Tài liệu Báo cáo khoa học: An intermediate step in the evolution of ATPases – a hybrid F0–V0 rotor in a bacterial Na+ F1F0 ATP synthase pdf

An intermediate step in the evolution of ATPases – a
hybrid F
0
–V
0
rotor in a bacterial Na
+
F
1
F
0
ATP synthase
Michael Fritz
1,
*, Adriana L. Klyszejko
2,
*, Nina Morgner
3,
*, Janet Vonck
4
, Bernd Brutschy
3
,
Daniel J. Muller
2
, Thomas Meier
4
and Volker Mu
¨
ller
1

1 Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt ⁄ Main, Germany
2 BioTechnological Center, University of Technology Dresden, Germany
3 Microkinetic, Clusterchemistry, Mass- and Laserspectroscopy, Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe
University Frankfurt ⁄ Main, Germany
4 Max-Planck-Institute of Biophysics, Frankfurt, Germany
ATP synthases are key elements in bioenergetics [1]. In
bacteria, ATP synthesis is catalyzed by F
1
F
0
ATP syn-
thase, which uses the electrochemical H
+
(or in some
species Na
+
) potential to drive the synthesis of ATP
[2]. ATP synthases are rotary machines that work as a
pair of coupled motors, a chemically driven motor (F
1
)
and a membrane-embedded, ion gradient-driven motor
(F
0
) [3]. The membrane-embedded motor comprises a
stator and a rotor. The stator is formed by subunits a
and b
2
, and the rotor is formed from 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 membrane
[4–6].
Subunit c of the F
1
F
0
ATP synthases has a molecular
mass of approximately 8 kDa, and folds in the mem-
brane like a hairpin, with two transmembrane helices
connected by a cytoplasmic loop [7]. Each monomer
contains an ion-binding site (H
+
or Na
+
) [8,9]. Recent
studies have demonstrated that the c ring stoichiometry
in different organisms ranges between 10 and 15 mono-
mers (see Discussion). Assuming that each subunit
takes up one ion, each c ring revolution induces
the synthesis of three molecules of ATP. This gives a
Keywords
Acetobacterium; acetogen; ATP-synthase;
c ring; F
0
-V
0
hybrid rotor
Correspondence
V. Mu

¨
ller, Molecular Microbiology &
Bioenergetics, Institute of Molecular
Biosciences, Johann Wolfgang Goethe
University, Max-von-Laue-Straße 9,
D-60438 Frankfurt, Germany
Fax: +49 69 79829306
Tel: +49 69 79829507
E-mail:
*These authors contributed equally to this
study
(Received 18 December 2007, revised 15
February 2008, accepted 22 February 2008)
doi:10.1111/j.1742-4658.2008.06354.x
The Na
+
F
1
F
0
ATP synthase operon of the anaerobic, acetogenic bacte-
rium Acetobacterium woodii is unique because it encodes two types of
c subunits, two identical 8 kDa bacterial F
0
-like c subunits (c
2
and c
3
),
with two transmembrane helices, and a 18 kDa eukaryal V

0
-like (c
1
)
c subunit, with four transmembrane helices but only one binding site. To
determine whether both types of rotor subunits are present in the same
c ring, we have isolated and studied the composition of the c ring. High-
resolution atomic force microscopy of 2D crystals revealed 11 domains,
each corresponding to two transmembrane helices. A projection map
derived from electron micrographs, calculated to 5 A
˚
resolution, revealed
that each c ring contains two concentric, slightly staggered, packed rings,
each composed of 11 densities, representing 22 transmembrane helices.
The inner and outer diameters of the rings, measured at the density bor-
ders, are approximately 17 and 50 A
˚
. Mass determination by laser-
induced liquid beam ion desorption provided evidence that the c rings
contain both types of c subunits. The stoichiometry for c
2
⁄ c
3
: c
1
was
9 : 1. Furthermore, this stoichiometry was independent of the carbon
source of the growth medium. These analyses clearly demonstrate, for the
first time, an F
0

–V
0
hybrid motor in an ATP synthase.
Abbreviations
AFM, atomic force spectroscopy; LILBID-MS, laser-induced liquid beam ion desorption mass spectroscopy.
FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 1999
theoretical H
+
(Na
+
) ⁄ ATP ratio of 3.5–5, which is the
value required for ATP synthesis given a transmem-
brane electrochemical ion gradient (Dl
H
+
⁄ Na
+
)of
around )200 mV and a phosphorylation potential
(DG
p
)of60kJÆmol
)1
according to the equation:
DG
p
¼ nFDl
Na
þ
where n is the number of translocated ions and F is

the Faraday constant.
The c subunit of the eukaryal V
1
V
0
ATPases present
in organelles arose by duplication and fusion of the
bacterial c subunit, giving rise to a protein of approxi-
mately 16 kDa with four transmembrane helices that
form two covalently linked hairpins in the membrane
[10]. Importantly, the ion-binding site is not conserved
in hairpin one. If one assumes the same number of
transmembrane helices in V
0
and F
0
, the rotor of
eukaryal V
1
V
0
ATPases has only half the number
of ion-binding sites compared to F
1
F
0
ATP syntheses.
This low H
+
(Na

+
) ⁄ ATP ratio is apparently the reason
for the inability of eukaryal V
1
V
0
ATPases to catalyze
ATP synthesis in vivo [11,12]. On the other hand, this
low ratio strongly favors the generation of steep ion
gradients driven by ATP hydrolysis, and indeed the
cellular function of V
1
V
0
ATPases is to energize endo-
cytoplasmatic membranes in the eukaryotic cell [13].
The Na
+
F
1
F
0
ATP synthase operon from the
anaerobic, acetogenic bacterium Acetobacterium woodii
differs from all other F
1
F
0
ATP synthases by the
presence of one V

0
subunit c gene (atpE
1
) and two
genes (atpE
2
⁄ atpE
3
) encoding identical F
0
c subunits.
The gene atpE
1
encodes an 18 kDa protein with two
predicted hairpins, and, like its eukaryotic counter-
part, is missing one ion binding site (in hairpin two).
The genes atpE
2
and atpE
3
encode two identical
8 kDa subunits with one ion-binding site each. The
three genes are encoded in the same operon and their
products are present in the same enzyme preparation
[14–16]. Here, we have addressed the question
whether both types of c subunit assemble into one
ring, and whether the c ring composition changes
with the growth conditions. We present data that
unequivocally demonstrate a V
0

–F
0
hybrid rotor, the
first found in nature.
Results
Purification of the c ring from A. woodii
The c rings of the F
1
F
0
ATP synthase from A. woodii
were isolated according to the method developed for
Ilyobacter tartaricus [17]. The purified c ring migrated
as a single band on SDS–PAGE, with an apparent
molecular mass of 57–59 kDa, depending on the acryl-
amide concentration used (Fig. 1A). Western blotting
analyses of the intact as well as the denatured c rings
revealed the presence of subunit c
1
as well as c
2 ⁄ 3
in
the isolated c ring. Densitometric analysis of the
c monomers visualized by silver staining (Fig. 1)
revealed a stoichiometry of approximately 1 : 8.6 for
c
1
: c
2 ⁄ 3
. As observed before for the c rings of entire

Na
+
F
1
F
0
ATP synthases [18,19] as well as isolated
c rings [20], the A. woodii c ring was highly stable and
did not dissociate by boiling in 20 mm Tris ⁄ HCl, 5%
SDS (pH 8.0) for up to 30 min, but did dissociate by
autoclaving (120 °C) in the presence of 5% SDS for
5 min or in the presence of 40 mm trichloroacetic acid.
The c rings from different preparations always showed
the same migration behaviour in SDS–PAGE, and the
isolated c ring migrated to a position identical to that
of the c ring present in native ATP synthase solubi-
lized in the same detergent (Fig. 1).
Fig. 1. Isolation and subunit composition of
the c rings from the Na
+
F
1
F
0
ATP synthase
from A. woodii. Samples of isolated enzyme
(lane 1) and isolated c rings (lanes 3, 4 and
7) were boiled at 80 °C for 20 min and
applied to a 10.0% (lanes 1–3) or 13.5%
(lanes 4–9) polyacrylamide gel. The c ring

was disintegrated by treatment with
trichloroacetic acid (lanes 6, 8 and 9), and
individual subunits were detected by silver
staining (lane 6) or immunoblotting using an
antibody against subunit c
1
(lanes 7 and 8)
or subunit c
2 ⁄ 3
(lane 9). The antibody
against c
2 ⁄ 3
also reacts with c
1
.
Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase M. Fritz et al.
2000 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS
High-resolution AFM imaging
c rings reconstituted into 1-palmitoyl-2-oleoyl-sn-glyce-
ro-3-phosphocholine at lipid-to-protein ratios of 0.5
and 1.0 assembled into 2D crystals that were exam-
ined by high-resolution atomic force microscopy

(AFM). AFM topographs revealed crystalline and
paracrystalline membrane patches surrounded by the
lipid bilayer (Fig. 2). c rings with an outer diameter
of 5.8 ± 0.4 nm (n = 125) were surrounded by smal-
ler c rings of diameter 5.4 ± 0.4 nm (n = 150). In
agreement with previous measurements on c rings
from other F
1
F
0
ATP synthases [21–23], the occur-
rence of two diameters indicated that the reconsti-
tuted c rings had an ‘upside-down’ orientation in the
membrane and that we imaged both ring surfaces. In
further agreement with previous measurements, the
smaller c rings exhibited central protrusions that were
shown to represent lipid headgroups [24] and to
reflect the extracellular side of the ring [8,24].
Whereas the lipid bilayer exhibited a height of
4.5 ± 0.5 nm (n = 10), the proteins protruded
7.7 ± 0.5 nm (n = 10) from the supporting mica sur-
face. At a lateral resolution of approximately 1 nm,
the subunits of individual ring-shaped c oligomers
became visible (Fig. 2A,B). Cross-correlation averages
applied to further enhance common structural fea-
tures showed different assemblies of the c rings, each
being composed of 11 equally sized domains
(Fig. 2C,D). Similarly, the reference-free averages
generated by translational and rotational alignment
of single c rings showed the same stoichiometry

(Fig. 2D,E). From both the raw data and averages of
c rings, it was clear that they were composed of 11
domains each, corresponding to 22 transmembrane
helices.
Structural investigations of the A. woodii c ring
The same 2D crystals of the A. woodii c ring used for
AFM were also used for structural investigations by
electron microscopy. The A. woodii c ring sample con-
sisted of vesicles containing crystalline areas with
dimensions up to 0.5 lm. The 2D crystals are of
plane group p22
1
2
1
(Fig. 3). The unit cell has dimen-
sions of 100 · 108 A
˚
and contains four c rings, each
with 11 densities. The crystals are tightly packed, and
each ring is in contact with at least four neighbouring
c rings. The projection map, calculated to 5 A
˚
resolu-
tion (Fig. 3), shows that each c ring comprises two
concentric, slightly staggered, packed rings, each com-
posed of 11 densities. Whereas the inner ring of den-
sities is tightly packed, the outer one is more loosely
arranged, and the densities correspond to the N- and
C-terminal helices of the c ring, respectively. The
C-terminal helices show a clear handedness, and two

of the rings face in the opposite direction in the
membrane to the other two, forming the same pattern
as in the AFM surface representation of Fig. 2A. By
comparison with the 3D structure of the I. tartaricus
c ring [24], the black rings represent the view from
the cytoplasm (open rings in AFM) and the red ones
the view from the extracellular side (smaller, closed
rings in Fig. 3). The inner and outer diameters of the
rings, measured at the density borders, are approxi-
mately 17 and 50 A
˚
. However, the resolution
obtained did not enable us to distinguish c
1
from
c
2 ⁄ 3
.
Subunit composition of the c ring from A. woodii
The above structural analyses clearly assigned 22
transmembrane helices to the c ring of A. woodii.To
unravel the c
1
and c
2 ⁄ 3
subunit composition of the
potential hetero-oligomeric ring, we used laser-induced
Fig. 2. High-resolution AFM topographs of reconstituted c rings.
(A, B) Crystalline assemblies of c rings. Although the number of
subunits forming the rings can be seen, the signal-to-noise ratio

may be further enhanced by calculating their averages. (C, D)
Nonsymmetrized correlation averages of both crystalline assem-
blies reveal 11 masses forming the c ring. Each ring is neighbored
by rings exposing either their wide or narrow ends. (E, F)
Reference-free correlation averages for the two assemblies
revealed 11 masses forming the wide and narrow ends of the
rings. AFM topographs were recorded in dialysis buffer and exhib-
ited gray levels correspond to a vertical scale of 3 nm.
M. Fritz et al. Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase
FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2001
liquid beam ion desorption mass spectroscopy (LIL-
BID-MS), a recently established method to determine
subunit stoichiometries in membrane protein com-
plexes such as the cytochrome oxidase from Para-
coccus denitrificans [25] and c rings from various
organisms [26]. Figure 4 shows MS measurements of
the A. woodii c ring purified from cells grown on
fructose, taken under various desorption conditions.
The mass spectrum in Fig. 4A shows an m ⁄ z distribu-
tion of the complex with charges varying from 1 to
5. Individual peaks broadened towards higher masses,
due to detergent and water molecules that stayed

attached to the ring under the ultra-soft desorption
process [27]. The overall mass of the c ring was deter-
mined to be 93.5 ± 0.1 kDa. Harsher desorption con-
ditions, achieved by increasing the laser intensity, led
to the detachment of detergent and water molecules.
Moreover, additional energy was transferred into the
system, and the c rings (partly) dissociated into single
subunits and subcomplexes. The mass spectrum in
Fig. 4B was used to determine the c
1
to c
2 ⁄ 3
stoichio-
metry. The peak distribution contains two series of
subcomplexes. One series corresponds to subcomplex-
es containing only c
2 ⁄ 3
units of the form (c
2 ⁄ 3
)
n
,
where n = 1–5, the other is built up from c
1
and c
2 ⁄ 3
units in the form c
1
(c
2 ⁄ 3

)
n
, where n = 0–9. No sub-
complexes that contain two or more c
1
subunits were
detectable. The mass of the c
1
monomer was deter-
mined to be 18.7 ± 0.1 kDa, and that for the c
2 ⁄ 3
monomer was 8.3 ± 0.1 kDa. Comparison of the
spectra of the complete ring and the fragments
revealed a stoichiometry for c
1
: c
2 ⁄ 3
of 1 : 9, leading
to a mass of 93.4 kDa and hence 22 transmembrane
helices for the c ring.
Fig. 3. Electron microscopy of 2D crystals from A. woodii c rings. Projection map of 13 merged images at 5 A
˚
resolution. One unit cell
of plane group p22
1
2
1
with its symmetry elements (two-fold rotation axes and screw axes) is indicated. The unit cell measures
100.3 · 108.5 A
˚

and contains four c rings.
Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase M. Fritz et al.
2002 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS
Does the subunit composition of the c ring from
A. woodii vary with the carbon source?
As outlined above, the unique presence of a eukaryal
V
0
-like c subunit in a bacterial ATP synthase raised
the question whether the stoichiometry of V
0
:F
0
-like
subunits may be flexible and thus a mechanism to
change the action of the enzyme from ATP synthase
to ATPase. To address potential variation in c ring
subunit composition depending on the growth condi-
tions, cells were grown under autotrophic conditions
(ATP synthase required) or heterotrophic fermenting
conditions (ion-pumping ATPase function required),
and c rings were purified and subjected to LILBID

analysis. The c rings of cells grown on fructose
(20 mm), methanol (60 mm) or betaine (40 mm) (all
heterotrophic) and on formate (80 mm) (autotrophic)
showed an identical stoichiometry for c
1
: c
2 ⁄ 3
of 1 : 9,
thus excluding the possibility of carbon source-depen-
dent variation.
Discussion
A critical and long-standing question in (bacterial) bio-
energetics is whether the ratio of translocated ions to
ATP for a given ATP synthase is a fixed value. This
value depends on the one hand on the number of cata-
lytic sites, which seems to be invariable as all the
enzymes analyzed so far have a a
3
b
3
(F
1
F
0
)orA
3
B
3
(A
1

A
0
,V
1
V
0
) stoichiometry [28]. The uncertainty lay
in the number of ion-translocating subunits in the
membrane-embedded rotor. Recently, the atomic struc-
ture of a c ring from the F
1
F
0
ATP synthase from
I tartaricus was solved and revealed 11 monomers [8].
Interestingly, on the basis of structural, biochemical
and genetic studies, the c ring stoichiometry in F
1
F
0
ATP synthases is apparently variable among species.
Ten monomers are found in c rings from the F
1
F
0
ATP synthases from yeast, Escherichia coli or Bacillus
PS3 [29–31], undecameric rings were found in I. tar-
taricus [22], Propionigenium modestum [17] and Clos-
tridium paradoxum [32] Na
+

F
1
F
0
ATP synthases, a
tridecameric c ring was found in the thermoalkaliphilic
Bacillus sp. strain TA2.A1 [26], 14 subunits were found
in the ATP synthase from spinach chloroplasts [21],
and 13–15-meric c rings were identified in various
cyanobacterial ATP synthases [23,33]. Less is known
about the c subunit stoichiometries in the evolution-
arily related V
1
V
0
ATPases, with only one structure
solved, from Enterococcus hirae, which revealed 10
monomers [9].
The Na
+
F
1
F
0
ATP synthase operon of A. woodii is
so far the only F
1
F
0
ATP synthase operon that has

been found to encode F
0
and V
0
c subunit genes [14].
The genes have been found to be expressed [15] and
the subunits have been found in the purified enzyme
[16]. However, a critical question that was solved here
was whether both subunits are part of one rotor or
whether there are two populations of enzymes, one
having only the F
0
-like c subunit and the other only
Fig. 4. Mass spectra of the c ring taken
under various laser desorption conditions.
Under ultrasoft desorption conditions (A),
the c ring is detected unfragmented with a
charge distribution of one to four as indi-
cated by red vertical bars. The broadening
of the peaks towards higher masses is due
to the attachment of detergent and water
molecules. Under harsh desorption condi-
tions (B), the c ring is fragmented, which
leads to two series of subcomplexes con-
taining only c
2 ⁄ 3
subunits (indicated by blue
vertical bars) or one c
1
subunit and 1–9 c

2 ⁄ 3
subunits (indicated by red vertical bars). No
subcomplex contains more than one c
1
sub-
unit (theoretical masses of a c
1
series are
indicated by green vertical bars). These find-
ings and comparison of the two spectra
reveal a c
1
: c
2 ⁄ 3
stoichiometry of 1 : 9 for
the A. woodii c ring.
M. Fritz et al. Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase
FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2003
the V
0
-like c subunit. Here, we have unequivocally
excluded the latter possibility. The LILBID analyses

showed no peaks that contained two or more c
1
sub-
units, excluding the existence of more than one
c
1
unit per ring and of course rings formed from c
1
only. No mass was detectable corresponding to a ring
made by c
2 ⁄ 3
subunits only or more than nine c
2 ⁄ 3
subunits.
The stoichiometry of the subunits in the c ring of
A. woodii was determined to be 1 : 9 (c
1
: c
2 ⁄ 3
), with a
total of 22 transmembrane helices. This value is identi-
cal to the value obtained for the other Na
+
F
1
F
0
ATP
synthases. Additionally, the size of the A. woodii
c rings (approximately 58 A

˚
by AFM, approximately
50 A
˚
by electron microscopy) is comparable to those
from I. tartaricus, P. modestum and C. paradoxum
[17,32]. However, the major difference is that sub-
unit c
1
not only lacks the conserved Na
+
binding site
but also the essential negative charge (glutamate or
aspartate) in transmembrane helix four as part of the
ion-binding site. Therefore, the c ring of A. woodii has
only 10 membrane-buried negative charges that are
essential for binding the ion and also for the rotational
mechanism of the ring. The c ring of I. tartaricus has
11 negative charges that are equally distributed along
the horizontal axis of the rotor [8]. A positive charge
on the stator attracts one of the negative charges on
the ring and thus keeps the ring in place [34–36]. How-
ever, the system is not stiff but instead the ring idles in
front of the positive charge. This site is accessible to
the outside, and ions (H
+
,Na
+
) flow from the outside
to the binding site (driven by the electrical potential

across the membrane) and occasionally bind to and
thus compensate the negative charge on the rotor. The
freed positive charge on the stator attracts the next
negative charge on the rotor, thus leading to rotation
of the ring.
As most of the c rings investigated so far have a
number of monomers that cannot be divided by three,
this implies that the translocated ion to ATP ratio is
not an integer. It has been suggested that an elastic
power transmission between F
1
and F
0
is important
for operation of the enzyme under symmetry mismatch
conditions [37]. In the enzyme from A. woodii, rotation
of the c ring over each phase of 120° is coupled to at
least two different numbers of ions. Obviously, the
force that has to be applied to overcome the spatial
difference between three hairpins (c
2 ⁄ 3
)
–c
1,N-term
)
c
1,C-term
neutral
– c
2 ⁄ 3

)
) is more than that required to dis-
locate just one hairpin. How this is achieved is
unknown but is a challenging task for future studies.
A variable number of identical c subunits in the ring
was suggested to be a regulatory mechanism in E. coli
[38], but this could not be confirmed experimentally in
spinach chloroplast ATP synthase [39] or in the pres-
ent study using the Na
+
F
1
F
0
ATP synthase from
A. woodii. Rather, the stoichiometry seems to be fixed
within a certain species and is determined by the geom-
etry of the individual subunit [40–42]. The determined
stoichiometry of 1 : 9 (c
1
: c
2 ⁄ 3
) gives an Na
+
⁄ ATP
stoichiometry of 3.3, compared to 3.6 for the enzymes
from P. modestum and I. tartaricus. It makes the
enzyme from A. woodii a slightly better ATP-driven
ion pump than an ATP synthase; whether this mar-
ginal difference is of physiological relevance is ques-

tionable but remains to be addressed experimentally.
The electrochemical ion potential across the cyto-
plasmic membrane of A. woodii has not yet been
determined due to high nonspecific binding of the
radioactive probes, but it is reasonable to assume that
is similar to that in other bacteria, i.e. in the range of
)180 to )200 mV. Therefore, the enzyme will work as
an ATP synthase under physiological conditions. Its
capability to synthesize ATP despite the presence of
the V
0
-like c subunit has been demonstrated very
recently in a proteoliposome system [16].
Experimental procedures
Growth of cells and isolation of membranes
A. woodii (DSM 1030) was grown in 20-1iter fermentors to
mid-exponential growth phase as described previously [43].
Fructose (20 mm), betaine (40 mm), methanol (60 mm)or
formate (80 mm) were used as carbon and energy sources.
The NaCl concentration was 20 m m, unless otherwise
stated. The ATP synthase was purified to apparent homo-
geneity by solubilization with 1% dodecyl- b -d-maltoside
followed by chromatography as described previously [16].
All preparations were routinely analyzed by SDS–PAGE
using the buffer system described by Scha
¨
gger and von
Jagow [44]. Polypeptides were visualized by staining with
Coomassie brilliant blue [45] or silver [46]. The protein
concentration of samples was determined according to the

Lowry method [47], with BSA as a standard.
Purification of c rings from A. woodii F
1
F
0
ATP
synthases
The ATP synthase from A. woodii was purified as previ-
ously described [16], and the c ring was purified as
described previously [17] with some modifications. The
purified enzyme was incubated with 1.5% N-lauroylsarco-
sine at 68 °C for 20 min. After cooling to 20 °C,
(NH
4
)
2
SO
4
was added to a saturation of 68%. After 2 h of
incubation at 20 °C, the precipitated protein was removed
Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase M. Fritz et al.
2004 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS

in a first step by filtration (filter paper, 2.6 lm pore size,
Schleicher & Schuell, Dassel, Germany) followed by filtra-
tion through a 0.2 lm filter (4 mm syringe filters, Nalgene,
Rochester, NY, USA). The filtrate was dialyzed overnight
at 4 °C against 10 mm Tris ⁄ HCl, 200 mm NaCl, pH 8.0,
followed by addition of b-octylglycoside (Biomol, Mu
¨
n-
chen, Germany) to a final concentration of 1.5%. To fur-
ther concentrate the c rings and to remove excess salt, the
sample was loaded onto an Amicon Ultra-4 tube
(30 000 Da molecular mass cut-off; Amicon, Hanover,
Germany) and concentrated to about 2–4 mgÆmL
)1
.
Western blot analysis
After separation by SDS–PAGE, the ATP synthase subun-
its were blotted onto a nitrocellulose membrane as
described previously [48]. Western blot enhanced chemilu-
minescence (ECL) detection reagents were either purchased
from Perkin Elmer Life Sciences (Boston, MA, USA) or
produced in our laboratory. Blot membranes were incu-
bated in a mixture of 4 mL of solution A (0.1 m Tris ⁄ HCl,
pH 6.8, 50 mg luminol in a total volume of 200 mL),
400 lL of solution B (11 mg p-hydroxycoumaric acid in
10 mL dimethylsulfoxide) and 1.2 lLofH
2
O
2
for 2 min

before exposure to WICORex film (Typon Imaging AG,
Burgdorf, Switzerland).
Two-dimensional crystallization of the c ring
For crystallization in 2D according to the method described
previously [22], a sample of c ring (2 mgÆmL
)1
) was
mixed with 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine
(Avanti Polar Lipids Inc., Alabaster, AL, USA) at lipid-to-
protein ratios of 0.5, 1.0 and 1.5 w ⁄ w. The mixture was
dialyzed in 50 lL buttons (Hampton Research, Aliso Viejo,
CA, USA) against 50 mL of 10 mm Tris ⁄ HCl (pH 8.0),
200 mm NaCl and 3 mm NaN
3
for 24 h at 25 °C and
another 24 h at 37 ° C. The 2D crystals were stored at 4 °C
until further analysis.
Atomic force microscopy
An atomic force microscope (Nanoscope IIIa; DI-Veeco,
Santa Barbara, CA, USA), equipped with a 100 lm X–Y
piezo scanner, was optimized for observing single molecules
in the buffer solution. The 100 lm-long silicon nitride
AFM cantilevers (Olympus, Tokyo, Japan) had nominal
spring constants of 0.9 N ⁄ m. To adsorb the protein mem-
branes, 20 mL of the sample buffer (approximately
10 lgÆmL
)1
reconstituted c rings, 10 mm Tris ⁄ HCl, 200 mm
NaCl, 0.02% NaN
3

, 10% glycerol, pH 7.8) was placed onto
freshly cleaved mica for about 30 min. Then the sample
was rinsed with dialysis buffer to remove weakly attached
material. Contact-mode AFM topographs were recorded in
dialysis buffer at 25 °C, with a loading force of approxi-
mately 100 pN and a line frequency of 4–6 Hz. No differ-
ences between topographs recorded in the trace and retrace
directions were observed, indicating that the scanning pro-
cess did not influence the appearance of the sample. For
image processing, individual particles of the AFM topo-
graphs were subjected to reference-free alignment and
averaging using the SPIDER image processing system
(Wadsworth Labs, New York, NY, USA). Correlation
averages were calculated using the SEMPER image process-
ing system (Synoptics Ltd, Cambridge, UK). To assess the
rotor symmetry, the rotational power spectra of reference-
free averages and of single rotors were calculated.
Electron microscopy and image processing
Two-dimensional crystal samples were prepared in 4.5%
w ⁄ v trehalose on molybdenum grids (Pacific Grid-Tech,
San Diego, CA, USA) by the back-injection method. Grids
were examined in a JEOL 3000 SFF helium-cooled electron
microscope (JEOL Ltd., Tokyo, Japan) at 4 K at an accel-
erating voltage of 300 kV. Images were recorded by a spot-
scanning procedure, using 24 spots by 30 spots per image
on Kodak SO-163 film (Kodak, Stuttgart, Germany) at a
magnification of 53 000 · and with an electron dose of 20–
30 electrons ⁄ A
˚
2

. The films were developed for 12 min in
full-strength Kodak D-19 developer. Images selected by
optical diffraction were digitized on a Zeiss SCAI scanner
(Zeiss, Jena, Germany) using a pixel size of 7 lm, corre-
sponding to 1.3 A
˚
on the specimen. Images were processed
using MRC [49] and CCP4 [50]. Data were merged to a
resolution of 5 A
˚
.
LILBID
LILBID-MS [25,27] works with liquid sample targets.
Therefore, microdroplets of the sample solution (50 lm
diameter, volume 65 pL) were introduced into a vacuum
using an on-demand droplet generator at a frequency of
10 Hz. The droplets are irradiated one by one by IR laser
pulses, tuned to the absorption maximum of water at
around 2.8 lm. The laser energy is transferred into the
stretching vibrations of water, leading to a supercritical
state of the liquid. The droplets explode and the charged
biomolecules in the solution are set free. Those that escape
the following charge neutralization are accelerated and
mass-analyzed in a time-of-flight reflectron mass spectrome-
ter constructed in our laboratory.
Acknowledgements
This work was supported by the Deutsche Forschungs-
gemeinschaft (SFB 472 to VM and Werner Ku
¨
hlbrandt,

M. Fritz et al. Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase
FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2005
SFB 579 to BB, Cluster of Excellence ‘Macromolecular
Complexes’ Project EXC 115 to TM), the Fonds der
chemischen Industrie (to BB), and the EU (grant
NEST2004 PathSYS29084 to DM). We thank Deryck
Mills for assistance with electron microscopy and
Werner Ku
¨
hlbrandt (MPi of Biophysics, Frankfurt,
Germany) for comments on the manuscript.
References
1 Boyer PD (1997) The ATP synthase – a splendid molec-
ular machine. Annu Rev Biochem 66, 717–749.
2 Walker JE (1994) The regulation of catalysis in ATP
synthase. Curr Opin Struct Biol 4, 912–918.
3 Yoshida M, Muneyuki E & Hisabori T (2001) ATP
synthase – a marvellous rotary engine of the cell. Nat
Rev Mol Cell Biol 2, 669–677.
4 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.
5 Hirata T, Nakamura N, Omote H, Wada Y & Futai M
(2000) Regulation and reversibility of vacuolar H
+
-
ATPase. J Biol Chem 275, 386–389.
6 Tsunoda SP, Aggeler R, Yoshida M & Capaldi RA
(2001) Rotation of the c subunit oligomer in fully func-
tional F
1
F
0
ATP synthase. Proc Natl Acad Sci USA 98,
898–902.
7 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.
8 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.
9 Murata T, Yamato I, Kakinuma Y, Leslie AG & Walker
JE (2005) Structure of the rotor of the V-type Na
+
-AT-
Pase from Enterococcus hirae. Science 308, 654–659.
10 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.
11 Nelson N & Taiz L (1989) The evolution of H
+
-ATPas-
es. Trends Biochem Sci 14, 113–116.
12 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.
13 Forgac M (1999) Structure and properties of the vacuo-
lar H
+
-ATPases. J Biol Chem 274, 12951–12954.
14 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.
15 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 c1, c2, and c3 constitute a
mixed c-oligomer. J Biol Chem 275, 33297–33301.
16 Fritz M & Mu
¨

ller V (2007) An intermediate step in the
evolution of ATPases – the F
1
F
0
-ATPase from Aceto-
bacterium woodii contains F-type and V-type rotor
subunits and is capable of ATP synthesis. FEBS J 274,
3421–3428.
17 Meier T, Matthey U, von Ballmoos C, Vonck J, Krug
von Nidda T, Ku
¨
hlbrandt W & Dimroth P (2003) Evi-
dence for structural integrity in the undecameric c-rings
isolated from sodium ATP synthases. J Mol Biol 325,
389–397.
18 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.
19 Laubinger W & Dimroth P (1989) The sodium translo-
cating adenosintriphosphatase of Propionigenium mode-

stum pumps protons at low sodium ion concentrations.
Biochemistry 28, 7194–7198.
20 Meier T & Dimroth P (2002) Intersubunit bridging by
Na
+
ions as a rationale for the unusual stability of the
c-rings of Na
+
-translocating F
1
F
0
ATP synthases.
EMBO Rep 3, 1094–1098.
21 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.
22 Stahlberg H, Mu
¨
ller DJ, Suda K, Fotiadis D, Engel A,
Meier T, Matthey U & Dimroth P (2001) Bacterial
Na
+
-ATP synthase has an undecameric rotor. EMBO
Rep 2, 229–233.
23 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.
24 Vonck J, von Nidda TK, Meier T, Matthey U, Mills
DJ, Ku
¨
hlbrandt W & Dimroth P (2002) Molecular
architecture of the undecameric rotor of a bacterial
Na
+
-ATP synthase. J Mol Biol 321, 307–316.
25 Morgner N, Kleinschroth T, Barth HD, Ludwig B &
Brutschy B (2007) A novel approach to analyze mem-
brane proteins by laser mass spectrometry: from protein
subunits to the integral complex. J Am Soc Mass Spec-
trom 18, 1429–1438.
26 Meier T, Morgner N, Matthies D, Pogoryelov D,
Keis S, Cook GM, Dimroth P & Brutschy B (2007)
A tridecameric c ring of the adenosine triphosphate
(ATP) synthase from the thermoalkaliphilic Bacillus
sp. strain TA2.A1 facilitates ATP synthesis at low
Hybrid V
0

–F
0
rotor in a F
1
F
0
ATP synthase M. Fritz et al.
2006 FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS
electrochemical proton potential. Mol Microbiol 65,
1181–1192.
27 Morgner N, Barth HD & Brutschy B (2006) A new way
to detect noncovalently bonded complexes of biomole-
cules from liquid micro-droplets by laser mass spec-
trometry. Aust J Chem 59, 109–114.
28 Gru
¨
ber G, Wieczorek H, Harvey WR & Mu
¨
ller V
(2001) Structure–function relationships of A-, F- and
V-ATPases. J Exp Biol 204, 2597–2605.
29 Stock D, Leslie AG & Walker JE (1999) Molecular
architecture of the rotary motor in ATP synthase.
Science 286, 1700–1705.
30 Jiang W, Hermolin J & Fillingame RH (2001) The pre-
ferred stoichiometry of c subunits in the rotary motor
sector of Escherichia coli ATP synthase is 10. Proc Natl
Acad Sci USA 98, 4966–4971.
31 Mitome N, Suzuki T, Hayashi S & Yoshida M (2004)
Thermophilic ATP synthase has a decamer c-ring: indi-

cation of noninteger 10:3 H
+
⁄ ATP ratio and permissive
elastic coupling. Proc Natl Acad Sci USA 101, 12159–
12164.
32 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.
33 Pogoryelov D, Reichen C, Klyszejko AL, Brunisholz R,
Muller DJ, Dimroth P & Meier T (2007) The oligo-
meric state of c rings from cyanobacterial F-ATP synth-
ases varies from 13 to 15. J Bacteriol 189, 5895–5902.
34 Oster G & Wang H (1999) ATP synthase: two motors,
two fuels. Structure 7, R67–R72.
35 Dimroth P, von Ballmoos C, Meier T & Kaim G (2003)
Electrical power fuels rotary ATP synthase. Structure
11, 1469–1473.
36 Junge W, Lill H & Engelbrecht S (1997) ATP synthase:
an electrochemical transducer with rotatory mechanics.
Trends Biochem Sci 22, 420–423.
37 Junge W, Pa
¨
nke O, Cherepanov DA, Gumbiowski K,
Mu
¨
ller M & Engelbrecht S (2001) Inter-subunit rotation
and elastic power transmission in F
0
F

1
-ATPase. FEBS
Lett 504, 152–160.
38 Schemidt RA, Qu J, Williams JR & Brusilow WS (1998)
Effects of carbon source on expression of F
0
genes and
on the stoichiometry of the c subunit in the F
1
F
0
ATPase of Escherichia coli. J Bacteriol 180, 3205–3208.
39 Meyer zu Tittingdorf JM, Rexroth S, Scha
¨
fer E, Sch-
lichting R, Giersch C, Dencher NA & Seelert H (2004)
The stoichiometry of the chloroplast ATP synthase oli-
gomer III in Chlamydomonas reinhardtii is not affected
by the metabolic state. Biochim Biophys Acta 1659, 92–
99.
40 Mu
¨
ller DJ, Dencher NA, Meier T, Dimroth P, Suda K,
Stahlberg H, Engel A, Seelert H & Matthey U (2001)
ATP synthase: constrained stoichiometry of the trans-
membrane rotor. FEBS Lett 504, 219–222.
41 Arechaga I, Butler PJ & Walker JE (2002) Self-assem-
bly of ATP synthase subunit c rings. FEBS Lett 515,
189–193.
42 Meier T, Yu J, Raschle T, Henzen F, Dimroth P &

Muller DJ (2005) Structural evidence for a constant c
11
ring stoichiometry in the sodium F-ATP synthase.
FEBS J 272, 5474–5483.
43 Heise R, Mu
¨
ller V & Gottschalk G (1993) Acetogenesis
and ATP synthesis in Acetobacterium woodii are cou-
pled via a transmembrane primary sodium ion gradient.
FEMS Microbiol Lett 112, 261–268.
44 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.
45 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.
46 Blum H, Beier H & Gross HJ (1987) Improved silver
staining of plant proteins, RNA, and DNA in poly-
acrylamide gels. Electrophoresis 8, 93–98.
47 Lowry OH, Rosebrough NJ, Farr AL & Randall RJ
(1951) Protein measurement with the folin–phenol
reagent. J Biol Chem 193, 265–275.
48 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.

49 Crowther RA, Henderson R & Smith JM (1996)
MRC image processing programs. J Struct Biol 116,
9–16.
50 Collaborative Computational Project (1994) The CCP4
suite: programs for protein crystallography. Acta Crys-
tallogr D 50, 760–763.
M. Fritz et al. Hybrid V
0
–F
0
rotor in a F
1
F
0
ATP synthase
FEBS Journal 275 (2008) 1999–2007 ª 2008 The Authors Journal compilation ª 2008 FEBS 2007