INTRODUCTION

Vacuolar ATPases comprise a family of structurally and functionally related enzymes that translocate protons across organelles and plasma membranes of eukaryotic cells (1, 2). Proton movements linked to ATP hydrolysis acidify membrane-bound spaces and establish electrochemical proton potentials that serve as the driving force for diverse proton-coupled secondary transporters. Vacuolar ATPase consists of a water-soluble, catalytic V₁ moiety and a membrane-integrated V₀ portion that conducts protons (3-5); the vectorial and chemical reactions are coupled when the V₀V₁ complex is incorporated into membranes.

The V₁ portions and V₀V₁ complexes, some of which are reconstitutively active in proton pumping, have been purified from a variety of eukaryotic species, and the amino acid sequences of these components have been determined (4-6). The V₀V₁ complex has three major subunits: the A and B subunits of the V₁ portion and the 17-kDa proton-conducting proteolipid of the V₀ portion. The amino acid sequences of these subunits are highly conserved among species and are known to resemble the sequences of the , , and c subunits of the F₀F₁-ATPase (another family of proton-translocating ATPases). It has been proposed that these two ATPase families are phylogenetically related and that the 17-kDa proteolipid of the vacuolar ATPase arose by tandem duplication of the gene for the 8-kDa proteolipid of the F₀F₁-ATPase (5-7). The amino acid sequences of other subunits of vacuolar ATPases are also well conserved among species, but the resemblance of these subunits to those of F₀F₁-ATPases is not conspicuous. Characterization of the subunits of vacuolar ATPase is most advanced in Saccharomyces cerevisiae, based on molecular genetics (4, 8). Recently two new proteolipids, Vma11p and Vma16p, were found to be subunits of the vacuolar ATPase (9). Thus, the vacuolar ATPase complex of yeast appears to contain at least 13 polypeptides, and purification of the holo enzyme has been attempted (8, 10).

Vacuolar-type ATPases also occur in bacteria (5, 6, 11); archaebacterial proton-translocating ATPases are thought to mediate ATP synthesis (12). An important variant among vacuolar ATPases is the enzyme from Enterococcus hirae, which transports Na⁺ rather than H⁺ under physiological conditions (13, 14). Our group has purified the catalytic V₁ moiety of this ATPase (15) and cloned the genes for the complex; the genes form an operon (designated ntp) consisting of 11 genes, ntpFIKECGABDHJ (16-18). The molecular properties of purified V₁ and the sequence analysis of these ntp gene products indicate that Enterococcus Na⁺-ATPase belongs to the vacuolar ATPase family. Three major subunits of Na⁺-ATPase, NtpA, NtpB, and NtpK (16-kDa proteolipid), resemble those of the eukaryotic ones; the other genes, ntpD, ntpE, ntpF, and ntpG, encode the polypeptides that resemble the Vma8p, Vma4p, Vma10p, and Vma7p subunits of yeast vacuolar ATPase, respectively, although the sequence similarities were only moderate (23-29% identity; 40-50% similarity). The similarities of the sequences of NtpC and NtpI to those of Vma6p and Vph1p of yeast vacuolar ATPase were less prominent (15-16% identity; 36-37% similarity). However, some amino acid clusters conserved among the corresponding subunits in eukaryotic vacuolar ATPases are conserved in these sequences; in case of NtpI, the sequence around the first membrane-spanning domain of Vph1p is conserved (18). The ntpJ gene product is a component of a K⁺ transport system linked to the Na⁺-ATPase, but it is not essential for the assembly or the operation of the ATPase (19). It appears, then, that this vacuolar-type Na⁺-ATPase consists of fewer than 10 polypeptides.

We report here the purification and reconstitution of the E. hirae Na⁺-ATPase complex as a stage in its biochemical characterization. The ATPase activity of the purified enzyme, which appears to consist of nine Ntp proteins, was inhibited by vacuolar ATPase inhibitors; ATP-driven ²²Na⁺ uptake by reconstituted proteoliposomes was blocked by nitrate and monensin but accelerated by valinomycin. These results suggest that the vacuolar Na⁺-ATPase of E. hirae is an electrogenic sodium pump.

MATERIALS AND METHODS

An Enterococcus-Escherichia coli shuttle vector pCem3 (4.6-kb¹) was constructed by ligating the 1.8-kb BamHI-HindIII fragment (the erythromycin resistance gene) of pJEM2 (19) with the 2.8-kb BglII-HindIII fragment of pC3 (20) carrying the replication origin for these bacteria; pCem3 has single XbaI site. The 18-kb pCemtp18 was constructed by ligation of the 13.4-kb XbaI-XbaI fragment of pKAZ171 (18), which extends from the promoter region of E. hirae Na⁺-ATPase (ntp) operon to the end of the ntpJ gene (see Fig. 2A) to the 4.6-kb XbaI cut of pCem3. pCemtp18 was introduced into E. hirae strain 25D, a mutant defective in production of F₀F₁, H⁺-ATPase (14). The transformed cells were grown on a complex medium (21) containing 0.5 M NaCl supplemented with 10 µg/ml erythromycin. Phospholipids were extracted from a large batch of Enterococcus faecalis cells, strain AD1001, grown on the same complex medium.

Membrane vesicles were prepared by disintegration of spheroplasts with a French press as described previously (21), suspended in buffer A (100 mM Tris-HCl, 10 mM MgSO₄, 1 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, pH 7.5) containing 10% glycerol, and stored at 80 °C. The Na⁺-ATPase was solubilized by incubation of the membrane vesicles (1.5 mg/ml) with 1.5% n-dodecyl -D-maltoside (DM) (Calbiochem) for 10 min at room temperature and recovered in the supernatant after centrifugation at 230,000 × g (30 min at 4 °C). The supernatant (0.6 ml) was applied to 10 ml of a linear glycerol gradient (10-30%) in buffer A containing 0.1% DM and 0.1 mg/ml dioleoylphosphatidylglycerol (Sigma) and was centrifuged at 175,000 × g for 12 h at 4 °C. After dividing into 18 fractions (0.6 ml each), the ATPase activity of the fractions was determined. One unit of the ATPase activity was defined as corresponding to 1 µmol of ATP hydrolyzed/min.

The phospholipids of E. faecalis AD1001 were extracted and purified from the cell mass by silica gel chromatography, as described elsewhere (22). Proteoliposomes were produced by a freeze-thaw and dilution method (22) as follows. 25 µl of the purified enzyme (0.05 mg/ml) was mixed with 100 µl of the liposome suspension (50 mg of phospholipid/ml) in buffer B (50 mM Tris-HCl, 100 mM KCl, 10 mM MgSO₄, 1 mM phenylmethylsulfonyl fluoride, and 2 mM dithiothreitol, pH 7.5) and supplemented with 11.4 µl of 15% CHAPS. After incubation for 10 min at 4 °C, the mixture was frozen in dry ice/acetone, thawed, and sonicated for 5 s. The mixture was finally diluted 200-fold into buffer B, and the proteoliposomes were recovered by centrifugation at 150,000 × g for 90 min. The liposomes were suspended in 0.1 ml of buffer B and stored at 4 °C.

The proteoliposomes were suspended in 2 ml of buffer B (0.6 µg of protein/ml); 0.1 mM ²²NaCl (6,500 cpm/nmol) was added to the mixture and allowed to equilibrate for 30 min. The uptake reaction was started by the addition of 5 mM ATP. Inhibitors or ionophores were added at 10 min before the addition of ATP. At intervals, 90 µl of the reaction mixture was filtered on a nitrocellulose filter (0.2-µm pore size, Toyo Roshi Co. Ltd., Tokyo) with suction and washed twice quickly with 4 ml of buffer B. The radioactivity trapped on the filter was measured with a liquid scintillation counter.

Rabbit antisera against various Ntp proteins were prepared by injection of a synthetic peptide for each protein conjugated to keyhole limpet hemocyanin. All antisera were purchased by Nippon Bio-Test Lab. Inc. (Tokyo) and Takara Shuzo Co. (Kyoto). The amino acid sequences of the oligopeptides synthesized were the following: CKTVEKYVNHKKK (near the COOH terminus of NtpI), GLIIDDAGIQYNFLF (the COOH terminus of NtpE), LEKEEEEIAKKKNL (the COOH terminus of NtpF), MMDYLITONGGMV (the NH₂ terminus of NtpK), and RAEANYKYPEESIM (near the COOH terminus of NtpJ).

The purified enzyme was subjected to SDS-PAGE on 12.5% gel and electroblotted to a polyvinylidene difluoride membrane (Bio-Rad). After staining with Coomassie Brilliant Blue, the bands corresponding to the 38-, 29-, and 8-kDa subunits were cut out, and the NH₂-terminal sequences of the polypeptides were analyzed with the HP G1005A protein sequencing system (Hewlet Packard) by Takara Shuzo Co.

SDS-PAGE was carried out using the system of Laemmli (23) and stained with Coomassie Brilliant Blue R-250 or silver. Western blotting was performed as described elsewhere (24); spots were visualized by using goat anti-rabbit IgG conjugated to alkaline phosphatase. The Na⁺-ATPase activity of the purified enzyme was determined in the presence of 25 mM NaCl as described previously (21); the reaction was started by the addition of 2 mM ATP, sometimes after a 10-min preincubation with various compounds. Protein was determined according to the method of Lowry et al. (25), with bovine serum albumin as standard. ²²NaCl was obtained from NEN Life Science Products (1.36 TBq/mmol).

RESULTS

The efficacy of various detergents for the solubilization of Na⁺-ATPase was examined first. Among the detergents tested (DM, Tween 80, octyl D-glucoside, Brij-58, Brij-35, CHAPS, cholate, C₁₂E₈, C₁₂E₉, C₁₂E₁₀, and Triton X-100), DM was the best for this enzyme. When the membrane vesicles were incubated with 1.5% DM, the activity of Na⁺-ATPase in the suspension doubled. This increase probably resulted from the disintegration of membrane structure because about half of the E. hirae vesicles prepared with the French press are everted (26). Under the conditions described in this paper, more than 80% of the membrane proteins were solubilized, and nearly 100% of total Na⁺-ATPase activity was recovered, suggesting that nearly all of the Na⁺-ATPase was solubilized in its native form (Fig. 1, inset). Further purification was accomplished by centrifugation through a linear glycerol gradient from 10 to 30% (Fig. 1). We observed a single peak of Na⁺-ATPase activity which was relatively sharp and also a peak of protein which coincided with that of ATPase activity; most of the applied protein was recovered in the upper fractions. Importantly, the specific activities of Na⁺-ATPase in fractions 4, 5, 6, and 7 were constant at 21 units/mg protein, which indicates a 10-fold purification from the DM extracts. About 80% of the Na⁺-ATPase solubilized with DM was recovered in these four fractions. The activity of solubilized enzyme was stable at 4 °C for at least 1 week and for 3 months at 80 °C.

Eight polypeptides with apparent molecular masses of 69, 52, 38, 27, 24, 15, 14, and 8 kDa were observed when the purified fractions (fractions 5, 6, and 7) were analyzed by SDS-PAGE (12.5% gel; Fig. 2B, lanes 3, 4, and 5). The 69-kDa protein band split into two bands of 69 and 65 kDa on 10% gel (Fig. 2B, lane 6), but no additional bands were observed by electrophoresis with different concentrations of acrylamide. The purified enzyme most probably consists of these nine polypeptides.

Determination of the NH₂-terminal sequences of the 38-kDa (MEYHELN), 27-kDa (MRLNVNP), and 8-kDa (TYKIGVV) bands indicated that these polypeptides correspond to the ntpC, ntpD, and ntpG gene products, respectively, of the Na⁺-ATPase operon (Fig. 2A). Fig. 2C shows Western blots of the fractions obtained from glycerol gradient centrifugation (Fig. 1), with various antisera. We have purified the V₁ catalytic moiety of this enzyme previously; it has a molecular mass of about 400 kDa and consists of three subunits, A, B, and D, with a stoichiometry of 3:3:1 (14). The 69- and 52-kDa polypeptides of the enzyme purified here were assigned to the A and B subunits, by Western blotting with antiserum against purified V₁ moiety (Fig. 2C). Using the antisera against the synthetic oligopeptides for the deduced amino acid sequences of the ntpE, ntpF, and ntpK gene products, the 24-, 15-, and 14-kDa polypeptides of the purified enzyme were assigned to the NtpE, NtpF, and NtpK proteins, respectively. These polypeptides reacting with anti-E, -F, and -K sera comigrated with NtpA and NtpB subunits in glycerol gradient centrifugation (Fig. 2C).

We also found that the 65-kDa band (Fig. 2B, lane 6), migrating slightly faster than the 69-kDa (A) subunit, reacted with anti-NtpI serum (Fig. 2C). This protein also comigrated with other Na⁺-ATPase subunits during centrifugation. Because the COOH-terminal half of the sequence of the ntpI gene product (M_r 75,619) is hydrophobic (18), the mobility of NtpI protein in SDS-PAGE may be affected. Thus, the purified enzyme probably consists of nine ntp gene products of the operon (Fig. 2A) but does not contain the ntpH and ntpJ gene products. The subunit ratio of purified enzyme was tentatively estimated by densitometric analysis of the amounts of Coomassie dye bound to these polypeptides. The apparent molar ratios of A, I, B, C, D, E, F, K, and G subunits were 3:1-2:3:1:1:3:1-2:3-4:1, although the exact estimation necessitates more detailed investigation.

Fig. 3A shows the activation of ATPase activity of the purified enzyme by Na⁺ or Li⁺ ions. The ATP hydrolysis rate of purified enzyme increased with the Na⁺ concentration, until saturation was reached at about 100 mM NaCl (Fig. 3A, inset). This is in accord with the proposal that the activity of the intact V₀V₁ complex of E. hirae Na⁺-ATPase requires stimulation by Na⁺ (27). Extrapolation of the activation curve to zero Na⁺ concentration suggested that as much as 20% of the maximal ATPase activity may be Na⁺-independent. However, because all assay media were contaminated with 5-10 µM Na⁺, we think that the purified enzyme is coupled tightly to Na⁺. ATPase activity was also stimulated by Li⁺ (Fig. 3A) but not by Cs⁺ or K⁺. The K_m value for ATP of the purified enzyme was about 0.5 mM (Fig. 3B), nearly identical to the value (K_m = 0.6 mM) measured for the Na⁺-ATPase of membrane vesicles (21).

The sensitivity of Na⁺-ATPase of the membrane vesicles to various reagents has been investigated (14, 15, 21). The Na⁺-ATPase activity of the membranes was inhibited by vacuolar ATPase inhibitors such as nitrate (K_i = 10 mM) and N-ethylmaleimide (K_i = 0.15 mM), but not by bafilomycin A₁, which strongly inhibits the eukaryotic vacuolar ATPase at a concentration below 1 µM (28). The sensitivity of the purified enzyme to these reagents was the same (Table I). The Na⁺-ATPase activity was inhibited by nitrate (K_i = 37 mM) and N-ethylmaleimide (K_i = 0.2 mM), but not by concanamycin A, another macrolide antibiotic that powerfully inhibits the vacuolar ATPase (28). The peptide antibiotic destruxin B, known to inhibit the vacuolar ATPase (29), is also effective against the purified Na⁺-ATPase at the same concentrations (K_i = 30 µM). N,N-Dicyclohexylcarbodiimide inhibited the purified enzyme, probably attacking the glutamic acid residue (Glu-139) of the NtpK proteolipid as proposed for other eukaryotic vacuolar ATPases (3-5). Azide and vanadate were without any significant effect on the Na⁺-ATPase of this bacterium. Thus, the effects of various compounds on purified Na⁺-ATPase are in good accord with the features of a vacuolar ATPase.

Table I. Effect of various ATPase inhibitors on the ATP hydrolytic activity of purified enzyme The Na+-ATPase activity of purified enzyme was determined as described under "Materials and Methods" after a 10-min incubation of the enzyme at 25 °C with the inhibitor at the concentrations indicated. 100% activity corresponds to 0.5 unit. Reagent Concentration Relative Na+-ATPase activity mM % Control 100 KNO3 30 56 N-Ethylmaleimide 0.2 52 Concanamycin A 0.005 98 Destruxin B 0.03 49 N,N-Dicyclohexylcarbodiimide 0.2 10 Vanadate 1 90 Azide 10 100

The purified ATPase was incorporated into liposomes by a freeze-thaw/dilution method, and Fig. 4 shows the time course of Na⁺ transport into such proteoliposomes. Virtually no Na⁺ was transported into the vesicles in the absence of ATP (Fig. 4A). Upon its addition, Na⁺ rapidly accumulated inside the liposomes, reaching a steady state in about 10 min. Accumulation of Na⁺ ions was prevented by incubation of the proteoliposomes with 50 mM nitrate or 50 µM destruxin B (Fig. 4A). Transport of Na⁺ was influenced markedly by the presence of certain ionophores (Fig. 4B). Na⁺ uptake into the proteoliposomes was abolished completely by the Na⁺-carrying ionophore monensin. On the other hand, valinomycin, in the presence of K⁺, stimulated the Na⁺ transport rate more than 2-fold, and the protonophore carbonyl cyanide m-chlorophenylhydrazone also promoted it significantly. These results are indicative of an electrogenic Na⁺ transport by purified Na⁺-ATPase: a membrane potential that limits the transport rate is dissipated by increasing the conductance of K⁺ or H⁺ across the membranes.

The initial rate of Na⁺ uptake (Fig. 4A) was about 0.1 µmol/min/mg of purified enzyme, which is only 1.4% of the enzyme's ATP hydrolytic activity (7 µmol/min/mg protein) measured under the same condition. However, we shall set aside this inconsistency for the present because we know nothing about the amount of Na⁺-ATPase incorporated into the liposomes, the orientation of the enzyme in the membrane, and, most possibly, the leakiness of proteoliposomes to Na⁺ ions. We would note, however, that this is the first clearly demonstrated example of vacuolar ATPase that acts as a primary Na⁺ pump.

E. hirae has an active K⁺ transport system designated KtrII; investigations with intact cells (21) led to the proposal that this is a primary system tightly linked to the action of the Na⁺-ATPase. The ntpJ gene, the tail end of the ntp operon (Fig. 2A), encodes a putative 49-kDa hydrophobic protein that resembles various K⁺ transporters of yeast and bacteria (18). Although this kind of polypeptide has not so far been found as a subunit of vacuolar proton ATPases in eukaryotes, the hypothesis that the NtpJ protein may be the part of the Na⁺-ATPase complex was attractive. We have suggested recently that the ntpJ gene product is a component of the KtrII K⁺ transport system, functionally independent of the Na⁺-ATPase activity (19). Western blotting with antiserum against the oligopeptide for the NtpJ sequence was done for the fractions from glycerol gradient centrifugation (Fig. 5). We found that the NtpJ protein, observed mainly in fractions 13 and 15 (Fig. 5B), was separable from the Na⁺-ATPase complex; only the A, B, C, and K subunits were observed here by silver staining in the fractions containing the peak of Na⁺-ATPase activity (Fig. 5A, fractions 5 and 7). Moreover, neither the activity nor the assembly of Na⁺-ATPase was impaired in an ntpJ-disrupted strain (19). These results suggest that the NtpJ protein is not part of the E. hirae Na⁺-ATPase complex.

DISCUSSION

Sodium ions regulate the induction of Na⁺-ATPase in E. hirae (30). Transcription of the Na⁺-ATPase operon is stimulated under conditions where the intracellular Na⁺ concentration is high (24). In Na⁺-limited medium, on the other hand, the sodium pumping activity via this ATPase is minimal. In the background to this work, we transformed pKAZ191 (18), harboring the whole ntp operon without its 5-promoter region, into an Na⁺-ATPase-defective mutant Nak1 (31); the operon is transcribed by the promoter activity of the vector. Cells of Nak1/pKAZ191 exhibited sodium pumping activity even on low Na⁺ medium. We also observed that the Na⁺-ATPase activity of strain Nak1 transformed with pKAZ192 (18), harboring the whole ntp operon including its promoter, was stimulated by the Na⁺ concentration in the medium, suggesting that active Na⁺-ATPase is expressed by the Na⁺-responsive ntp operon containing these ntp genes (Fig. 2A) (24).

Several aspects of the purification of Na⁺-ATPase should be emphasized. First, the enzyme was induced strongly by Na⁺; complex medium supplemented with 500 mM NaCl (final, 650 mM Na⁺) was used. The specific activity of the Na⁺-ATPase of the membrane vesicles was 0.15 unit/mg of protein. The amount of Na⁺-ATPase was enhanced further by introducing the operon on a multicopy shuttle vector pCem3, although the copy number was not determined exactly; the ATP hydrolytic activity of the membranes was 0.75 unit/mg of protein. Because the organism also contains the proton-translocating F₀F₁-ATPase (33), the Na⁺-ATPase was purified from a mutant (25D) defective in the production of the H⁺-ATPase (14). This avoided cross-contamination of the H⁺-ATPase during the purification of Na⁺-ATPase. It also diminished the proton-motive force of the cells, thus increasing their internal Na⁺ concentration, which is normally kept low by a Na⁺/H⁺ antiporter (30). Judging by the amount of the 65-kDa A subunit in the membrane proteins (Fig. 2B, lane 1), the Na⁺-ATPase made up more than 10% of total membrane proteins.

Second, phospholipids were added to all fractions during solubilization and purification of this enzyme. We have observed much lower activity of purified Na⁺-ATPase in the absence of the phospholipid. However, the enzyme seems to be stable even in this situation because the activity was recovered upon addition of phospholipid. Among the phospholipids examined, the total phospholipid of E. faecalis showed the best stimulation of the Na⁺-ATPase, although acidic phospholipids such as phosphatidylglycerol or phosphatidylserine were also stimulatory. Finally, the non-ionic detergent, dodecyl maltoside, was the best for solubilization and did not cause disintegration of the Na⁺-ATPase complex. The specific Na⁺-ATPase activity of the final sample was about 21 units/mg of protein, approximately 23 times higher than that of the cell membranes, suggesting that isolated enzyme is highly purified.

The kinetics of ATPase activation by Na⁺ or Li⁺ are biphasic (Fig. 3A). The K_m values for Na⁺ were estimated to be about 20 µM and 4 mM, and the K_m values for Li⁺ were 60 µM and about 3.5 mM. The high affinity for Na⁺ or Li⁺ ions is physiologically favorable when considering the intracellular ion concentration (30). On the other hand, the low affinity (K_m = 5-7 mM) of the ATP hydrolytic activity for Na⁺ has been estimated with membrane-bound ATPase (21), suggesting that the low affinity for Na⁺ takes part in the reaction of this enzyme. A definite role of these kinetic parameters in the ion-coupling mechanism of this enzyme should be elucidated although it is yet unknown.

SDS-PAGE and Western blotting revealed nine Ntp proteins in the E. hirae Na⁺-ATPase: the A, B, C, D, E, F, G, I, and K subunits. The ntpJ gene product, which is a component of the KtrII K⁺ transport system functionally independent of the Na⁺-ATPase, is apparently not the part of the complex (Fig. 5). We do not know if the ntpH gene product is a component of the Na⁺-ATPase. Because there is no strong Shine-Dalgarno sequence upstream of this minigene (18), we tentatively consider that ntpH is not an open reading frame, although proof of this notion requires further investigation. A search for homologs of the Ntp proteins in data bases indicated that nine putative genes of the genome of Methanococcus jannaschii correspond to the ntpA, -B, -C, -D, -E, -F, -G, -I, and -K genes for the E. hirae Na⁺-ATPase. These genes are likely to form an operon with the same gene order as that of the E. hirae operon, except for the ntpD-like gene (34). Taking into account the data reported for other archaebacterial ATPases (35), it is likely that the nine subunits constitute the basic complex of bacterial vacuolar ATPases.

The V₁ moiety is easily dissociated from the V₀ moiety by chelating Mg²⁺ (27). EDTA washing of the proteoliposomes suggested that the A, B, C, D, E, and G subunits are likely to make up the V₁ subunit. Densitometric analysis of purified enzyme stained with Coomassie dye suggested that the A, I, B, C, D, E, F, K, and G subunits of E. hirae Na⁺-ATPase occurred in a molar ratio of 3:1-2:3:1:1:3:1-2:3-4:1. The ratios of the A, B, and D subunits were identical with those of the purified catalytic moiety of this Na⁺-ATPase (15). Peng et al. (32) found higher molar ratio of E subunit/vacuolar ATPase complex in clathrin-coated vesicles. In this context, it is noteworthy that the ratio of E subunit/complex was reproducibly 3 by Coomassie staining in our estimation. We are now engaged in determining the exact molar ratio of Ntp subunits, especially focusing on the possibility that the E subunit may be another major subunit of the vacuolar ATPases.

The E. hirae sodium pump is the first demonstrated example of a Na⁺-translocating ATPase having a vacuolar ATPase structure; the purified ATPase retained the capacity for electrogenic Na⁺ pumping by the reconstituted proteoliposomes (Fig. 4). Although this Na⁺-ATPase, as well as archaebacterial ATPases, is insensitive to macrolide antibiotics such as bafilomycin A₁ or concanamycin A, the molecular resemblance to the H⁺-translocating vacuolar ATPase of many other organisms suggests that the fundamental mechanism of ion transport is equivalent in these phylogenetically related enzymes. This sodium-coupled E. hirae system has yielded new insights into the catalytic mechanism of these enzymes, and it has many advantages for the investigation of the energy coupling mechanism.