Characterization and Subunit Structure of the ATP Synthase of the Halophilic Archaeon Haloferax volcanii and Organization of the ATP Synthase Genes*

(Received for publication, August 21, 1996, and in revised form, November 19, 1996)

Kerstin Steinert Dagger , Volker Wagner , Peter G. Kroth-Pancic and Susanne Bickel-Sandkötter §

From the Institut für Biochemie der Pflanzen, Heinrich-Heine-Universität Düsseldorf, Universitätsstraße 1, D-40225 Düsseldorf, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The archaeal ATPase of the halophile Haloferax volcanii synthesizes ATP at the expense of a proton gradient, as shown by sensitivity to the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone, to the ionophore nigericin, and to the proton channel-modifying reagent N,N'-dicyclohexylcarbodiimide. The conditions for an optimally active ATP synthase have been determined. We were able to purify the enzyme complex and to identify the larger subunits with antisera raised against synthetic peptides. To identify additional subunits of this enzyme complex, we cloned and sequenced a gene cluster encoding five hydrophilic subunits of the A1 part of the proton-translocating archaeal ATP synthase. Initiation, termination, and ribosome-binding sequences as well as the result of a single transcript suggest that the ATPase genes are organized in an operon. The calculated molecular masses of the deduced gene products are 22.0 kDa (subunit D), 38.7 kDa (subunit C), 11.6 kDa (subunit E), 52.0 kDa (subunit B), and 64.5 kDa (subunit A). The described operon contains genes in the order D, C, E, B, and A; it contains no gene for the hydrophobic, so-called proteolipid (subunit c, the proton-conducting subunit of the A0 part). This subunit has been isolated and purified; its molecular mass as deduced by SDS-polyacrylamide gel electrophoresis is 9.7 kDa.


INTRODUCTION

The archaeon (archaebacterium) Haloferax volcanii belongs to the more moderate halophilic archaea, growing best in a medium containing 2 mol/liter NaCl and 0.25 mol/liter MgCl2 at pH 7 (1). The proton-conducting ATPase of this archaeon is a halophilic enzyme that shows maximum activity at 1.75 mol/liter NaCl. It was characterized recently with respect to its hydrolytic function (2). The membrane-bound ATPases from archaea share properties with both bacterial (formerly eubacterial) and eucaryal (formerly eukaryotic) ATPases. Like F0F1-ATPases, they are able to synthesize ATP from ADP and phosphate coupled to a proton flow. Genetically derived amino acid sequences of the large subunits of archaeal ATPases (2-6), immunological cross-reactivity (7, 8), and inhibition of the enzymes by specific inhibitors (1, 9-11) deliver the main arguments for the conclusion that the archaeal ATPases resemble eucaryal vacuolar ATPases (V-ATPases)1 more closely than bacterial F-ATPases.

The primary structures of subunits A and B of H. volcanii are closely related to those of Halobacterium salinarium (halobium) (6) and indeed more related to the class of the eucaryal V-ATPases. On the other hand, the function of eucaryal V-ATPases is to generate a Delta mu-tilde H+ at the expense of ATP hydrolysis; they are not able to synthesize ATP in a reversed reaction. More detailed investigations on the intermediate position of different archaeal ATPases are required.

Up to now, almost nothing was known about the small subunits of halophilic ATPases. F-ATPases have one copy each of three different small subunits, gamma , delta , and epsilon , which are involved in proton conduction and/or regulation (for review, see Refs. 12 and 13). V-ATPases, as far as we know, have three to four different small subunits, C, D, E, and F. We know almost nothing about the function of these subunits (for review, see Ref. 14). This paper reports on the proton-dependent ATP synthesis of the H. volcanii ATPase, the purification and immunodetection of subunits, and the nucleotide sequence of the ATPase operon encoding the A1 part of the enzyme that contains three small subunits as in F-ATPases.


EXPERIMENTAL PROCEDURES

Growth Conditions

H. volcanii WR 340 was grown in a medium containing (w/v each) 16.2% NaCl, 3% MgSO4, 1% KCl, 0.08% CaCl2, 40 mmol/liter Tris-Cl, pH 7.2, 0.43% peptone, and 0.26% yeast extract according to Ref. 15.

Preparation of Vesicles

H. volcanii cells were harvested by centrifugation at 10 °C in a Beckman centrifuge (Model J2-21) at 5000 × g for 30 min. The pellets were pooled, resuspended, and washed with a medium containing 1.75 mol/liter NaCl and 50 mmol/liter Tris-HCl, pH 7. After centrifugation, the cell concentration was adjusted to a value corresponding to 60 A520, followed by sonication on ice for 3 × 30 s (Branson sonifier with microtip). By this method, up to 80% inside-out vesicles were made as shown by the membrane-bound menadione-dependent NADH dehydrogenase reaction. After centrifugation (4 °C, 8000 × g, 30 min), the supernatants were collected and transferred to a Beckman ultracentrifuge (Model L8-70M) to remove the remaining unruptured cells, followed by ultracentrifugation for 60 min at 140,000 × g and 4 °C. The resulting pellet, containing the vesicles, was collected in a small volume of 1.75 mol/liter NaCl and 50 mmol/liter Tris-HCl, pH 7, and again centrifuged at 140,000 × g for 5 min. Aliquots of this suspension were used for measurements of ATP synthesis.

ATP Synthesis

Acid/base-dependent ATP synthesis (by alkalization of the outer medium) was measured by the addition of vesicles prepared at pH 7 (see above) in a medium comprising 20 mmol/liter MgCl2, 5 mmol/liter Pi, 5 mmol/liter ADP, 1.75 mmol/liter NaCl, and 50 mmol/liter Tris-Cl, pH 9. The final volume was 700 µl, and the temperature was adjusted to 40 °C, if not indicated otherwise. The reaction was started by the addition of 100 µl of prepared vesicles (see above) and stopped after the indicated times by adding 100-µl portions to 100 µl of perchloric acid (final concentration of 0.5 mol/liter). Immediate neutralization of the samples was followed by centrifugation and determination of the ATP concentration by the luciferin/luciferase reaction (Pharmacia Luminometer). ATP was determined in a medium containing 0.1 mol/liter Tris/acetic acid, pH 7.75, 2 mmol/liter EDTA, 5 mmol/liter MgSO4, 10 mmol/liter K2SO4, and 50 µl of luciferase reagent (ATP bioluminescence kit, CLS II, Boehringer, Mannheim, Germany). The reaction was started by the addition of 50 µl of neutralized supernatant (see above) to 500 µl of reaction medium.

Isolation of Proteolipid

The isolation procedure was performed according to Denda et al. (16). Membranes of H. volcanii (~400 mg of protein) were suspended in 60 ml of 50 mM Tricine, pH 7.5, followed by ultracentrifugation (140,000 × g, 55 min), and were washed twice. The precipitated membranes were resuspended in 60 ml of diethyl ether and precipitated by centrifugation (7000 × g, 10 min). We repeated the washing procedure twice. After resuspension of the pellet in 60 ml of chloroform/methanol (2:1, plus 5 mmol/liter dithiothreitol), the suspension was stirred overnight at 4 °C. The addition of 20 ml of water and centrifugation at 7000 × g for 15 min resulted in two phases. The lower organic phase was concentrated to a final volume of 2 ml and mixed with 8 ml of ice-cold (-20 °C) diethyl ether. We collected the resulting pellet by centrifugation and washed it with ice-cold acetone. This final pellet was dried under vacuum, followed by resuspension with ~100 µl of chloroform/methanol (2:1), and stored at -20 °C. Protein determinations were performed according to Bensadoun and Weinstein (17).

Cloning and Sequencing

Genomic DNA from H. volcanii WR 340 was prepared as described previously (2). RNA preparation was carried out according to Chomczynski and Sacchi (18).

Northern and Southern hybridization experiments were performed according to standard protocols (19). alpha -32P-Labeled gene probes were obtained by using the random-primed labeling kit from Boehringer. Restriction fragments were subcloned in pBluescript SK- and pUC19, respectively, using Escherichia coli XL-1 Blue as host. Sequencing was carried out by employing the dideoxy chain termination method (20) using the T7 sequencing kit (Pharmacia, Freiburg, Germany). Regions forming secondary structures during sequencing reactions or during electrophoresis were additionally sequenced with the TaQence kit (U. S. Biochemical Corp.) using Taq polymerase, both with and without 7-deaza-dGTP. Furthermore, the T7 sequencing kit was used together with 7-deaza-dGTP and 7-deaza-dATP.

Electrophoresis and Immunodetection

The protein distribution of crude membranes, isolated protein, and isolated subunit c of the ATPase fractions was studied on SDS-polyacrylamide gels (15%) (21) after silver staining (22). The molecular mass of the proteolipid was estimated from the positions of standard proteins. Polyclonal antisera against synthetic peptides of subunits A and B of the Haloferax ATPase were raised in rabbits and used as probes in Western blots to confirm that the purified protein represents the A1A0-ATPase from H. volcanii. The oligopeptides used were DEKWEFEPTVSEGDE (amino acids 123-137 of subunit A) and EDDYEDDAESVEAER (the final 15 amino acids of subunit B) (2). Western blot analysis was carried out as described earlier (2).


RESULTS

Characterization of the ATP Synthase in Membrane Vesicles

Archaea are able to convert a transmembrane potential into ATP using an ATP synthase that is in some respects similar to F- and V-type ATPases from other organisms. H. volcanii contains an A0A1-ATPase that has been characterized with respect to the ATP hydrolyzing capacity. Conditions for the ATPase function are high NaCl concentrations, a medium pH of 9, and the presence of divalent cations. The optimal temperature for ATP hydrolysis is 60 °C (1, 2). ATP synthesis performed by membrane vesicles of H. volcanii should be due to this enzyme. To confirm this, it was essential to analyze the optimal conditions for the ATP synthesis reaction.

ATP synthesis in washed membranes of H. volcanii was optimal at pH 9. Lower pH values decreased the activity very strongly, whereas at pH 10, 75% of the optimal activity was left. The optimal temperature for ATP synthesis is 60 °C, whereas at 25 °C, only 10% of the optimal activity was obtained (30% at 70 °C). In contrast to the ATP hydrolysis reaction, where Mn2+ and Mg2+ could be used almost equally, the ATP synthesis reaction strictly depends on Mg2+. Mn2+ can replace Mg2+, but with a dramatic loss of activity (Fig. 1). Like ATP hydrolysis, ATP synthesis could be performed in 1.75 mol/liter KCl instead of NaCl. Using Mg2+ and ADP as substrates, we determined a Michaelis-Menten constant of 0.67 mmol/liter (ADP) and a Vmax of 18.2 nmol of ATP/mg of protein/min; using Mn2+ instead of Mg2+ diminished the Vmax to 7.1 nmol of ATP/mg of protein/min (Fig. 1).


Fig. 1. Upper panel, ATP synthesis of H. volcanii vesicles (0.98 mg of protein/ml) as a function of the ADP/MgCl2 concentration (ratio of 1:1) (bullet ) and of the ADP/MnCl2 concentration (open circle ). Conditions were as follows: 1.75 mmol/liter NaCl and 50 mmol/liter Tris, pH 9, at 40 °C. The rates were determined by three-point kinetics (30, 90, and 150 s). Lower panel, Lineweaver-Burk plots of the curves. The Km (ADP) calculated from these experiments is 0.67 mmol/liter, and the Vmax values are 18.2 and 7.1 nmol of ATP/mg of protein/min using MgCl2 and MnCl2, respectively.
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The ATP synthase activity was inhibited neither by the F-ATPase inhibitors 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (up to 1 mmol/liter) and phlorizin (up to 2 mmol/liter) nor by the V-ATPase inhibitor bafilomycin (0.1 mmol/liter). N-Ethylmaleimide, a potent V-ATPase inhibitor, caused only a 5-10% loss of activity if the vesicles were preincubated for 2 h and a concentration of 10 mmol/liter was employed. These results are in accordance with our previous analysis of ATP hydrolysis by the Haloferax ATPase (2). Incubation of the membranes overnight at 4 °C in the presence of 1 mmol/liter N,N'-dicyclohexylcarbodiimide resulted in a residual activity of 18% of the ATP synthesis rate measured with vesicles that were stored under the same conditions without N,N'-dicyclohexylcarbodiimide. Lower concentrations of N,N'-dicyclohexylcarbodiimide and short incubation times had only negligible effects.

Carboxyl cyanide p-trifluoromethoxyphenylhydrazone, an uncoupler of photophosphorylation in photosynthesis, is able to diminish ATP synthesis very effectively if added in relatively high concentrations (final concentration of 200 µmol/liter carboxyl cyanide p-trifluoromethoxyphenylhydrazone in the experiment shown in Fig. 2). In contrast, the ionophore nigericin is effective at concentrations that are 2 orders of magnitude lower (50% inhibition at a concentration of 4 µmol/liter (data not shown)).


Fig. 2. Inhibition of ATP synthesis of H. volcanii vesicles (0.89 mg of protein/ml) by the uncoupler carboxyl cyanide p-trifluoromethoxyphenylhydrazone. Carboxyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; final concentration of 200 µmol/liter) was added at the indicated times. Incubation was carried out under standard conditions. The rate of ATP synthesis without inhibitor was 7.5 nmol of ATP/mg of protein/min.
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Immunodetection of the A1 Part of the ATP Synthase

H. volcanii ATP synthase was removed from the membranes by passing the membranes through a French press at 20,000 p.s.i. (23) and was purified by fractionated precipitation using polyethylene glycol. Most of the ATP hydrolysis activity was found in the 25% polyethylene glycol fraction. The respective pellet was applied to a Superose 6 HR 10/30 column (Pharmacia) and eluted with 1.75 mol/liter NaCl plus 50 mmol/liter Tricine, pH 7.5, and 200 mmol/liter MgCl2. After this procedure, a purification factor of 90 and a corresponding yield of 32% were achieved. The specific activity (ATP hydrolysis) of the purified enzyme is 31.4 µmol of Pi/mg of protein/min. When the eluate of the Superose column was subjected to SDS-PAGE, polypeptides with apparent molecular masses of 70, 63, 55, 43, and 37 kDa were observed in addition to smaller peptides. Employing further purification methods, e.g. ammonium sulfate-mediated HPLC (24), resulted in additional small bands and in a dramatic loss of activity (to ~10% of the original activity).

Examining the immunological reactivity of the membranes with antisera against oligopeptides that were constructed according to the deduced amino acid sequences of the large subunits A and B (2), we found that in the membrane fraction, one 70-kDa peptide (subunit A) and one 55-kDa peptide (subunit B) were labeled (Fig. 3). However, when fractions of the Superose gel filtration product were studied employing the antisera, a diminished 70-kDa band and an additionally labeled 51-kDa peptide (subunit A) were obtained (Fig. 3). Obviously, a fragment of 18.3 kDa was split from the 70-kDa protein employing gel filtration. The gel filtration product of subunit B showed immunological reactivity with the 55- and 70-kDa peptides (Fig. 3). At least two additional bands were labeled when the product of a subsequent HPLC column (ammonium sulfate-mediated chromatography) was studied with the help of Western blots (data not shown). Despite the high sodium chloride concentration maintained during every step of purification, degradation of the halophilic ATPase seems to be unavoidable.


Fig. 3. PAGE of membranes and Superose column product and Western blotting of the H. volcanii ATPase (membranes and Superose column product, respectively). Lanes 1 and 3, 10 µg of membranes reacted with antiserum against the synthetic peptide derived from subunits A and B of the H. volcanii A-ATPase, respectively; lanes 2 (subunit A) and 4 (subunit B), 45 µg of isolated and partially purified protein reacted with the same antisera. Immunization of rabbits with the coupled peptides was performed by Eurogentec (Seraing, Belgium). Lane 5, purified chloroplast ATPase (coupling factor 1, alpha - and beta -subunits) reacted with anti-coupling factor 1 (marker); lanes 6-8, ink-stained blot of SDS-PAGE of coupling factor 1, total membranes, and isolated enzyme partially purified by HPLC (Superose gel filtration), respectively.
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Structure of the atp Gene Cluster

To obtain more data concerning the structure of the archaeal ATPases, we cloned and sequenced the atpA and atpB genes of the archaeon H. volcanii (2). When sequencing atpA and atpB, we found that at least two more open reading frames exist upstream of atpA, whereas no reading frame can be found downstream of atpB. Moreover, Northern blot experiments using polymerase chain reaction products of subunits A and B (2) as probes resulted in a single labeled mRNA transcript of ~5 kilobase pairs in both cases, supporting the idea of an A1-encoding operon (Fig. 4). We cloned two genomic DNA fragments, both overlapping with the previously described fragment containing atpA and atpB, and sequenced these fragments completely in both directions. Fig. 5 shows the restriction map for this region.


Fig. 4. Northern hybridization analysis of total RNA from H. volcanii with the DNA probe for subunit A of the Haloferax ATPase that was prepared according to Steinert et al. (2). The RNA was separated on a 1% agarose gel containing 1.1% formaldehyde and blotted onto Biodyne A membrane, followed by overnight hybridization with the radioactive labeled probe for subunit A. The arrow indicates the 4.9-kilobase pairs (kbp) hybridizing mRNA.
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Fig. 5. Restriction map of the atp gene cluster encoding the subunits of the A1 part of the H. volcanii ATPase. Upper panel, positions of the cloned fragments A3 and A5 in relation to the previous cloned fragments AB4 and AB7 (2) and the respective restriction sites indicated by the restriction enzymes. Middle panel, restriction map of the complete gene cluster encoding subunits D, C, E, A, and B. The scale is bases. Lower panel, restriction map and sequencing strategy of the genes encoding the small ATPase subunits D, C, and E. Arrows show the direction and extent of DNA sequencing.
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Three additional reading frames were found upstream of atpA. The genes were designated atpD, atpC, and atpE according to their size and similarity to known ATPase subunits. Upstream of atpD, a 5'-untranslated region of 550 base pairs exists. The noncoding regions between all reading frames are extremely short: atpA is only four nucleotides apart from atpB; there is a spacer of 6 base pairs between atpA and atpE; and the start and stop codons of the reading frames in front of atpE even overlap by 4 base pairs each. The reading frames atpD, atpC, and atpE were translated into amino acid sequences of 194, 348, and 106 residues, respectively (Fig. 6). The calculated molecular masses of the ATPase subunits are 22.03 (subunit D), 38.76, (subunit C), and 11.58 (subunit E) kDa. There are three reasons to assume that the A1-ATPase genes are organized in a single operon. (i) A single transcript of ~5 kilobase pairs was detected by Northern hybridization; (ii) there is a typical promoter region (Fig. 6, boxed) together with a large 5'-untranslated region; and (iii) we found a pyrimidine-rich transcription termination sequence (Fig. 6, underlined).



Fig. 6. Nucleotide sequence of the H. volcanii atp operon and flanking regions. The atpA and atpB regions encoding both large subunits of the ATPase are published elsewhere (2) and are thus indicated by a dashed line. Deduced amino acid sequences are given in single letter code. Potential ribosome-binding sites are underlined with arrows, promoter regions are boxed, and termination sequences are underlined.
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Properties of the Respective Peptides and Amino Acid Sequence Identities

All five putative H. volcanii ATPase genes encode very hydrophilic proteins, with ~20% of their amino acids being aspartic acid and glutamic acid (Table I). This high amount of negatively charged amino acids is well known for halophilic proteins, which lose their native conformation and folding when studied under low salt conditions. Table I shows the amounts of hydrophobic, neutral, and hydrophilic residues of subunits C, D, and E as well as the respective calculated isoelectrical points. Subunit C is the most hydrophobic subunit of the A1 part of the ATPase, with an isoelectrical point of 4.13. 

Table I.

Amino acid composition of H. volcanii ATPase subunits and calculated isoelectrical points (pI) of the subunits


Amino acid Subunit
A
B
C
D
E
No. % No. % No. % No. % No. %

  Ala 53 9.0 33 7.1 42 12.1 22 11.3 7 6.6
  Arg 27 4.6 31 6.6 31 8.9 21 10.8 8 7.5
  Asn 14 2.3 13 2.8 10 2.9 5 2.6 2 1.0
  Asp 42 7.1 43 9.2 28 8.1 22 11.3 13 12.3
  Cys 4 0.6 1 0.2 0 0.0 1 0.5 0 0.0
  Gln 22 3.7 17 3.6 4 1.2 10 5.2 3 2.8
  Glu 73 12.4 56 12 40 11.2 34 17.5 11 10.4
  Gly 49 8.3 40 8.5 25 7.2 8 4.1 10 9.4
  His 6 1.0 4 0.9 0 0.0 0 0.0 2 1.9
  Ile 30 5.1 33 7.1 21 6.1 4 2.1 8 7.5
  Leu 40 6.8 40 8.5 42 12.1 20 10.3 9 8.5
  Lys 14 2.3 7 1.5 7 2.0 4 2.1 3 2.8
  Met 15 2.5 10 2.1 3 0.9 1 0.5 3 2.8
  Phe 16 2.7 15 3.2 14 4.0 4 2.1 3 2.8
  Pro 35 5.9 25 5.3 8 2.3 0 0.0 3 2.8
  Ser 27 4.6 23 4.9 22 6.3 13 6.7 7 6.6
  Thr 35 5.9 29 6.2 9 2.6 3 1.5 6 5.7
  Trp 10 1.7 1 0.2 2 0.6 1 0.5 0 0.0
  Tyr 20 3.4 18 3.8 16 4.6 3 1.5 0 0.0
  Val 54 9.2 29 6.2 24 6.9 18 9.3 8 7.5
Acidic amino acids 115 19.5 99 21.2 68 19.3 56 28.8 24 22.7
pI 3.84 3.85 4.13 3.92 4.00

atpD

The first reading frame of the H. volcanii operon (atpD) was translated into an amino acid sequence of 194 residues. The calculated molecular mass is 22.03 kDa. Data base searches revealed that the putative gene product of atpD resembles subunit E of the bovine kidney vacuolar ATPase (25). Alignment of the deduced amino acid sequence with different V-ATPase E subunits is shown in Fig. 7. Comparing subunit E of Saccharomyces cerevisiae (26) and subunit E of the bovine kidney V-ATPase (25) with the atpD gene product of H. volcanii resulted in 23% identical amino acids. Moreover, we found 16.5% identity to subunit E of the V-ATPase from Enterococcus hirae (formerly Streptococcus faecalis) (32). In the latter eubacterium, a H+-translocating F-ATPase resides besides a Na+-translocating V-ATPase that plays an important role at high pH, where the proton potential is not generated. Comparing the translated amino acid sequence for atpD with F-ATPase delta -subunits, which have similar molecular masses (spinach chloroplast ATPase delta -subunit, 20.5 kDa (27); and Odontella sinensis delta -subunit, 21.1 kDa (28)), resulted in very low similarities of 12 and 13%, respectively. We found no significant similarity comparing the atpD product with the gamma -subunit of the Sulfolobus ATPase. Although the overall sequence homology among the compared proteins is very poor, the N- and C-terminal regions revealed some degree of sequence conservation. Cross-link experiments have shown that the N-terminal region is involved in the connection of the delta -subunit to subunit I of the membrane part of chloroplast ATPase (29). The delta -subunit (subunit D) is supposed to be involved in proton conduction from the membrane-integral part to the catalytic subunits.


Fig. 7. Alignment of the deduced amino acid sequences of H. volcanii ATPase Subunits C, D, and E (H.vol C, D, and E) with different V-ATPase subunits. S.cer, Saccharomyces cerevisiae (43); B.cgr, bovine chromaffin granule (44); E.hir, Enterococcus hirae (45); B.kid, bovine kidney (25); M.sex, Manduca sexta (46). Asterisks indicate identical amino acids.
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atpC

The second reading frame, overlapping with the first one by four nucleotides, was translated into 348 amino acids, resulting in a polypeptide with a calculated molecular mass of 38.7 kDa. The calculated molecular mass of the atpC gene product corresponds to the size of the partially purified enzyme of 37 kDa as estimated by SDS-PAGE (Table II). The size of subunit C is comparable to various C subunits of V-ATPases, e.g. subunit C of E. hirae (Fig. 7). The similarity between subunit C of V-ATPases from different sources and/or the gamma -subunit of F-ATPases is generally not very strong. Only 17% identical residues could be found comparing H. volcanii subunit C with Enterococcus subunit C (Fig. 7).

Table II.

Molecular masses of the H+-ATPase subunits of H. volcanii


Subunit Nucleotides Amino acids Molecular mass
Sequencea SDS-PAGEb

A 1761 586 64.5 70c
B 1407 468 52.0 55c
C 1047 348 38.7 37
D 585 194 22.0 NDd
E 321 106 11.6 12
c ND ND ND 9.7

a As calculated from deduced amino acids.
b As deduced by denatured PAGE.
c Calculated from the Western blot shown in Fig. 2.
d NF, not found; ND, not done.

atpE

The third reading frame, translated into a sequence of 106 amino acids with a molecular mass 11.58 kDa, was compared with the 14-kDa subunit (subunit F) of the V-ATPase from the hawk moth Manduca sexta. The atpE gene product of H. volcanii showed 23% identical amino acids when compared with this only known subunit F of eucaryal V-ATPases. An amount of 16% identical amino acids was found when comparing subunit E with E. hirae subunit G (Fig. 7), but only 12% was found when comparing with epsilon -subunits of F-ATPases. Despite the poor similarity, the amino terminus of the sequence shows rather high identity to M. sexta subunit F and E. hirae subunit G: 10 of 22 amino acids are identical or conservative exchanges.

The Proton-conducting Membrane Part of the Haloferax ATPase

Since the H. volcanii ATPase operon does not contain a gene coding for the membrane-spanning proton channel or subunit c, we isolated this peptide from the membranes according to Denda et al. (16) and Kakinuma et al. (30). After purification, a single protein was obtained with a molecular mass of 9.7 kDa as determined by SDS-PAGE. To identify this protein, it was blotted onto polyvinylidene difluoride membrane and transferred to a gas-phase sequencer for automatic amino acid sequence analysis. The first 10 amino acids (Fig. 8) indicate that the peptide is related to the proteolipids (c subunits) of E. coli and spinach chloroplast ATPase, whereas there is almost no similarity to the N terminus of the respective peptide of Sulfolobus acidocaldarius (30).


Fig. 8. A, silver-stained polyacrylamide gel of the isolated proteolipid (0.5 µg) (lane 2) and molecular mass standards (2 µg; Merck) (lane 1); B, amino acid sequence of the amino terminus of the isolated proteolipid of H. volcanii (H.vol) compared with that of E. coli (E.co) and spinach chloroplast ATPase (spi. III).
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DISCUSSION

So far there is only one complete archaeal ATPase operon described in the literature: the acidothermophile archaeon S. acidocaldarius contains five genes encoding hydrophilic polypeptides (atpD, atpA, atpB, atpG, and atpE) that precede one gene (atpP) coding for a very hydrophobic subunit (c or "proteolipid") (31). The authors describe the small subunits to have no significant similarity either to F-ATPase or to eucaryal V-ATPase subunits, whereas the large alpha - and beta -subunits and the channel-building subunit c do have homologous counterparts in other ATPases.

The H. volcanii ATPase operon is the first reported halophilic archaeal ATPase gene cluster. In Fig. 9, the arrangement of the Haloferax genes is compared with the respective operon of the archaeon S. acidocaldarius (31). Furthermore, the gene arrangement coding for the V-ATPase (Na+-ATPase) from E. hirae (32) as well as the F-ATPase gene clusters from E. coli (33) and Rhodospirillum rubrum (34) are shown. The most surprising finding was that the Haloferax operon is quite different from the operon in the acidothermophilic archaeon S. acidocaldarius, whereas complete correspondence could be found when comparing the Haloferax operon with the eubacterial E. hirae Na+-ATPase gene cluster. Thus, our finding indicates a conserved operon structure for V- and A-ATPases. In a very recent paper, Wilms et al. (47) reported on the organization of a gene cluster encoding the archaeal ATP synthase from Methanosarcina mazei Gö1. This gene cluster combines the arrangement of that of H. volcanii with the one of S. acidocaldarius since it contains genes homologous to the Sulfolobus gamma - and epsilon -subunits. In Enterococcus, the proteolipid-encoding gene is atpK, positioned in front of atpE, which encodes the peptide homologous to H. volcanii subunit D. The operon of the S. acidocaldarius ATPase ends with the gene for the hydrophobic subunit c that builds up the proton-conducting, membrane-spanning part of the ATPase. In the photosynthetic bacterium R. rubrum, the F1- and F0-subunits are encoded by different operons, which are transcribed independently (34). This may also be true for H. volcanii since we could not find any coding region for the hydrophobic membrane part or for a related membrane-spanning protein.


Fig. 9. Arrangement of the genes in the atp operon of H. volcanii (archaeal H+-ATPase), S. acidocaldarius (archaeal H+-ATPase), E. hirae (eubacterial Na+-ATPase, V-type), E. coli (eubacterial H+-ATPase, F-type), and R. rubrum (purple photobacterium H+-ATPase, F-type). Related subunits are shaded alike. The dashed line (Rhodospirillum) indicates two independent operons, which are separately transcribed. The letters indicate the subunits encoded by the respective genes. Citations are given under "Discussion."
[View Larger Version of this Image (23K GIF file)]


The molecular mass of the so-called proteolipid in the F0 part of F-ATPases is ~8 kDa, whereas V-ATPases possess proteolipids with molecular masses of ~16 kDa, consisting of four membrane-spanning helices. A data bank search revealed that the first half and the last half of the protein are both similar to the F-ATPase proteolipid (35). Thus, both proteolipids are proposed to have originated from a common ancestral gene that underwent gene duplication (35). From the structures described above and the apparent similarities of F-, A-, and V-ATPases, the structure of the Haloferax ATP synthase has been predicted on the basis of the well known structure of F-ATPases (36).

Since the universal phylogenetic tree has been restructured by Woese et al. (37), the halophilic bacteria belong to the Euryarchaeota together with the methanogens and their relatives. The second archaebacterial kingdom, named Crenarchaeota, contains the thermoacidophiles. Together, they form the domain Archaea. This new order was mainly based upon 16 S RNA sequence comparisons (38, 39). The presence of a number of eucaryal features in archaea, including the relationship between the large subunits of the A-ATPases and eucaryal V-ATPases, has led to a closer grouping of archaea and eucarya than of archaea and eubacteria (37). Whereas the origin of the F-ATPases is rooted in eubacteria, which evolved into the cell organelles mitochondria and chloroplasts, according to Nelson and Taiz (40) and Gogarten et al. (41), the origin of V-ATPases is proposed to be related to archaebacteria. Nelson (42) proposed that evolution of the V-ATPases to become exclusively proton pumps is related to the duplication of their gene encoding the proteolipid.

Comparing the operon structure of the E. hirae V-ATPase gene with the Haloferax ATPase operon, we were able to confirm the proposed relationship concerning the A1 and V1 parts of archaeal and vacuolar ATPases. Yet, the membrane-embedded proteolipid seems to be more closely related to the respective protein of F-ATPases, as shown by its size and N-terminal sequence. It has been proposed that the gene clusters for the catalytic portion of the ATPases initially have evolved separately (for review, see Ref. 42). As an example for separated operons, the gene clusters of R. rubrum are shown in Fig. 9. The split between the operons is between the delta - and beta -subunits. The H. volcanii gene cluster ends with atpD, encoding the delta -homolog subunit D. The same position can be observed for the gene encoding the delta -subunit of S. acidocaldarius, although the arrangement of the genes is different from that of Haloferax. Work to obtain more information about the second ATPase gene cluster that codes for the A0 part of the Haloferax ATPase is in progress.


FOOTNOTES

*   This work was supported in part by Deutsche Forschungsgemeinschaft Sonderforschungsbereich 189.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X79516[GenBank].


Dagger    Supported by a fellowship from Noidrhein-Westfalen (GrFG-NW).
§   To whom correspondence should be addressed. Tel.: 211-81-12381; Fax: 211-81-13706; E-mail: bickel{at}uni_duesseldorf.de.
1   The abbreviations used are: V-ATPases, vacuolar ATPases; AATPases, archaeal ATPases; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; HPLC, high pressure liquid chromatography.

Acknowledgments

Protein sequencing was performed by D. Kienzle (Forschungszentrum Jülich). We thank Dr. Jörg Soppa (Max-Planck-Institute Martinsried) for the generous gift of H. volcanii strain WR 340 and for help. We thank Dr. David Mozley and Martina Kroth for critical reading of the manuscript and for linguistic corrections.


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