Effects of Genome Position and the DNA Damage Checkpoint on the Structure and Frequency of sod2 Gene Amplification in Fission Yeast

The Schizosaccharomyces pombe sod2 gene, located near the telomere on the long arm of chromosome I, encodes a Na+ (or Li+)/H+ antiporter. Amplification of sod2 has previously been shown to confer resistance to LiCl. We analyzed 20 independent LiCl-resistant strains and found that the only observed mechanism of resistance is amplification of sod2. The amplicons are linear, extrachromosomal elements either 225 or 180 kb long, containing both sod2 and telomere sequences. To determine whether proximity to a telomere is necessary for sod2 amplification, a strain was constructed in which the gene was moved to the middle of the same chromosomal arm. Selection of LiCl-resistant strains in this genetic background also yielded amplifications of sod2, but in this case the amplified DNA was exclusively chromosomal. Thus, proximity to a telomere is not a prerequisite for gene amplification in S. pombe but does affect the mechanism. Relative to wild-type cells, mutants with defects in the DNA damage aspect of the rad checkpoint control pathway had an increased frequency of sod2 amplification, whereas mutants defective in the S-phase completion checkpoint did not. Two models for generating the amplified DNA are presented.     

Identification of conserved polar residues important for salt tolerance by the Na+/H+ exchanger of Schizosaccharomyces pombe

The Na+/H+ exchanger is a ubiquitous protein that transports Na+ and H+ in opposite directions across cell membranes. In fission yeast, the Na+/H+ exchanger sod2 plays a major role in the removal of excess detrimental intracellular sodium. The effect of mutagenesis of conserved polar amino acids of sod2 was examined by expressing 10 different mutant forms of sod2 in sod2 deficient S. pombe and characterizing salt tolerance. Asp145, 266, 267, and Glu173 were critical for proper function of sod2. Asp241 had an intermediate effect on sod2 function while mutation of Asp178 did not impair sod2 function. Simultaneous mutation of the Asp266, 267 pair impaired sod2 function. Mutation of each individual residue demonstrated that both were critical for sod2 function. Conservative mutations (Asp to Glu) of Asp266 and 267 failed to restore sod2 function. The results suggest that acidic residues associated with transmembrane segments are important in function, possibly being important in binding and coordinating cations.     

AtNHX8, a member of the monovalent cation: proton antiporter-1 family in Arabidopsis thaliana, encodes a putative Li/H antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.     

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.     

The Schizosaccharomyces pombe Pzh1 protein phosphatase regulates Na+ ion influx in a Trk1-independent fashion.

We have previously shown that fission yeast encodes a PPZ-like phosphatase, designated Pzhl, which is an important determinant of cation homeostasis. pzh1 delta mutants display increased tolerance to Na+ ions, but they are hypersensitive to KC1 [Balcells, L., Gómez, N., Casamayor, A., Clotet, J. & Ari?o, J. (1997) Eur. J. Biochem. 250, 476-483]. We have immunodetected Pzh1 in yeast extracts and found that this phosphatase is largely associated with particulate fractions. Cells defective in Pzh1 do not show altered efflux of Na+ or Li+ ions, but they accumulate these cations more slowly than wild-type cells. K+ ion content of pzh1 delta cells is about twice that of wild-type cells, and this can be explained by decreased efflux of K+. Therefore, Pzh1 may regulate both Na+ influx and K+ efflux in fission yeast. To test the possible relationship between K+ uptake, Na+ tolerance and Pzh1 function, we deleted the trk1+ gene, which encodes a putative high-affinity transporter of K+ ions. trkl delta mutants grew well even at relatively low concentrations of KCl and did not show significantly altered content or influx of K+ ions. However, they showed a Na(+)-sensitive phenotype which was greatly intensified by deletion of the sod2+ gene (which encodes the major determinant for efflux of Na+ ions), and clearly ameliorated by deletion of the pzh1 phosphatase, as well as by moderate concentrations of KCl in the medium. These results suggest that Trk1 does not mediate the effect of Pzh1 on NaCl tolerance and that fission yeast contains efficient systems, other than Trk1, for uptake of K+ ions.     

Vacuolar cation/H+ exchange, ion homeostasis, and leaf development are altered in a T-DNA insertional mutant of AtNHX1, the Arabidopsis vacuolar Na+/H+ antiporter

The function of vacuolar Na+/H+ antiporter(s) in plants has been studied primarily in the context of salinity tolerance. By facilitating the accumulation of Na+ away from the cytosol, plant cells can avert ion toxicity and also utilize vacuolar Na+ as osmoticum to maintain turgor. As many genes encoding these antiporters have been cloned from salt-sensitive plants, it is likely that they function in some capacity other than salinity tolerance. The wide expression pattern of Arabidopsis thaliana sodium proton exchanger 1 (AtNHX1) in this study supports this hypothesis. Here, we report the isolation of a T-DNA insertional mutant of AtNHX1, a vacuolar Na+/H+ antiporter in Arabidopsis. Vacuoles isolated from leaves of the nhx1 plants had a much lower Na+/H+ and K+/H+ exchange activity. nhx1 plants also showed an altered leaf development, with reduction in the frequency of large epidermal cells and a reduction in overall leaf area compared to wild-type plants. The overexpression of AtNHX1 in the nhx1 background complemented these phenotypes. In the presence of NaCl, nhx1 seedling establishment was impaired. These results place AtNHX1 as the dominant K+ and Na+/H+ antiporter in leaf vacuoles in Arabidopsis and also suggest that its contribution to ion homeostasis is important for not only salinity tolerance but development as well.     

Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter

A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway

For plants growing in highly saline environments, accumulation of sodium in the cell cytoplasm leads to disruption of metabolic processes and reduced growth. Maintaining low levels of cytoplasmic sodium requires the coordinate regulation of transport proteins on numerous cellular membranes. Our previous studies have linked components of the Salt-Overly-Sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana and demonstrated that the activity of the plasma membrane Na+/H+ exchanger (SOS1) is regulated by SOS2 (a protein kinase) and SOS3 (a calcium-binding protein). Current studies were undertaken to determine if the Na+/H+ exchanger in the vacuolar membrane (tonoplast) of Arabidopsis is also a target for the SOS regulatory pathway. Characterization of tonoplast Na+/H+ exchange demonstrated that it represents activity originating from the AtNHX proteins since it could be inhibited by 5-(N-methyl-N-isobutyl)amiloride and by anti-NHX1 antibodies. Transport activity was selective for sodium (apparent Km=31 mm) and electroneutral (one sodium ion for each proton). When compared with tonoplast Na+/H+-exchange activity in wild type, activity was significantly higher, greatly reduced, and unchanged in sos1, sos2, and sos3, respectively. Activated SOS2 protein added in vitro increased tonoplast Na+/H+-exchange activity in vesicles isolated from sos2 but did not have any effect on activity in vesicles isolated from wild type, sos1, or sos3. These results demonstrate that (i) the tonoplast Na+/H+ exchanger in Arabidopsis is a target of the SOS regulatory pathway, (ii) there are branches to the SOS pathway, and (iii) there may be coordinate regulation of the exchangers in the tonoplast and plasma membrane.     

Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts

Net H+ fluxes across the plasma membrane of Chinese hamster lung fibroblasts (CC139) were monitored by pH-stat titration. Na+-depleted cells release H+ upon addition of Na+. Conversely Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+,Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride and does not occur in digitonin-permeabilized cells. A similar Na+/H+ exchanger was identified in vascular smooth muscle cells, corneal and aortic endothelial cells, lens epithelial cells of bovine origin, and human platelets. Kinetic studies carried out with CC139 cells indicate the following properties: 1) half-saturation of the system is observed at pH = 7.8, in the absence of Na+; 2) external Na+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki = 2-3 mM); 3) amiloride is a competitive inhibitor for Na+ (Ki congruent to 1 microM) and a noncompetitive inhibitor for H+; 4) a coupling ratio of 1.3 +/- 0.3 for the H+/Li+ exchange suggests a stoichiometry of 1:1. We conclude that CC139 cells possess in their plasma membrane a reversible, electroneutral, and amiloride-sensitive Na+/H+ antiporter, with two distinct and mutually exclusive binding sites for Na+ and H+. The rapid stimulation of the Na+/H+ antiporter in G0/G1-arrested CC139 cells upon addition of growth factors, together with the fact that intracellular H+ concentration is, under physiological conditions, around the apparent K0.5 of the system, strongly suggests a key role of this antiport in pHi regulation and mitogen action.     

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.     

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.     

Serum regulates Na+/H+ exchange in Caco-2 cells by a mechanism which is dependent on F-actin.

Regulation of Na+/H+ exchange by fetal bovine serum was studied in Caco-2 cells, an established cell line derived from a human colon carcinoma. Cells were grown as polarized monolayers on collagen-coated filters and intracellular pH measured fluorometrically with 2',7'-bis(2-carboxymethyl)-5,6-carboxyfluorescein. Na+/H+ exchange was reduced 64% when cells were deprived of serum for 4 h. In contrast to other cell types, readdition of serum for 10 min did not activate Na+/H+ exchange; however, readdition of serum for 4 h restored Na+/H+ exchange to control values. This long-term effect of serum on Na+/H+ exchange activity could not be explained by changes in intracellular buffering capacity or intracellular [Na+]. 4-h serum deprivation reduced the K(t) of the exchanger for external Na+ from 21 to 6 mM, and reduced the V(max) by 57%, but did not alter the IC50 for amiloride in the presence of 140 mM Na+. Inhibition of protein synthesis with cycloheximide (5 microM) did not alter the effect of serum removal or readdition on Na+/H+ exchange. Low temperature (13 degrees C) completely prevented the inhibition of Na+/H+ exchange caused by the removal of serum. In addition, once Na+/H+ exchange was inhibited by serum removal at 37 degrees C, maintaining cells at 13 degrees C also blocked the recovery of Na+/H+ exchange caused by serum readdition. Conversely, cytochalasin D (0.1-20 microM) blocked the reduction of Na+/H+ exchange which occurred due to 4-h serum deprivation, but did not block the restoration of Na+/H+ exchange when the cells were re-exposed to serum for a further 4 h. Colchicine (20 microM) did not alter the effect of serum removal or readdition. These data suggest that serum regulates Na+/H+ exchange activity by a posttranslational mechanism which is dependent on F-actin.     

Rice Na<Superscript>+</Superscript>/H<Superscript>+</Superscript>-antiporter Nhx1 partially complements the alkali-metal-cation sensitivity of yeast strains lacking three sodium transporters

A triple mutant strain of Saccharomyces cerevisiae lacking its own Na+-ATPases and Na+/H+ antiporters (enal-4delta nha1delta nhx1delta) was used for the expression of the Oryza sativa NHX1 gene encoding a putative vacuolar Na+/H+ exchanger. Upon expression in yeast cells, the OsNhx 1p is not a transport system specific only for sodium cations but it has a broad substrate specificity for at least four alkali metal cations (Na+, Li+, K+ and Rb+) and is able to substitute for the endogenous yeast ScNhx1 antiporter. Its activity contributes to sequestration of alkali metal cations in intracellular vesicles.     

Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils. Involvement of a pertussis toxin-sensitive G protein

Activation of neutrophils by most soluble stimuli is associated with a marked increase in intracellular free Ca2+ ([Ca2+]i). However, under physiological conditions (Na+-rich media), the potent activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes no change or a decrease in [Ca2+]i. We report here that the [Ca2+]i response to phorbol esters varies depending on the ionic composition of the medium. A marked increase in [Ca2+]i was detected in Na+-free solutions. Maximal effects were observed when N-methyl-D-glucammonium+ or choline+ were substituted for Na+, whereas an intermediate response was recorded in K+ medium. The increase in [Ca2+]i was substantially (approximately 65%) inhibited by removal of external Ca2+. A [Ca2+]i increase was also elicited by other beta-phorbol diesters and by diacylglycerol, but not by unesterified phorbol or by alpha-phorbol diesters, indicating involvement of protein kinase C. The increase in [Ca2+]i observed in Na+-free media is not due to inhibition of Na+/Ca2+ exchange, since no change in [Ca2+]i in response to TPA was observed in: 1) cells suspended in Li+, which is not countertransported for Ca2+; 2) cells preloaded with Na+ to eliminate the driving force for Na+/Ca2+ exchange; and 3) cells treated with 3',4'-dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange. Similarly, the [Ca2+]i increase in Na+-free media is not linked to the absence of Na+/H+ exchange and the associated cytoplasmic acidification since: 1) it was not observed in Na+ media in the presence of inhibitors of the Na+/H+ antiport and 2) it was not mimicked by inducing acidification with nigericin. Pretreatment with pertussis toxin largely inhibited the phorbol ester-induced change in [Ca2+]i, while activation of protein kinase C under these conditions was unaffected. It is concluded that in the absence of extracellular Na+ (or Li+), activation of protein kinase C leads to a net Ca2+ influx into the cytoplasm through a process mediated by a GTP-binding or G protein. Opening of a Na+-sensitive Ca2+ channel could partially explain these observations. Alternatively, the nature of the monovalent cation could conceivably affect the conformation of a G protein or of an associated receptor, inducing the appearance of a site susceptible to an activating phosphorylation by protein kinase C.     

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.     

Production of<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Nha2 Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter improves the salt tolerance of<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Yarrowia lipolytica plasma-membrane Na+/H+ antiporter, encoded by the YlNHA2 gene, is a very efficient exporter of surplus sodium from the cytosol. Its heterologous expression in Saccharomyces cerevisiae wild-type laboratory strains increased their sodium tolerance more efficiently than the expression of ZrSod2-22 antiporter from the osmotolerant yeast Zygosaccharomvces rouxii.     

Characterization of the ion transport activity of the budding yeast Na+/H+ antiporter, Nha1p, using isolated secretory vesicles

The Saccharomyces cerevisiae Nha1p, a plasma membrane protein belonging to the monovalent cation/proton antiporter family, plays a key role in the salt tolerance and pH regulation of cells. We examined the molecular function of Nha1p by using secretory vesicles isolated from a temperature sensitive secretory mutant, sec4-2, in vitro. The isolated secretory vesicles contained newly synthesized Nha1p en route to the plasma membrane and showed antiporter activity exchanging H+ for monovalent alkali metal cations. An amino acid substitution in Nha1p (D266N, Asp-266 to Asn) almost completely abolished the Na+/H+ but not K+/H+ antiport activity, confirming the validity of this assay system as well as the functional importance of Asp-266, especially for selectivity of substrate cations. Nha1p catalyzes transport of Na+ and K+ with similar affinity (12.7 mM and 12.4 mM), and with lower affinity for Rb+ and Li+. Nha1p activity is associated with a net charge movement across the membrane, transporting more protons per single sodium ion (i.e., electrogenic). This feature is similar to the bacterial Na+/H+ antiporters, whereas other known eukaryotic Na+/H+ antiporters are electroneutral. The ion selectivity and the stoichiometry suggest a unique physiological role of Nha1p which is distinct from that of other known Na+/H+ antiporters.     

Cation coupling to melibiose transport in Salmonella typhimurium.

Melibiose transport in Salmonella typhimurium was investigated. Radioactive melibiose was prepared and the melibiose transport system was characterized. Na+ and Li+ stimulated transport of melibiose by lowering the Km value without affecting the Vmax value; Km values were 0.50 mM in the absence of Na+ or Li+ and 0.12 mM in the presence of 10 mM NaCl or 10 mM LiCl. The Vmax value was 140 nmol/min per mg of protein. Melibiose was a much more effective substrate than methyl-beta-thiogalactoside. An Na+-melibiose cotransport mechanism was suggested by three types of experiments. First, the influx of Na+ induced by melibiose influx was observed with melibiose-induced cells. Second, the efflux of H+ induced by melibiose influx was observed only in the presence of Na+ or Li+, demonstrating the absence of H+-melibiose cotransport. Third, either an artificially imposed Na+ gradient or membrane potential could drive melibiose uptake in cells. Formation of an Na+ gradient in S. typhimurium was shown to be coupled to H+ by three methods. First, uncoupler-sensitive extrusion of Na+ was energized by respiration or glycolysis. Second, efflux of H+ induced by Na+ influx was detected. Third, a change in the pH gradient was elicited by imposing an Na+ gradient in energized membrane vesicles. Thus, it is concluded that the mechanism for Na+ extrusion is an Na+/H+ antiport. The Na+/H+ antiporter is a transformer which converts an electrochemical H+ gradient to an Na+ gradient, which then drives melibiose transport. Li+ was inhibitory for the growth of cells when melibiose was the sole carbon source, even though Li+ stimulated melibiose transport. This suggests that high intracellular Li+ may be harmful.