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:
(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.
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