力把Na^ 排出细胞或在液泡中区隔化以消除Na^ 的毒害。主要讨论植物中Na^ /H^ 逆向转运蛋白研究在分子水平的最新进展。     

SNARE proteins in membrane trafficking

SNAREs are the core machinery mediating membrane fusion. In this review, we provide an update on the recent progress on SNAREs regulating membrane fusion events, especially the more detailed fusion processes dissected by well‐developed biophysical methods and in vitro single molecule analysis approaches. We also briefly summarize the relevant research from Chinese laboratories and highlight the significant contributions on our understanding of SNARE‐mediated membrane trafficking from scientists in China.      

Na+,K+/H+ antiporters regulate the pH of endoplasmic reticulum and auxin‐mediated development

AtNHX5 and AtNHX6 are endosomal Na⁺,K⁺/H⁺ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H⁺ leak, control auxin homeostasis and auxin‐mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin‐related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the endoplasmic reticulum (ER)‐localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were colocalized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H⁺‐leak pathway provides a fine‐tuning mechanism that controls cellular auxin fluxes.     

The Na+,K+/H+ -antiporter Nha1 influences the plasma membrane potential of Saccharomyces cerevisiae

There are three different sodium transport systems (Ena1-4p, Nha1p, Nhx1p) in Saccharomyces cerevisiae. The effect of their absence on the tolerance to alkali-metal cations and on the membrane potential was studied. All three sodium transporters were found to participate in the maintenance of Na+, Li+, K+ and Cs+ homeostasis. Measurements of the distribution of a fluorescent potentiometric probe (diS-C3(3) assay) in cell suspensions showed that the lack of all three transporters depolarizes the plasma membrane. The overexpression of the Na+,K+/H+ antiporter Nha1 resulted in the hyperpolarization of the plasma membrane and consequently increased the sensitivity to Cs+, Tl+ and hygromycin B. This is the first evidence that the activity of a Na+,K+/H+ antiporter could play a role in the homeostatic regulation of the plasma membrane potential in yeast cells.     

pH homeostasis and bioenergetic work in alkalophiles

Abstract A Na⁺/H⁺ antiporter catalyses coupled Na⁺ extrusion and H⁺ uptake across the membranes of extremely alkalophilic bacilli. This exchange is electrogenic, with H⁺ translocated inward > Na⁺ extruded. It is energized by the Δψ ² component of the ΔμH⁺ that is established during primary proton pumping by the alkalophile respiratory chain complexes. These complexes abound in the membranes of extreme alkalophiles. Combined activity of the respiratory chain, the antiporter, and solute transport systems that are coupled to Na⁺ re-entry, allow the alkalophiles to maintain a cytoplasmic pH that is several pH units more acidic than optimal external pH values for growth. There is no compelling evidence for a specific and necessary role for any ion other than sodium in pH homeostasis, and although there is very high cytoplasmic buffering capacity in the alkaline range, active mechanisms for pH homeostasis are crucial. Energization of the antiporter as well as the proton translocating F ₁ F ₀-ATPase that catalyses ATP synthesis in the extreme alkalophiles must accommodate the problem of the low net ΔμH⁺ and the very low concentrations of protons, per se, in the external medium. This problem is by-passed by other bioenergetic work functions, such as solute uptake or motility, that utilize sodium ions for energy-coupling in the place of protons.     

质膜Na^＋／H^＋逆向转运蛋白与植物耐盐性

土壤盐碱化是造成农作物减产的主要原因之一。质膜Na^＋／H^＋逆向转运蛋白能够介导植物根部Na^＋的外排和体内Na^＋的长距离运输, 并能够调控细胞K＋的稳态平衡及细胞内pH值和Ca^2＋的转运, 因此其在植物耐盐性方面具有重要作用。该文概述了植物质膜Na^＋／H^＋逆向转运蛋白的分子结构、功能、表达调控及其与植物耐盐性关系等方面的研究进展, 并对今后有关该蛋白的主要研究方向作了分析和展望。     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Schizosaccharomyces pombe possesses two plasma membrane alkali metal cation/H antiporters differing in their substrate specificity

The Schizosaccharomyces pombe plasma membrane Na(+)/H(+) antiporter, SpSod2p, has been shown to belong to the subfamily of yeast Na(+)/H(+) antiporters that only recognize Na(+) and Li(+) as substrates. Nevertheless, most of the studied plasma membrane alkali metal cation/H(+) antiporters from other yeasts have broader substrate specificities, exporting K(+) and Rb(+) as well. Such antiporters probably play two roles in the physiology of cells: the elimination of surplus toxic cations, and the regulation of stable intracellular K(+) content, pH and cell volume. The systematic sequencing of the Sch. pombe genome revealed the presence of an as-yet uncharacterized homolog of the Spsod2 gene (designated Spsod22). Spsod22 and Spsod2 were expressed in Saccharomyces cerevisiae cells lacking their own alkali metal cation efflux systems, and the transport properties of both Sch. pombe antiporters were compared to those of the Sac. cerevisiae Nha1 antiporter expressed under the same conditions. Here we show that SpSod22p has broad substrate specificity upon heterologous expression in Sac. cerevisiae cells and contributes to cell tolerance to high external levels of K(+). Thus, the Sch. pombe genome encodes two plasma membrane alkali metal cation/H(+) antiporters that play different roles in the physiology of the yeast.     

Partial immunochemical identity between (Na+ + K+)-ATPase and a membrane component extractable with chloroform: methanol

An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)-ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically-related antigen when a plasma membrane-enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically-related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

Function and dysfunction of the PI system in membrane trafficking

The phosphoinositides (PIs) function as efficient and finely tuned switches that control the assembly–disassembly cycles of complex molecular machineries with key roles in membrane trafficking. This important role of the PIs is mainly due to their versatile nature, which is in turn determined by their fast metabolic interconversions. PIs can be tightly regulated both spatially and temporally through the many PI kinases (PIKs) and phosphatases that are distributed throughout the different intracellular compartments. In spite of the enormous progress made in the past 20 years towards the definition of the molecular details of PI–protein interactions and of the regulatory mechanisms of the individual PIKs and phosphatases, important issues concerning the general principles of the organisation of the PI system and the coordination of the different PI-metabolising enzymes remain to be addressed. The answers should come from applying a systems biology approach to the study of the PI system, through the integration of analyses of the protein interaction data of the PI enzymes and the PI targets with those of the ‘phenomes' of the genetic diseases that involve these PI-metabolising enzymes.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

Emerging roles of phosphoinositide-associated membrane trafficking in plant stress responses

Eukaryotic cells are confined by membranes that create hydrophobic barriers for substance and information exchange between the inside and outside of the cell. These barriers are formed by assembly of lipids and protein in aqueous environments. Lipids not only serve as building blocks for membrane construction, but also possess regulatory functions in cellular activities. These regulatory lipids are non-uniformly distributed in membrane systems; their temporal and spatial accumulation in specific membranes decodes environmental cues and changes cellular activity accordingly. Phosphoinositides (PIs) are phospholipids that exert regulatory effects. In recent years, research on PIs roles in regulating plant growth, development, and responses to environmental stress is increasing. Several reviews have been published on the composition of PIs, intermolecular transferring of PIs by lipid kinases (phosphatases) or PI-PLCs, subcellular localization, and specially their functions in plant developments. Herein, we review the crucial regulatory functions of PIs in plant stress responses, with a particular focus on PIs involved in membrane trafficking.     

The gene sll0273 of the cyanobacterium Synechocystis sp. strain PCC6803 encodes a protein essential for growth at low Na+/K+ ratios

A mutant of Synechocystis sp. strain PCC6803 was obtained by random cartridge mutagenesis, which could not grow at low sodium concentrations. Genetic analyses revealed that partial deletion of the sll0273 gene, encoding a putative Na ⁺ /H ⁺ exchanger, was responsible for this defect. Physiological characterization indicated that the sll0273 mutant exhibited an increased sensitivity towards K ⁺ , even at low concentrations, which was compensated for by enhanced concentrations of Na ⁺ . This enhanced Na ⁺ demand could also be met by Li ⁺ . Furthermore, addition of monensin, an ionophore mediating electroneutral Na ⁺ /H ⁺ exchange, supported growth of the mutant at unfavourable Na ⁺ /K ⁺ ratios. Measurement of internal Na ⁺ and K ⁺ contents of wild‐type and mutant cells revealed a decreased Na ⁺ /K ⁺ ratio in mutant cells pre‐incubated at a low external Na ⁺ /K ⁺ ratio, while it remained at the level of the wild type after pre‐incubation at a high external Na ⁺ /K ⁺ ratio. We conclude that the Sll0273 protein is required for Na ⁺ influx, especially at low external Na ⁺ concentrations or low Na ⁺ /K ⁺ ratios. This system may be part of a sodium cycle and may permit re‐entry of Na ⁺ into the cells, if nutrient/Na ⁺ symporters are not functional or operating.