Báo cáo khoa học: An intermediate step in the evolution of ATPases ) the F1F0-ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable of ATP synthesis Michael Fritz and Volker Muller ¨ docx

An intermediate step in the evolution of ATPases ) the F 1 F 0 -ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable of ATP synthesis Michael Fritz and Volker Mu ¨ ller Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University, Frankfurt am Main, Germany Membrane-bound, multisubunit, ion-translocating ATP synthases ⁄ ATPases are present in every domain of life. They arose from a common ancestor, but evolved into three distinct classes of ATP synthases ⁄ ATPases: the F 1 F 0 -ATP synthase present in bacteria, mitochondria and chloroplasts, the A 1 A 0 -ATP synthase present in archaea, and the V 1 V 0 -ATPase present in eukarya [1,2]. A common feature of ATP synthases ⁄ ATPases is their organization into two domains, a soluble and a membrane-bound domain, which are connected by (at least) two stalks, one central and one to two peripheral [3–5]. The hydrophilic, cytoplasmic domain catalyzes ATP hydrolysis [6,7], whereas the membrane domain translocates ions from one side of the membrane to the other against their electrochemical gradient [8,9]. ATP synthases⁄ ATPases are rotary machines that work as a pair of coupled motors, a chemically driven (F 1 ⁄ A 1 ⁄ V 1 ) motor and a membrane-embedded, ion gradient-driven motor (F 0 ⁄ A 0 ⁄ V 0 ) [10–12]. The membrane-embedded motor is composed of a stator and a rotor. The stator is composed of subunits a and b, and the rotor is composed of multiple copies of subunit c. They form an oligomeric ring of non- covalently linked subunits, and rotation of the c ring is obligatorily coupled to ion flow across the mem- brane [13,14]. Subunit c of F 1 F 0 -ATP synthases has a molecular mass of around 8 kDa, and folds in the membrane like a hairpin, with two transmembrane helices that are connected by a cytoplasmic loop [15]. Each monomer contains an ion-binding site, and as 10–15 subunits constitute the rotor (depending on the spe- cies), it has a total of 10–15 ion-binding sites [12,16– 19] This gives a H + (Na + ) ⁄ ATP stoichiometry of 3.3–5, a value required for ATP synthesis, given a Keywords Acetobacterium woodii; ATP synthase; F-type; rotor subunits; V-type Correspondence V. Mu ¨ ller, Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Max-von-Laue- Str. 9, 60438 Frankfurt, Germany Fax: +49 69 79829306 Tel: +49 69 79829507 E-mail: vmueller@bio.uni-frankfurt.de (Received 23 March 2007, revised 2 May 2007, accepted 8 May 2007) doi:10.1111/j.1742-4658.2007.05874.x Previous preparations of the Na + F 1 F 0 -ATP synthase solubilized by Triton X-100 lacked some of the membrane-embedded motor subunits [Reidlinger J&Mu ¨ ller V (1994) Eur J Biochem 233, 275–283]. To improve the subunit recovery, we revised our purification protocol. The ATP synthase was solu- bilized with dodecylmaltoside and further purified to apparent homogeneity by chromatographic techniques. The preparation contained, along with the F 1 subunits, the entire membrane-embedded motor with the stator subunits a and b, and the heterooligomeric c ring, which contained the V 1 V 0 -like subunit c 1 and the F 1 F 0 -like subunits c 2 and c 3 . After incorporation into liposomes, ATP synthesis could be driven by an electrochemical sodium ion potential or a potassium ion diffusion potential, but not by a sodium ion potential. This is the first demonstration that an ATPase with a V 0 –F 0 hybrid motor is capable of ATP synthesis. Abbreviations DY, membrane potential; DlNa + , electrochemical sodium ion potential; DpNa, sodium ion potential. FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS 3421 transmembrane electrochemical ion gradient of around  200 mV. The c subunit of V 1 V 0 -ATPases arose by duplication and fusion of the bacterial c subunit, giving rise to a  16 kDa protein with two hairpins [20]. Most important, the ion-binding site is not conserved in hairpin 1. If one assumes the same number of hairpins in V 0 and F 0 , the rotor of V 1 V 0 - ATPases has only half the number of ion-binding sites. This is seen as the reason for the apparent inab- ility of V 1 V 0 -ATPases to catalyze ATP synthesis in vivo. Indeed, V 1 V 0 -ATPases have evolved to be effi- cient ion pumps, a function required by the physiol- ogy of the eukaryotic cell [21]. The operon encoding the N a + F 1 F 0 -ATP synthase from the a naerobic, a cetogenic bacterium Acetob acterium woodii is unique and encodes nine F 1 F 0 -like subunits along with one gene encoding a V 1 V 0 -like subunit. The atp operon has one homolog each of a gene encoding the F 1 F 0 subunits a, b, c, d, e, a, and b, but it has three genes encoding differently sized c subunits [22]. Subunits c 2 and c 3 have a molecular mass of 8.18 kDa, are identical at the amino acid level, and are similar to c subunits from F 1 F 0 -ATP synthases. Like other F 1 F 0 - ATP synthase c subunits, they are predicted to span the membrane like a hairpin and to have one ion-bind- ing site. In contrast, subunit c 1 is similar to the c sub- units of V 1 V 0 -ATPases and predicted to have four transmembrane helices with only one ion-binding site. A. woodii is, so far, the only organism known with V 1 V 0 - and F 1 F 0 -like c subunit genes in one ATPase operon. This poses the obvious questions of whether the heterooligomeric c ring can promote ATP synthe- sis, whether the c subunit stoichiometry is variable, and, if so, whether a variation of the c 1 ⁄ c 2 ⁄ 3 ratio may change the function of the enzyme from an ATP syn- thase to an ATPase [23]. These questions have not so far been addressed, due to the lack of a purified, intact ATP synthase. Despite the clear genetic evidence for different c subunits, as well as for the presence of subunits a and b, they were not detected in previous preparations of the enzyme [24,25]. Later, separation of gently solubilized mem- brane protein complexes by blue native PAGE and N-terminal sequencing of polypeptides present in the gel revealed both types of c subunit, as well as sub- units a and b, in the ATPase complex [26]. These stud- ies prompted us to revise our purification scheme, with the aim of obtaining an intact ATP synthase from A. woodii. We here present a protocol yielding a com- plete Na + F 1 F 0 -ATP synthase complex, including the entire membrane motor. Most important, both types of c subunits were present. This made it possible to readdress the longstanding question of whether an ATPase with F 0 - and V 0 -type rotor subunits is able to synthesize ATP. Results Purification of a complete Na + F 1 F 0 -ATP synthase from A. woodii The Na + F 1 F 0 -ATP synthase purified previously was solubilized from membranes with Triton X-100 [24,25]. To improve the recovery of subunits, we analyzed the effectiveness of dodecyl-b-d-maltoside in solubilizing the entire ATP synthase. When used at 1% (w ⁄ v) and 1 mg of detergent per mg of protein, dodecyl-b-d- maltoside solubilized about 85% of the membrane- bound ATPase activity. The ATP synthase was then purified by gel filtration to apparent homogeneity. This procedure resulted in a 16-fold enrichment, but was accompanied by loss of 70% of the activity (Table 1). The molecular mass of the complex as determined by gel filtration was 590 kDa. As can be seen from Fig. 1, the enzyme preparation contained 12 polypeptides. The identity of the peptides was established using MALDI-TOF or western blot analyses. These studies revealed that the 58 kDa fragment corresponds to sub- unit a, the 54 kDa fragment to subunit b, the 35 kDa fragment to subunit c, the 19 kDa fragment to subunit d, the 18 kDa fragment to a mixture of subunits a and b, the 16 kDa fragment to subunit e, the 14 kDa frag- ment to subunit c 1 , and the 10 kDa fragment to sub- unit c 2 ⁄ 3 (Fig. 1A). The 42 kDa fragment reacted with antibobies against c 2 ⁄ c 3 (which also recognize c 1 ) and with antibodies against c 1 (which do not recognize c 2 ⁄ 3 ; Fig. 1B). These data demonstrate that the 42 kDa fragment represents the SDS-resistant, heterooligomeric c ring of the Na + F 1 F 0 -ATP synthase. When the pre- paration was heated to 120 °C for 5 min, the c oligo- mer was disrupted, and the monomers could be detected immunologically (Fig. 1B). In summary, these experiments clearly demonstrated the presence of sub- units a, b, c 1 and c 2 ⁄ 3 in the membrane-embedded rotor of the purified Na + F 1 F 0 -ATP synthase from A. woodii. Table 1. Purification of the Na + F 1 F 0 -ATP synthase from A. woodii. Step Protein (mg) Volume (mL) Activity (U) Activity (U ⁄ mg) Purification (fold) Yield (%) Membranes 430 50 214 0.6 1 100 Solubilizate 41 47 188 4.6 8.3 87 Concentrated solubilizate 16 15 98 6.2 11 45 Gel filtration 6.9 10 67 9.7 16 30 Na + F 1 F 0 -ATP synthase from A. woodii M. Fritz and V. Mu ¨ ller 3422 FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS Characterization of the Na + F 1 F 0 -ATP synthase from A. woodii The specific ATPase activity of the complete enzyme was in the range 5–9.9 UÆmg )1 protein, depending on the batch. This is in the same range as the one deter- mined previously with the incomplete enzyme [25]. Next, we compared the enzymatic properties of the complete enzyme with those of the preparations stud- ied previously. The basic biochemical parameters, such as temperature and pH dependence, as well as the kinetic data for ATP hydrolysis were identical (data not shown). Of special interest was the effect of Na + on activity, as Na + is known to interact with the membrane-embedded motor. As seen before, ATP hydrolysis was clearly Na + dependent (Fig. 2), and Na + could be substituted by Li + . Furthermore, the K m for Na + or Li + (0.5 mm, 2.0 mm) was compar- able to the values determined before. As described before, the stimulation by Na + was less pronounced at more acidic pH values, indicating competition of Na + and H + for a common binding site (data not shown). Furthermore, inhibition by N¢,N¢-dicyclo- hexylcarbodiimide was abolished in the presence of Na + (Fig. 3). In summary, the biochemical parame- ters of the complete preparation were indistinguish- able from those of the preparation described previously [25]. α β γ c-oligomer c 2/3 ε c 1 δ a/b 3 4 5 6 7 1 2 AB a citn 1 tna ai i t na c 2 / 3 -66 - -45 - -30 - -20 - -14 - 66 - 30 - 20 - 14 - 45 - 94 - -66 -45 -30 -20 -14 Fig. 1. Subunit composition of the Na + F 1 F 0 -ATP synthase from A. woodii. Proteins were separated by SDS ⁄ PAGE and stained with SERVA Blue G (Serva GmbH, Heidelberg, Germany) (A) or blotted against specific antibodies (B). Lane 1: molecular mass marker. Lane 2: ATP syn- thase preparation was denatured by incubation at 80 °C for 10 min. Lane 3: ATP synthase was heated for 5 min at 120 °C prior to SDS ⁄ PAGE to disrupt the c oligomer and blotted against c 1 antibodies. Lane 4: ATP synthase was incubated for 10 min at 80 °C, and blotted against c 1 antibodies. Lane 5: the sample was incubated for 10 min at 80 °C and hybridized with antibody specific for the a subunit. Lane 6: ATP synthase was incubated for 5 min at 120 °C and hybridized with antibodies against subunit c 2 ⁄ 3 (which also detect subunit c 1 ). Lane 7: ATP synthase was denatured by boiling for 15 min, and blotted against c 2 ⁄ 3 antibodies. NaCl (mM) ATPase activity (U/mg) A LiCl (mM) ATPase activity (U/mg) 0 2 4 6 8 10 12 14 0 1 2 3 4 5 -3 -2 -1 0 1 2 3 4 5 6 7 8 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1/ATPase activity 0 2 4 6 8 10 12 0 1 2 3 4 5 1.0 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1/ATPase activity B Fig. 2. Ion dependence of ATP hydrolysis by the Na + F 1 F 0 -ATP synthase from A. woodii. ATPase activity was measured at 30 °C using the assay described in Experimental procedures. NaCl (A) or LiCl (B) was added from stock solutions to the concentrations indicated. The insert shows the same data plotted by the method of Lineweaver–Burk. M. Fritz and V. Mu ¨ ller Na + F 1 F 0 -ATP synthase from A. woodii FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS 3423 ATP synthesis catalyzed by the Na + F 1 F 0 -ATP synthase from A. woodii Next, we investigated whether the enzyme is capable of ATP synthesis despite its heterooligomeric c ring. Liposomes were prepared from lipids extracted from chicken egg, and the complete Na + F 1 F 0 -ATPase was reconstituted into these liposomes. The rate of ATP hydrolysis as catalyzed by these proteoliposomes was 2.8 UÆmg )1 . To analyze whether the enzyme was reconstituted in a functionally coupled state that allows for ATP synthesis, the following experiments were performed. In a fully coupled system, ATP hydrolysis is accompanied by ion transport into the proteoliposomes, and the membrane potential estab- lished slows down or even inhibits further ion trans- port and thus ATPase activity. This thermodynamic control can be overcome by addition of ionophores. After addition of the Na + ionophore N¢,N¢,N¢,N-tetra- cyclohexyl-1,2-phenylenedioxydiacetamide to the proteo- liposomes, ATP hydrolysis was stimulated seven-fold. Stimulation, but to a lower extent, was also observed with the protonophore tetrachlorosalicylanilide. These experiments demonstrated coupling of ATP hydrolysis to the generation of a membrane potential in our pro- teoliposome system. Next, we applied artificial driving forces to the pro- teoliposomes. The general strategy is shown in Fig. 4. When the proteoliposomes were loaded with 200 mm NaCl and incubated in the presence of 200 mm KCl, thus creating a sodium ion potential (DpNa), there was no ATP synthesis (Fig. 5). Upon addition of 2 lm vali- nomycin, a membrane potential (DY, inside positive) was created in addition by the influx of K + into the liposomes (potassium ion diffusion potential) and in the presence of both DY and DpNa, ATP was synthes- ized at a rate of about 40 mol ATPÆ(mol pro- tein) )1 Æmin )1 . When a DY was applied separately, ATP was synthesized at a rate comparable to that with electrochemical sodium ion potential (DlNa + ) as the driving force. ATP synthesis was strictly dependent on the presence of ADP, the coupling ion Na + , and the presence of a DY (data not shown). In summary, these data demonstrate not only that subunits a and b are required to confer the ability to synthesize ATP, but equally important, that the pres- ence per se of subunit c 1 does not abolish ATP synthe- sis by the Na + F 1 F 0 -ATP synthase from A. woodii. Discussion The Na + F 1 F 0 -ATP synthase from the anaerobic, ace- togenic bacterium A. woodii purified here contained all the subunits deduced from the operon sequence. Most importantly, it contained the membrane motor sub- units a and b, which were absent in previous prepara- tions. Because the ATP synthase preparations from the close relatives Moorella thermoautotrophicum and Moo- rella thermoacetica were also devoid of subunits a and b, it was suggested in the literature that the ATP synthases of acetogenic bacteria may be simpler in architecture than other F 1 F 0 -ATP synthases [27,28]. DCCD (µM) ATPase activity (%) 0 25 50 75 100 0 25 50 75 100 Fig. 3. Inhibition of the Na + F 1 F 0 -ATP synthase from A. woodii by N ¢,N ¢-dicyclohexylcarbodiimide and relief of inhibition by Na + . ATPase activity was measured at 30 °C and pH 7.5 using the assay described in Experimental procedures. The samples were incubated with 5 m M (m) or 100 lM NaCl (j) for 30 min. N¢,N¢-Dicyclohexyl- carbodiimide was then added, and the samples were incubated for another 25 min. The reaction was started by addition of 5 m M ATP. One hundred per cent activity corresponds to 9 UÆmg )1 . Fig. 4. General scheme for the application of artificial driving forces to the proteoliposomes. For explanation, see text. Na + F 1 F 0 -ATP synthase from A. woodii M. Fritz and V. Mu ¨ ller 3424 FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS However, this argument was difficult to understand, as the genes encoding subunits a and b were embedded in the atp operons of M. thermoacetica and A. woodii [22,29], and one would have to envision a mechanism that post-trancriptionally or post-translationally specif- ically removes these subunits. Furthermore, these sub- units are essential for motor function. From the results presented here, it is evident that the critical step in purification is the solubilization procedure. The Triton X-100 used in previous studies apparently did not solu- bilize the stator subunits a and b. Whether or not our previous preparation contained both types of c subunit is difficult to retrace. However, Fig. 3 in Reidlinger & Mu ¨ ller [25] shows a faint band at about 17 kDa, which appeared when the enzyme was autoclaved. At that time, c 1 was unknown, and an N-terminal sequence of this fragment was not obtained. Therefore, it is not only not excluded that it was the c 1 subunit but likely, as the c ring is rather stable, and the use of Triton X- 100 should not lead to removal of c 1 only. The basic biochemical properties of the complete enzyme are similar to those of the enzyme studied before. It is Na + dependent, and Na + and N¢,N¢-di- cyclohexylcarbodiimide compete for binding to a com- mon site. It should be noted that the incomplete enzyme isolated previously was capable of ATP-driven Na + transport. As ATP-driven Na + transport would also require the stator subunits, one has to assume that previous preparations had substoichiometric amounts of subunits a and b. This is in accordance with the low Na + ⁄ ATP ratio determined previously. The important difference is that the stator-depleted enzyme was unable to synthesize ATP in a proteoliposome system [30], whereas the entire enzyme used here was competent in ATP synthesis. This underlines the essential function of the stator subunits in driving ion gradient-driven ATP synthesis. As observed before with the Na + F 1 F 0 -ATP synthase from Propionigenium modestum, DlNa + as well as DY but not DpNa were sufficient as driving forces [31–33]. Most importantly, the Na + F 1 F 0 -ATP synthase from A. woodii was able to catalyze ATP synthesis despite its different c subunits. Although the c subunit stoichiometry has not yet been solved, at least one copy of c 1 must have been present. Further discus- sion of the coupling efficiencies has to await the deter- mination of the rotor subunit stoichiometry. In summary, we have solved a longstanding problem, the purification of the Na + F 1 F 0 -ATP synthase from A. woodii, including the membrane-embedded motor. Most importantly, we have demonstrated that the enzyme as isolated from fructose-grown cells is compet- ent in ATP synthesis, despite its unusual and unique membrane-embedded motor. This is the starting point for a detailed analysis of the stucture and function of the rotor of the Na + F 1 F 0 -ATP synthase from A. woo- dii, the first containing V 0 - and F 0 -like c subunits. Experimental procedures Growth of cells and isolation of membranes A. woodii (DSM 1030) was grown in 20 L vessels to midex- ponential growth phase as described previously [34]. Fruc- tose (20 mm) was used as the carbon and energy source. The cells were harvested by continuous centrifugation (Heraeus centrifuge, Stratos, HCF 22.300 rotor), and stored at ) 70 °C until used. For the isolation of membrane vesi- cles, 20–25 g of cells (wet mass) was suspended in 200 mL of 50 mm Tris ⁄ HCI, 10 mm MgCI 2 , and 420 mm sucrose (pH 7.5) (buffer A). After addition of 1 g of lysozyme (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), the suspension was incubated at 37 °C for 60 min. All sub- sequent steps were carried out at 4 °C unless otherwise indicated. The resulting protoplasts were collected by cen- trifugation (16 000 g, 10 min, Beckman Avanti J25, JA14 rotor) and suspended in one volume of buffer A containing a few crystals of DNase (Sigma-Aldrich Chemie GmbH) and phenylmethanesulfonyl fluoride (final concentration 0.5 mm). The protoplasts were disrupted by two passages through a French pressure cell at 42 MPa. Cell debris and unbroken cells were removed by two sequential centrifuga- tion steps at 6000 g for 15 min (Beckman Avanti J25, JA14 rotor). The supernatant was diluted with one volume of 0 0 1 0 2 0 3 0 4 0 5 0 6 0 7 08 09 001 0 1 1 021 0 3 1 0 01 0 2 0 3 0 4 05 06 07 08 0 9 mite)s( mol ATP / mol enzyme Fig. 5. ATP synthesis by Na + F 1 F 0 -ATP synthase-containing proteo- liposomes. The artificial driving forces DlNa + (j), DY (m)orDpNa (r) were applied to the proteoliposomes as described in Experi- mental procedures. In one assay (d), a DlNa + was applied but ADP was omitted. M. Fritz and V. Mu ¨ ller Na + F 1 F 0 -ATP synthase from A. woodii FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS 3425 50 mm Tris ⁄ HCl (pH 7.5), 10 mm MgCl 2 , and 17% (v ⁄ v) glycerol (buffer B), and centrifuged at 130 000 g for 60 min (Beckman L100K, 50.2 Ti rotor) to collect the membranes. The membranes were washed once with buffer B, and resuspended in 60 mL of buffer B. Purification of ATP synthase The membranes were diluted to 10 mgÆmL )1 with buffer B, and solubilization was accomplished by the addition of 1% dodecyl-b-d-maltoside (w ⁄ v) (Sigma-Aldrich Chemie GmbH). After 60 min on ice, the extract was centrifuged at 130 000 g for 30 min (Beckman L100K, 50.2 Ti rotor). The solubilized ATP synthase was concentrated to a volume of 15 mL (Vivaspin 20 columns, 300 kDa cutoff; Vivascience, No ¨ rten-Hardenberg, Germany), and applied to a gel filtra- tion column (Sephacryl S-400, 2.6 ⁄ 100 cm; GE-Healthcare, Freiburg, Germany). The column was equilibrated with col- umn buffer (50 mm imidazole, 50 mm NaCl, 25 mm MgSO 4 , 0.5 mm phenylmethanesulfonyl fluoride, 0.1% reduced Tri- ton X-100 (pH 7.5) (Sigma-Aldrich Chemie GmbH), at a flow rate of 0.5 mL Æ min )1 . The purified ATP synthase was found in three fractions. These fractions were pooled and concentrated to 5 mL (Vivaspin 20 columns, 100 kDa cutoff; Vivascience). All preparations were routinely analyzed by SDS ⁄ PAGE, using the buffer system of Scha ¨ gger & von Jagow [35]. Polypeptides were visualized by staining with SERVA blue G250 (Sigma-Aldrich Chemie GmbH) [36] or silver [37]. Determination of ATPase activity The ATPase activity was assayed in buffer C (100 mm Tris base, 100 mm maleic acid, 5 mm MgCl 2 ). The pH was adjus- ted to 7.5 with KOH. The characterization of the enzyme was performed at 30 °C by a discontinuous assay following the ATP-dependent formation of inorganic orthophosphate, according to the method of Heinonen & Lahti [38] as des- cribed previously [39]. The assay contained 5 mm MgCI 2 when carried out at pH 7.5, and 50 mm MgCl 2 at pH 5.3. For inhibitor studies, the samples were incubated with the inhibitor for 30 min before the reaction was started by addi- tion of ATP. N¢,N¢-Dicyclohexylcarbodiimide (Sigma-Ald- rich Chemie GmbH) was added as an ethanolic solution, and controls received solvents only. Western blot analysis After separation by SDS ⁄ PAGE, the ATP synthase sub- units were blotted onto a nitrocellulose membrane as des- cribed previously [40]. Western blot ECL detection reagents were either purchased from PerkinElmer Life Sci- ences (Boston, MA, USA) or made in-house [solution A (200 mL containing 0.1 m Tris ⁄ HCl, pH 6.8, 50 mg of luminol), and solution B (10 mL of dimethylsulfoxide con- taining 11 mg of p-hydroxycoumaric acid)]. Blot mem- branes were incubated in a mixture of 4 mL of solution A, 400 lL of solution B and 1.2 lLofH 2 O 2 for 2 min before exposure to WICORex film (Typon Imaging AG, Burgdorf, Switzerland). Reconstitution of ATPase into proteoliposomes A suspension of 60 mgÆmL )1 l-a-phosphatidylcholine type II-S (Sigma-Aldrich Chemie GmbH) in buffer D (100 mm Tris, 100 mm maleic acid, 20 mm NaCl, 5 mm MgCl 2 , pH 7.5) was sonicated on ice at 120 W and 20% (Ultra- sonic Disintegrator, type MK2, Crawley, England) until the creamy suspension became translucent. To the purified ATP synthase, the liposomes were added to a final lipid concentration of 30 mgÆmg protein )1 . The proteoliposomes were prepared by the method of Knol et al. [41], and the detergent was removed by stirring in the presence of Bio- Beads (Bio-Rad, Mu ¨ nchen, Germany) for 12 h at 4 °C. The proteoliposomes were collected by gel filtration using a 10 mL column filled with Sephadex 25 (Bio-Rad) matrix equilibrated with buffer D and driven by gravity. More than 85% of the ATPase activity applied for the reconstitu- tion experiments was found in the liposome fraction (0.7 mg proteinÆmL )1 ), indicating almost complete incor- poration into the proteoliposomes. ATP synthesis was determined via a standard luciferin ⁄ luciferase assay, monit- oring the emitted light with a chemiluminometer (Lumac, AC Landgraaf, The Netherlands). To generate aDlNa + , the proteoliposomes were first loaded with Na + to create a DpNa. The vesicles were incu- bated in buffer D containing, in addition, 200 mm NaCl for 12 h at 4 °C. After this, the Na + -loaded vesicles were collected by gravity-driven gel filtration using a 10 mL pip- ette filled with Sephadex 25 matrix and equilibrated with buffer D containing, in addition, 200 mm KCl and 5 mm KH 2 PO 4 . The synthesis reactions were carried out at 30 °C with 2 mL of proteoliposome solution from gel filtration and by adding 5 mm ADP. The synthesis reaction was star- ted by addition of 2 mL of valinomycin (Sigma-Aldrich Chemie GmbH) to induce a DY. Samples (10 mL) were withdrawn every 30 s and immediately added to 250 mL of an ATP determination buffer (5 mm NaHAsO 4 ,4mm MgSO 4 ,20mm glycylglycine, pH 8). After the addition of 5 mL of firefly lantern crude extract (Lumac), light emis- sion was measured. Calibration was done with standards of a known ATP content. To apply a DY only, the proteoliposomes were incubated for 12 h with buffer D. After this, the vesicles were collected by gravity-driven gel filtration using a 10 mL pipette filled with Sephadex 25 matrix and equilibrated with buffer D containing, in addition, 200 mm KCl and 5 mm KH 2 PO 4 . The synthesis reactions were carried out at 30 °C with 2 mL Na + F 1 F 0 -ATP synthase from A. woodii M. Fritz and V. Mu ¨ ller 3426 FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS of proteoliposome solution from gel filtration and by adding 5mm ADP. The synthesis reaction was started by the addi- tion of 2 mL of valinomycin to induce a DY. Furthermore, when only a DpNa was to be applied, the vesicles were incubated in buffer D containing, in addition, 200 mm NaCl for 12 h at 4 °C. After this, the Na + -loaded vesicles were collected by gravity-driven gel filtration using a 10 mL pipette filled with Sephadex 25 matrix and equili- brated with buffer D containing, in addition, 200 mm KCl and 5 mm KH 2 PO 4 . The synthesis reactions were carried out at 30 °C with 2 mL of proteoliposome solution from gel filtration, and started by addition of 5 mm ADP. MALDI-TOF analysis Proteins were separated by SDS ⁄ PAGE, and all bands vis- ible by Coomassie staining were entirely cut out and subjec- ted to in-gel digestion protocols [42,43], which were adapted for use on a Microlab Star digestion robot (Bonaduz, Switzerland). Samples were reduced, alkylated and subsequently digested overnight using bovine trypsin (sequencing grade; Roche, Mannheim, Germany). The gel pieces were extracted, and the extracts were dried in a vacuum centrifuge and stored at ) 20 °C until use ⁄ analysis. MALDI-TOF MS experiments were performed on an Ultraflex TOF ⁄ TOF mass spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). The samples were prepared as described previously [44]. Spectra were externally calibrated with a Sequazyme Peptide Mass Standards Kit (Applied Biosystems, Foster City, CA, USA), and internally calibra- ted on a tryptic auto digestion peptide (m ⁄ z 2163.0564). The spectra were processed in flexanalysis version 2.2 (Bruker Daltonics) using the SNAP algorithm (signal-to- noise threshold 3; maximal number of peaks 150; quality factor threshold 80). Proteins were identified by mascot (Matrix Science, Boston, MA, USA) (peptide mass toler- ance 50 p.p.m.; maximum missed cleavages 1) using the NCBInr database (2 543 645 sequences; date 6 July 2005). Proteins with a score of 77 or higher were considered to be significant (P<0.05). Acknowledgements This work was supported by a grant from the Deut- sche Forschungsgemeinschaft (SFB472). The help of Dr O. Klimmek in preparing the proteoliposomes is gratefully acknowledged. References 1 Hilario E & Gogarten JP (1998) The prokaryote-to- eucaryote transition reflected in the evolution of the V ⁄ F ⁄ A-ATPase catalytic and proteolipid subunits. J Mol Evol 46, 703–715. 2 Cross RL & Mu ¨ ller V (2004) The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H + ⁄ ATP stoichiometry. FEBS Lett 576, 1–4. 3 Boekema EJ, van Breemen JF, Brisson A, Ubbink-Kok T, Konings WN & Lolkema JS (1999) Connecting stalks in V-type ATPase. Nature 401, 37–38. 4 Karrasch S & Walker JE (1999) Novel features in the structure of bovine ATP synthase. J Mol Biol 290, 379– 384. 5 Coskun U ¨ , Chaban YL, Lingl A, Mu ¨ ller V, Keegstra W, Boekema EJ & Gru ¨ ber G (2004) Structure and subunit arrangement of the A-type ATP synthase com- plex from the archaeon Methanococcus jannaschii visu- alized by electron microscopy. J Biol Chem 279, 38644–38648. 6 Forgac M (2000) Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H + - ATPases. J Exp Biol 203, 71–80. 7 Cross RL (1981) The mechanism and regulation of ATP synthesis by F 1 -ATPases. Annu Rev Biochem 50, 681–714. 8 Fillingame RH (1992) H + transport and coupling by the F 0 sector of the ATP synthase ) insights into the molecular mechanism of function. J Bioenerg Biomembr 24, 485–491. 9 Schneider E & Altendorf K (1987) Bacterial adenosine 5¢-triphosphate synthase (F 1 F 0 ): purification and recon- stitution of F 0 complexes and biochemical and func- tional characterization of their subunits. Microbiol Rev 51, 477–497. 10 Yoshida M, Muneyuki E & Hisabori T (2001) ATP syn- thase ) a marvellous rotary engine of the cell. Nat Rev Mol Cell Biol 2, 669–677. 11 Nakanishi-Matsui M & Futai M (2006) Stochastic pro- ton pumping ATPases: from single molecules to diverse physiological roles. IUBMB Life 58, 318–322. 12 Stock D, Leslie AG & Walker JE (1999) Molecular architecture of the rotary motor in ATP synthase. Science 286, 1700–1705. 13 Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto- Kihara A, Ueda I, Yanagida T, Wada Y & Futai M (1999) Mechanical rotation of the c subunit oligomer in ATP synthase (F 0 F 1 ): direct observation. Science 286, 1722–1724. 14 Hirata T, Nakamura N, Omote H, Wada Y & Futai M (2000) Regulation and reversibility of vacuolar H + - ATPase. J Biol Chem 275, 386–389. 15 Fillingame RH (1992) Subunit c of F 1 F 0 ATP synthase ) structure and role in transmembrane energy transduc- tion. Biochim Biophys Acta 1101, 240–243. 16 Seelert H, Dencher NA & Mu ¨ ller DJ (2003) Fourteen protomers compose the oligomer III of the proton-rotor in spinach chloroplast ATP synthase. J Mol Biol 333, 337–344. M. Fritz and V. Mu ¨ ller Na + F 1 F 0 -ATP synthase from A. woodii FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS 3427 17 Meier T, Ferguson SA, Cook GM, Dimroth P & Vonck J (2006) Structural investigations of the membrane- embedded rotor ring of the F-ATPase from Clostridium paradoxum. J Bacteriol 188, 7759–7764. 18 Meier T, Polzer P, Diederichs K, Welte W & Dimroth P (2005) Structure of the rotor ring of F-type Na + - ATPase from Ilyobacter tartaricus. Science 308, 659–662. 19 Pogoryelov D, Yu J, Meier T, Vonck J, Dimroth P & Mu ¨ ller DJ (2005) The c 15 ring of the Spirulina platensis F-ATP synthase: F 1 ⁄ F 0 symmetry mismatch is not obligatory. EMBO Rep 6, 1040–1044. 20 Mandel M, Moriyama Y, Hulmes JD, Pan Y-CE, Nel- son H & Nelson N (1988) cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of H + -ATPases. Proc Natl Acad Sci USA 85, 5521–5524. 21 Forgac M (1999) Structure and properties of the vacuo- lar H + -ATPases. J Biol Chem 274, 12951–12954. 22 Rahlfs S, Aufurth S & Mu ¨ ller V (1999) The Na + -F 1 F 0 - ATPase operon from Acetobacterium woodii. Operon structure and presence of multiple copies of atpE which encode proteolipids of 8- and 18-kDa. J Biol Chem 274, 33999–34004. 23 Mu ¨ ller V, Aufurth S & Rahlfs S (2001) The Na + cycle in Acetobacterium woodii: identification and characteri- zation of a Na + translocating F 1 F 0 -ATPase with a mixed oligomer of 8 and 16 kDa proteolipids. Biochim Biophys Acta 1505, 108–120. 24 Spruth M, Reidlinger J & Mu ¨ ller V (1995) Sodium ion dependence of inhibition of the Na + -translocating F 1 F 0 -ATPase from Acetobacterium woodii. Probing the site(s) involved in ion transport. Biochim Biophys Acta 1229, 96–102. 25 Reidlinger J & Mu ¨ ller V (1994) Purification of ATP synthase from Acetobacterium woodii and identification as a Na + -translocating F 1 F 0 -type enzyme. Eur J Bio- chem 223, 275–283. 26 Aufurth S, Scha ¨ gger H & Mu ¨ ller V (2000) Identification of subunits a, b, and c 1 from Acetobacterium woodii Na + -F 1 F 0 -ATPase. Subunits c 1 , c 2 , and c 3 constitute a mixed c-oligomer. J Biol Chem 275, 33297–33301. 27 Das A, Ivey DM & Ljungdahl LG (1997) Purification and reconstitution into proteoliposomes of the F 1 F 0 ATP synthase from the obligately anaerobic gram- positive bacterium Clostridium thermoautotrophicum. J Bacteriol 179, 1714–1720. 28 Ivey DM & Ljungdahl LG (1986) Purification and char- acterization of the F 1 -ATPase from Clostridium thermo- aceticum. J Bacteriol 165, 252–257. 29 Das A & Ljungdahl LG (1997) Composition and pri- mary structure of the F 1 F 0 ATP synthase from the obli- gately anaerobic bacterium Clostridium thermoaceticum. J Bacteriol 179, 3746–3755. 30 Reidlinger J (1994) Reinigung und Charakterisierung einer Na + -translozierenden F 1 F 0 -ATPase aus Acetobac- terium woodii. Dissertation, University of Go ¨ ttingen, Germany. 31 Dimroth P, Kaim G & Matthey U (2000) Crucial role of the membrane potential for ATP synthesis by F 1 F 0 ATP synthases. J Exp Biol 203, 51–59. 32 Kaim G & Dimroth P (1999) ATP synthesis by F-type ATP synthase is obligatorily dependent on the trans- membrane voltage. EMBO J 18, 4118–4127. 33 Dmitriev O, Deckershebestreit G & Altendorf K (1993) ATP synthesis energized by DpNa and DY in proteoliposomes containing the F 0 F 1 -ATPase from Propionigenium modestum. J Biol Chem 268, 14776– 14780. 34 Heise R, Mu ¨ ller V & Gottschalk G (1993) Acetogenesis and ATP synthesis in Acetobacterium woodii are coupled via a transmembrane primary sodium ion gradient. FEMS Microbiol Lett 112, 261–268. 35 Scha ¨ gger H & von Jagow G (1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 369–379. 36 Weber K & Osborne M (1969) The reliability of the molecular weight determination by dodecyl sulfate poly- acrylamide gel electrophoresis. J Biol Chem 244, 4406– 4412. 37 Blum H, Beier H & Gross HJ (1987) Improved silver staining of plant proteins, RNA, and DNA in poly- acrylamide gels. Electrophoresis 8, 93–98. 38 Heinonen JE & Lahti RJ (1981) A new and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophos- phatase. Anal Biochem 113, 313–317. 39 Heise R, Mu ¨ ller V & Gottschalk G (1992) Presence of a sodium-translocating ATPase in membrane vesicles of the homoacetogenic bacterium Acetobacterium woodii. Eur J Biochem 206, 553–557. 40 Kyhse-Andersen J (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellu- lose. J Biochem Biophys Methods 10, 203–209. 41 Knol J, Sjollema K & Poolman B (1998) Detergent- mediated reconstitution of membrane proteins. Biochem- istry 37 , 16410–16415. 42 Shevchenko A, Wilm M, Vorm O & Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850–858. 43 Rosenfeld J, Capdevielle J, Guillemot JC & Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after 1-dimensional or 2-dimensional gel elec- trophoresis. Anal Biochem 203, 173–179. 44 Burre J, Beckhaus T, Scha ¨ gger H, Corvey C, Hofmann S, Karas M, Zimmermann H & Volknandt W (2006) Analysis of the synaptic vesicle proteome using three gel-based protein separation techniques. Proteomics 6, 6250–6262. Na + F 1 F 0 -ATP synthase from A. woodii M. Fritz and V. Mu ¨ ller 3428 FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS . An intermediate step in the evolution of ATPases ) the F 1 F 0 -ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable. RL & Mu ¨ ller V (200 4) The evolution of A-, F-, and V-type ATP synthases and ATPases: reversals in function and changes in the H + ⁄ ATP stoichiometry. FEBS