Identification of Subunits a, b, andc 1 from Acetobacterium woodiiNa+-F1F0-ATPase

The Na+-F1F0-ATPase operon ofAcetobacterium woodii was recently shown to contain, among eleven atp genes, those genes that encode subunita and b, a gene encoding a 16-kDa proteolipid (subunit c 1), and two genes encoding 8-kDa proteolipids (subunits c 2 andc 3). Because subunits a,b, and c 1 were not found in previous enzyme preparations, we re-determined the subunit composition of the enzyme. The genes were overproduced, and specific antibodies were raised. Western blots revealed that subunits a,b, and c 1 are produced and localized in the cytoplasmic membrane. Membrane protein complexes were solubilized by dodecylmaltoside and separated by blue native-polyacrylamide gel electrophoresis, and the ATPase subunits were resolved by SDS-polyacrylamide gel electrophoresis. N-terminal sequence analyses revealed the presence of subunitsa, c 2, c 3,b, δ, α, γ, β, and ε. Biochemical and immunological analyses revealed that subunitsc 1, c 2, andc 3 are all part of the c-oligomer, the first of a F1F0-ATPase that contains 8- and 16-kDa proteolipids.

hair pin contains an ion-binding site (10,14,15). AtpE 1 (c 1 ) most likely arose by duplication of an ancestral gene and subsequent fusion of the gene copies. Subunit c 1 is predicted to have four transmembrane helices, but the ion binding motif is conserved only in hair pin one, but not two. Therefore, subunit c 1 of A. woodii is similar to the so-called 16-kDa proteolipids of V 1 V 0 -ATPases, which also arose by gene duplication accompanied by loss of the protonbinding residue in hair pin one. The loss of the proton-binding residue was believed to be the reason for the apparent inability of V 1 V 0 -ATPases to function as ATP synthases under in vivo conditions (16). Western blot analyses verified that atpE 1 was expressed and that the product was not posttranslationally split into two 8-kDa proteolipids (11). However, AtpE 1 was not found in the enzyme purified previously (6). The Na ϩ -F 1 F 0 -ATPase of A. woodii purified previously not only lacked subunit c 1 but also the gene products AtpB (subunit a) and AtpF (subunit b). Subunits a and b were also not present in the ATPase of Moorella thermoacetica, although the encoding genes were present. These findings led to the hypothesis that atpB and atpF are transcribed, but the messages are not translated (17,18). The finding of the genes atpE 1 , atpB, and atpF in the F 1 F 0 -ATPase operon of A. woodii raised the question whether subunits c 1 , a, and b are true subunits of the F 1 F 0 -ATP synthase of A. woodii. We demonstrate here that the genes encoding subunits a and b of A. woodii are expressed and that subunits a, b, and c 1 are assembled into the ATPase complex. This is the first demonstration of an F 1 F 0 -ATPase containing a heterooligomer of subunit c consisting of both 8-and 16-kDa proteolipids.

EXPERIMENTAL PROCEDURES
Materials-All chemicals used were reagent grade and purchased from Merck AG (Darmstadt, Germany). Antibodies were prepared by Bioscience (Göttingen, Germany).
Chloroform/Methanol Extraction of A. woodii Membranes-Membranes were prepared as described previously (6). Chloroform/methanol extraction of A. woodii membranes was performed as described (21) with 160 mg of membrane protein dissolved in 8 ml of 50 mM Tris, pH 8. The extracts were precipitated twice with four volumes of diethylether as described in Ref. 22. The 8-kDa proteolipid was electroeluted from an SDS-polyacrylamide gel and used for immunization of a rabbit.
Immunoblotting-SDS-PAGE 1 and Western blotting were performed as described previously (11,23). Transfer of proteins from blue native-PAGE to polyvinylidene difluoride membranes was essentially as described (24).
Blue Native-PAGE-Washed membranes were first pelleted by centrifugation at 140,000 ϫ g for 1 h and resuspended in 50 mM imidazole (pH 7.0), 50 mM NaCl, 2 mM aminocaproic acid, 1 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride. Membrane proteins were then solubilized with dodecylmaltoside (1 g/g of protein) for 20 min on ice. Thereafter, membranes were pelleted by centrifugation at 140,000 ϫ g for 30 min. The supernatant was subjected to blue native-PAGE as described (24), except that the cathode buffer contained 7.5 mM imidazole (pH 7.0), 50 mM tricine, and 0.02% Serva Blue and the anode buffer contained 50 mM imidazole (pH 7.0).

RESULTS
Expression of atpB, atpE 1 , atpF, and atpD in E. coli-To generate antibodies against subunits a, b, and c 1 , the genes atpB, atpF, and atpE 1 of A. woodii were fused to malE and expressed in E. coli, and the fusion proteins were used to immunize rabbits. Because attempts to express full-length a, b, and c 1 fusions in E. coli were unsuccessful, deletion derivatives were made. From atpB the 3Ј-terminal 230 base pairs were fused to malE. Expression was low in this case, but after purification the quantity of MalE-AtpB* was sufficient for immunization of rabbits. A malE-atpD fusion gave high expression yields. In case of atpE 1 a sequence of 66 base pairs, coding for the first hydrophilic loop of subunit c 1 , was fused to malE. This sequence was chosen to minimize cross-reactions of the antiserum with subunit c 2/3 , because only 10 of 22 amino acids in this sequence are identical in subunit c 1 and c 2/3 . This construct was expressed in appreciable amounts. 362 base pairs of atpF, coding for a part of the hydrophilic domain, were fused to malE, and the fusion gene was also expressed in sufficient amounts.
Immunological Detection of Subunits a and b in the Cytoplasmic Membrane of A. woodii-A. woodii was grown on 20 mM fructose to an A 600 of 0.8 (logarithmic growth phase) and harvested, and cytoplasmic and membrane fractions were prepared. After SDS-PAGE, the proteins were blotted on nitrocellulose membranes and probed with different polyclonal antisera.
The antiserum against subunit ␤ reacted, as expected, with a protein having an apparent molecular mass of 51 kDa ( Fig.  1), which is identical to the deduced molecular mass of subunit ␤. Subunit ␤ was found predominantly in the membrane fraction but also in the cytoplasm. The strongest reaction of the antiserum against subunit a was with a 29-kDa membrane protein ( Fig. 1). At higher protein concentrations (Ͼ25 g) 18and 15-kDa membrane proteins also reacted with the antisubunit a antiserum. The deduced molecular mass of subunit a is 24.5 kDa. Because subunit a from E. coli and Propionigenium modestum migrates in SDS-PAGE at molecular masses lower than expected from the deduced sequence (25,26), it is unlikely that the predominant 29-kDa signal corresponds to subunit a. Concomitantly, the N-terminal sequence analyses presented revealed that the 18-kDa protein is subunit a. The antiserum raised against subunit b reacted with a protein having an apparent molecular mass of 19 kDa, which is quite similar to the deduced molecular mass of 20.8 kDa. Subunit b was also found predominantly in the cytoplasmic membrane. These experiments demonstrate that subunits a and b are produced and located in the cytoplasmic membrane of A. woodii.
Specificity of the Antisera against c 1 and c 2/3 -For further studies it was important to clearly establish the specificity of the antisera generated against the different proteolipids. The antiserum against subunit c 1 reacted with subunit c 1 (apparent molecular mass of 16 kDa) in membranes of A. woodii, as observed before, but in addition a band at 43 kDa was obtained (Fig. 2). This band represents the c-oligomer, as determined by N-terminal sequencing (see below). Apparently, the anti-c 1 antiserum does not cross-react with subunits c 2/3 . The anti-c 2/3 antiserum reacted with subunits c 2/3 and the c-oligomer but not with subunit c 1 . Only at very high protein concentrations (Ͼ100 g) was there a weak cross-reaction of the anti-c 2/3 antiserum with c 1 (data not shown). These results show that subunits c 1 and c 2/3 are present in membranes of A. woodii in monomeric and oligomeric forms. In silver-stained SDS-polyacrylamide gels of chloroform/methanol extracts, two major proteins having apparent molecular masses of 16 and 7 kDa were observed. In Western blots, the anti-c 2/3 antiserum reacted with the 7-kDa polypeptide, which was identified as subunit c 2/3 by N-terminal sequencing, and to a much lesser extent with the 16-kDa polypeptide. The anti-c 1 antiserum reacted only with the 16-kDa polypeptide (Fig. 3). These studies verified that the anti-c 1 antiserum does not react with subunit c 2/3 . The reaction of the anti-c 2/3 antiserum with subunit c 1 is expected, because 60 and 72% of the amino acids of subunits c 2/3 are conserved in the first and second half of subunit c 1 , respectively (11).
Subunit Composition of the Native ATPase-Membrane proteins were solubilized with Triton X-100, dodecylmaltoside, and laurylmaltoside in different concentrations, ranging from 1 to 24 g of detergent/g of protein. The solubilized protein complexes were then separated by blue native-PAGE. Independent of the nature and concentration of the detergent used, a predominant protein band with an apparent molecular mass of 590 kDa was observed (Fig. 4). In addition, bands with much lower intensi- 1 The abbreviation used is: PAGE, polyacrylamide gel electrophoresis. ties were observed at 300 and 150 kDa. The apparent molecular mass of the 590-kDa complex corresponds well to the molecular mass of the F 1 F 0 -ATPase from A. woodii. That this complex indeed represents the ATPase was verified by Western blot analyses using the anti-␤ antiserum as probe (data not shown).
When the membrane protein complexes were separated in the first dimension by blue native-PAGE and in the second dimension by SDS-PAGE, the subunits of the ATPase complex were resolved (Fig. 4). Eight polypeptides with apparent molecular masses of 58, 55, 43, 37, 21, 19, 18, and 16.5 kDa were detected by silver staining. N-terminal sequencing of these proteins gave clear evidence that they are subunits ␣ and ␤, the c-oligomer, and subunits ␥, ␦, b, a, and ⑀, respectively. This experiment gives clear evidence that subunit a and b are present in the enzyme complex. However, c 1 and c 2/3 monomers could not be detected; only the c-oligomer could be detected. N-terminal sequencing of the polypeptides in the oligomer clearly revealed the presence of c 2/3 , but c 1 was not detected. To detect c 1 in the complex, an immunological approach was chosen.
Immunological Detection of c 1 in the Native Enzyme-To identify subunit c 1 in the native ATPase, the ATPase complex was resolved by blue native-PAGE and SDS-PAGE as described above, blotted on nitrocellulose membranes, and probed with anti-c 1 and anti-c 2/3 antiserum. Both antisera reacted with the c-oligomer but not with monomeric subunits c 1 and c 2/3 (Fig. 5). Nevertheless, this proves that subunit c 1 , besides subunit c 2/3 , is present in the c-oligomer. The c-oligomer of the A. woodii ATPase can be disrupted by autoclaving it at 120°C for 3 min (6). As can be seen from Fig. 5, this treatment leads to the disruption of the c-oligomer. Concomitantly, two polypeptides of 7 and 16 kDa appeared. The 7-kDa polypeptide was identified both immunologically and by N-terminal sequencing as subunit c 2/3 . Unfortunately, the concentration of the 16-kDa polypeptide was too low for N-terminal sequencing, but the Western blot analyses clearly identified it as subunit c 1 . Taken together, these experiments gave clear evidence that c 1 is assembled into the ATPase complex and is part of the coligomer. This demonstrates, for the first time, the presence of a duplicated proteolipid in an F 1 F 0 -ATPase. DISCUSSION We have now isolated the Na ϩ -F 1 F 0 -ATPase in its native state and found nine polypeptides. These were identified by N-terminal sequencing and immunological methods as subunits a, c 1 , c 2/3 , b, ␦, ␣, ␥, ␤, and ⑀. The N-terminal sequences now available allow us to identify unequivocally the start codons of the respective genes. With the exception of atpF, the experimentally determined start codons match the ones deduced from the DNA sequences. The start codon of atpF is actually 45 nucleotides downstream from the previously assumed start site (12). Translation of atpF starts with the unusual start codon TTG; the same is true for atpA (11). Nformylated N-terminal methionines were found in subunits a, b, and c 2/3 , whereas subunit ␣ has a deformylated methionine. The N-terminal methionine was removed from subunits ␤, ␥, ␦, and ⑀. Removal of the first methionine was also reported for subunits ␤, ␥, and ⑀ of the E. coli enzyme (27) and for subunits ␥ and ⑀ of the P. modestum enzyme (26).
ATPase preparations from A. woodii described previously lacked subunits a and b. The same was observed in M. thermoacetica and Moorella thermoautotrophica, although the encoding genes were present in the atp operons (17,18). atpB and atpF of M. thermoacetica were transcribed, but because antisera against synthetic polypeptides derived from the sequences of subunit a and b of M. thermoacetica did not cross-react with cell free extract (18), it was concluded that the messages are not translated. From the findings presented here it is clear that in A. woodii subunits a and b are produced. By using the gentle blue native-PAGE procedure, we were able to isolate the ATPase complex in its native state. Therefore, we have to conclude that subunits a and b were lost in the course of the purification procedure employed in a previous study. Even with the use of blue native-PAGE, subunit a was not detectable in every preparation.
Another striking and unique feature of the Na ϩ -F 1 F 0 -ATPase of A. woodii is its duplicated proteolipid, subunit c 1 . This is without precedence in bacteria. Duplicated proteolipids were, for a long time, seen as an exclusive feature of eucaryal V 1 V 0 -ATPases (28). In archaea, duplication and triplication of proteolipid-encoding genes with subsequent fusion of the genes was described very recently (16,29). With the experiments described here we add another argument, now derived from a bacterial species, that multiplied and fused proteolipid-encoding genes are not exclusively present in eucarya, but also in the other domains of life. oligomer is unknown, it appears from the Western blots and SDS-PAGEs that subunit c 1 is only a minor component. In this connection it should be mentioned that the migration behavior of the c-oligomer is dependent on the acrylamide concentration. In 10% SDS-polyacrylamide gels, the c-oligomer runs at 43 kDa, but in 16% gels, it runs at 61 kDa. Therefore, we cannot speculate about the number of monomers in the complex.
Subunit c 1 is not only duplicated, but Glu-162 in hair pin two is also substituted by a glutamine residue. The glutamate is part of the proposed sodium ion-binding site (Pro-Gln-Glu-Thr) (10,15,30) in subunit c. Although the free electron pair of the amino group of Gln-162 could in principal bind the sodium ion (as does Gln-46 in helix one and Gln-129 in helix three), the substitution might have consequences for the rotation of the motor of the ATPase. Current views on the function of the motor assume an electrostatic attraction of Na ϩ (H ϩ ) by Glu (Asp) (31,32). Due to the neutralization of the charge of Glu (Glu-62 in c 2/3 and Glu-79 in c 1 ) after coordinating a sodium ion, the c-ring may cross the electric barrier and rotate into the hydrophobic zone, driven by the electrostatic interaction of a highly conserved Arg (Arg-158 in A. woodii) in subunit a with another free Glu on the next monomer of the c-ring. This would lead to a rotation of the c-ring relative to subunits a and b. The lesser the number of carboxylates per ring, the worse is the coupling efficiency. In the worst case, the V 1 V 0 -ATPases, ATP synthesis (under physiological conditions) is abolished, but proton pumping capacity is increased. For the F 1 F 0 -ATPase of E. coli it was demonstrated that the ATPase can tolerate the exchange of one Asp-61 with an Asn residue without losing its capability to translocate H ϩ (33). The ATPase from Methanococcus jannaschii contains a triplicated proteolipid with only two proton-translocating groups, but this enzyme still functions as an ATP synthase. In view of this discussion, the determination of the exact stoichiometry of the subunits of the c-oligomer of A. woodii is essential; this remains a challenging task for the future.
What could be the function of the two different proteolipids in the ATPase of A. woodii? Although it is hard to speculate at present, an attractive idea is the regulation of the function of the enzyme by the relative stoichiometry of c 2/3 and c 1 . As pointed out above, the higher the ratio of c 2/3 over c 1 , the better the enzyme functions as an ATP synthase, whereas a high c 1 :c 2/3 ratio favors pump activity. During growth on fermentable substrates such as sugars, the enzyme may function as an ATP-driven ion pump used to regulate intracellular pH and/or Na ϩ concentration, whereas during growth on H 2 ϩ CO 2 the enzyme has to drive ATP synthesis by means of the electrochemical Na ϩ potential across the membrane. Regulation of the coupling efficiency in ATPases by varying the number of c-subunits per oligomer was originally suggested by Brusilow and co-workers (34). Verification of this interesting idea remains a challenging task for future experiments.