an intermediate step in the evolution of atpases − the f1f0-atpase from acetobacterium woodii...

An intermediate step in the evolution of ATPases ) theF1F0-ATPase from Acetobacterium woodii contains F-typeand V-type rotor subunits and is capable of ATP synthesisMichael Fritz and Volker Muller

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

F1F0-ATP synthase present in bacteria, mitochondria

and chloroplasts, the A1A0-ATP synthase present in

archaea, and the V1V0-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 (F1 ⁄A1 ⁄V1) motor and a membrane-embedded,

ion gradient-driven motor (F0 ⁄A0 ⁄V0) [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 F1F0-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. Muller, 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: [email protected]

(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+ F1F0-ATP synthase solubilized by Triton

X-100 lacked some of the membrane-embedded motor subunits [Reidlinger

J & Muller 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

F1 subunits, the entire membrane-embedded motor with the stator subunits

a and b, and the heterooligomeric c ring, which contained the V1V0-like

subunit c1 and the F1F0-like subunits c2 and c3. 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 V0–F0

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 V1V0-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 V0 and F0, the rotor of V1V0-

ATPases has only half the number of ion-binding

sites. This is seen as the reason for the apparent inab-

ility of V1V0-ATPases to catalyze ATP synthesis

in vivo. Indeed, V1V0-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 Na+ F1F0-ATP synthase

from the anaerobic, acetogenic bacterium Acetobacterium

woodii is unique and encodes nine F1F0-like subunits

along with one gene encoding a V1V0-like subunit. The

atp operon has one homolog each of a gene encoding

the F1F0 subunits a, b, c, d, e, a, and b, but it has

three genes encoding differently sized c subunits [22].

Subunits c2 and c3 have a molecular mass of 8.18 kDa,

are identical at the amino acid level, and are similar to

c subunits from F1F0-ATP synthases. Like other F1F0-

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 c1 is similar to the c sub-

units of V1V0-ATPases and predicted to have four

transmembrane helices with only one ion-binding site.

A. woodii is, so far, the only organism known with

V1V0- and F1F0-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 c1 ⁄ c2 ⁄ 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+ F1F0-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 F0- and V0-type rotor subunits is able to

synthesize ATP.

Results

Purification of a complete Na+ F1F0-ATP synthase

from A. woodii

The Na+ F1F0-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) and1 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 c1, and the 10 kDa fragment to sub-

unit c2 ⁄ 3 (Fig. 1A). The 42 kDa fragment reacted with

antibobies against c2 ⁄ c3 (which also recognize c1) and

with antibodies against c1 (which do not recognize

c2 ⁄ 3; Fig. 1B). These data demonstrate that the 42 kDa

fragment represents the SDS-resistant, heterooligomeric

c ring of the Na+ F1F0-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, c1 and c2 ⁄ 3 in the membrane-embedded

rotor of the purified Na+ F1F0-ATP synthase from

A. woodii.

Table 1. Purification of the Na+ F1F0-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+ F1F0-ATP synthase from A. woodii M. Fritz and V. Muller

3422 FEBS Journal 274 (2007) 3421–3428 ª 2007 The Authors Journal compilation ª 2007 FEBS

Characterization of the Na+ F1F0-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

Km 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].

αβ

γ

c-oligomer

c2/3

ε

c1

δa/b

3 4 5 6 7

1 2A B

acitn

1tna

ai itnac 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+ F1F0-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 c1 antibodies. Lane 4: ATP synthase was incubated for 10 min at 80 �C, and blotted

against c1 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 c2 ⁄ 3 (which also detect subunit c1). Lane 7:

ATP synthase was denatured by boiling for 15 min, and blotted against c2 ⁄ 3 antibodies.

NaCl (mM)

AT

Pas

e ac

tivi

ty (

U/m

g)

A

LiCl (mM)

AT

Pas

e ac

tivi

ty (

U/m

g)

0 2 4 6 8 10 12 140

1

2

3

4

5

-3 -2 -1 0 1 2 3 4 5 6 7 8

0.10.20.30.40.5

0.60.70.80.9

1/A

TP

ase

acti

vity

0 2 4 6 8 10 120

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

0.10.20.30.40.50.60.70.80.91.0

1/A

TP

ase

acti

vity

B

Fig. 2. Ion dependence of ATP hydrolysis by

the Na+ F1F0-ATP synthase from A. woodii.

ATPase activity was measured at 30 �Cusing 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. Muller Na+ F1F0-ATP synthase from A. woodii


ATP synthesis catalyzed by the Na+ F1F0-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+ F1F0-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))1Æmin)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 c1 does not abolish ATP synthe-

sis by the Na+ F1F0-ATP synthase from A. woodii.

Discussion

The Na+ F1F0-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 F1F0-ATP synthases [27,28].

DCCD (µM)

AT

Pas

e ac

tivity

(%

)

0 25 50 75 1000

25

50

75

100

Fig. 3. Inhibition of the Na+ F1F0-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 mM (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 mM 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.



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 &

Muller [25] shows a faint band at about 17 kDa, which

appeared when the enzyme was autoclaved. At that

time, c1 was unknown, and an N-terminal sequence of

this fragment was not obtained. Therefore, it is not

only not excluded that it was the c1 subunit but likely,

as the c ring is rather stable, and the use of Triton X-

100 should not lead to removal of c1 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+ F1F0-ATP

synthase from Propionigenium modestum, DlNa+ as

well as DY but not DpNa were sufficient as driving

forces [31–33]. Most importantly, the Na+ F1F0-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 c1 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+ F1F0-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+ F1F0-ATP synthase from A. woo-

dii, the first containing V0- and F0-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 MgCI2, 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 01 02 03 04 05 06 07 08 09 001 011 021 0310

01

02

03

04

05

06

07

08

09

mit e )s(

mo

l AT

P /

mo

l en

zym

e

Fig. 5. ATP synthesis by Na+ F1F0-ATP synthase-containing proteo-

liposomes. The artificial driving forces DlNa+ (j), DY (m) or DpNa

(r) were applied to the proteoliposomes as described in Experi-

mental procedures. In one assay (d), a DlNa+ was applied but

ADP was omitted.



50 mm Tris ⁄HCl (pH 7.5), 10 mm MgCl2, 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,

Norten-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 MgSO4,

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 mLÆmin)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 Schagger & 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 MgCl2). 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 MgCI2when carried out at pH 7.5, and 50 mm MgCl2 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 lL of H2O2 for 2 min

before exposure to WICORex film (Typon Imaging AG,

Burgdorf, Switzerland).

Reconstitution of ATPase into proteoliposomes

A suspension of 60 mgÆmL)1l-a-phosphatidylcholine type

II-S (Sigma-Aldrich Chemie GmbH) in buffer D (100 mm

Tris, 100 mm maleic acid, 20 mm NaCl, 5 mm MgCl2,

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, Munchen, Germany) for 12 h at 4 �C. Theproteoliposomes 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

KH2PO4. The synthesis reactions were carried out at 30 �Cwith 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 NaHAsO4, 4 mm

MgSO4, 20 mm 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 KH2PO4.

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 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 KH2PO4. 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.

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an intermediate step in the evolution of atpases − the f1f0-atpase from acetobacterium woodii...

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