Structural evidence for a constant c
11
ring stoichiometry
in the sodium F-ATP synthase
Thomas Meier
1
, Jinshu Yu
2
, Thomas Raschle
1
, Fabienne Henzen
1
, Peter Dimroth
1
and Daniel J. Muller
2
1 Institut fu
¨
r Mikrobiologie, Eidgeno
¨
ssische Technische Hochschule, Zu
¨
rich, Switzerland
2 Center of Biotechnology, University of Technology, Dresden, Germany
F-type ATP synthases are multisubunit protein com-
plexes lo cated i n the membrane of mitochondria, chloro-
plasts and bacteria. These enzymes use an electrochemical
H
+
or Na
+

the binding change mechanism [3], this arrangement
suggests a catalytic mechanism in which the c subunit
rotates within the a
3
b
3
cylinder. Elegant experiments
have subsequently visually verified this rotation [4].
The F
o
part, a rotary motor by itself, is fueled by the
electrochemical H
+
or Na
+
gradient and, upon rota-
tion, translocates these ions across the membrane.
Recent studies of the F
o
motor have focused on the
ion path through the membrane and the coupling
Keywords
atomic force microscopy; c ring
stoichiometry; F-ATP synthase; Ilyobacter
tartaricus; Propionigenium modestum
Correspondence
T. Meier, Institut fu
¨
r Mikrobiologie,
Eidgeno

gates of homogeneous c
11
rings and ⁄ or assemblies of c
11
rings and single
c monomers. Atomic force microscopy topographs of c rings reconstituted
into lipid bilayers showed that the c ring assemblies had identical diameters
and that stoichiometries throughout all rings resolved at high resolution.
This finding did not depend on whether the rings were assembled into crys-
talline or densely packed assemblies. Most of these rings represented com-
pletely assembled undecameric complexes. Occasionally, rings lacking a few
subunits or hosting additional subunits in their cavity were observed. The
latter rings may represent the aggregates between c
11
and c
1
, as observed
by SDS ⁄ PAGE. Our results are congruent with a stable c
11
ring stoichiom-
etry that seems to not be influenced by the expression level of subunit c in
the bacteria.
Abbreviations
AFM, atomic force microscopy.
5474 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS
between ion flux and torque generation [5–7]. Addi-
tionally, using various experimental approaches,
increasing evidence has accumulated on the overall
shape of the F
o

motor is not an
essential feature for function [14]. The number of
binding sites on the c ring determines the ATP to
proton ⁄ Na
+
ratio, and therefore this stoichiometry is
an important bioenergetic parameter for the cell. As
the number of subunits can vary among species, the
question was raised whether this stoichiometry could
also vary within one species in order to adapt to speci-
fic energetic requirements of the cells [15]. This proposi-
tion seemed to be supported by an effect of the carbon
source on the expression level of subunit c in Escheri-
chia coli. However, structural analyses of rotors from
I. tartaricus and from chloroplasts showed that their
stoichiometry seems to be constrained by the nearest
neighbor interaction between the subunits [16]. This
question has also been addressed with subunit c from
Escherichia coli [17], where annular shaped particles
were detected by electron microscopy after reconstitu-
tion from single c subunits. In agreement with the
above conclusions, it has been suggested that the
primary protein structure determines the ability of
subunit c to form rings. Furthermore, it was shown, by
gradient gel analysis, that the number of subunits in
the oligomer III isolated from the Chlamydomonas
reinhardtii chloroplast ATP synthase is not affected
by the metabolic state of the cells [18]. However, to
date, no structural methods have been applied to
clarify whether the stoichiometry of the c rings is

the strong T7 promoter produced large amounts of the
appropriate c subunit in the monomeric state, but also
sizeable amounts of oligomeric assemblies with a ratio
of  9:1 (c
1
:c
oligo
). For further analyses, these
assemblies were purified by sucrose density gradient
centrifugation and subjected to SDS ⁄ PAGE. The
results shown in Fig. 1 indicate that the assemblies
consisted not only of c
11
, but also of higher aggregates.
However, these aggregates are made up exclusively of
c subunits because they are converted completely into
the monomeric form by treatment with trichloroacetic
acid. A similar pattern of bands was observed with the
recombinant T67C mutant, with the exception of an
additional band corresponding to (c
11
)
2
and aggregates
at the top of the gel. For comparison, the c
11
ring pre-
paration of wild-type I. tartaricus cells is shown. Here,
the c
11

11
, and each supercomplex of higher
order was present in two- to threefold lower quantities
than the previous one. All of these supercomplexes dis-
assembled by SDS into the c
11
oligomer (and the SDS-
stable aggregates of c
11
, see below) as shown by the
equal mobility during SDS ⁄ PAGE (the second dimen-
sion in Fig. 2). The aggregation into supercomplexes
was prevented if the detergent octylglucoside was
replaced by Triton X-100 (Fig. 2A,B). The formation
of supercomplexes was also investigated in the recomb-
inantly synthesized T67C mutant. Here, in addition to
higher aggregates, a (c
11
)
2
form was observed which
Fig. 1. SDS gel electrophoresis of purified c ring preparations. The
c rings from Ilyobacter tartaricus and Propionigenium modestum
were heterologously expressed in Escherichia coli and purified as
described in the Experimental procedures. Two to three micro-
grams of each sample was subjected to SDS ⁄ PAGE and the gels
were stained with silver. The positions of the monomeric c subunit
(c
1
), the c ring (c

stained with silver. Intact c
11
ring and its supercomplexes were
marked with c
11
with the indexed numbers (n ¼ 1–4) correspond-
ing to the amount of complexed rings (c
11
)
n
. The monomeric c sub-
unit is marked with c
1
. A molecular mass standard is shown.
Structural evidence for a constant c
11
ring stoichiometry T. Meier et al.
5476 FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS
did not disintegrate into the c
11
oligomer by SDS, indi-
cating that a covalent disulfide bond had been formed
by the newly introduced cysteine residues.
Composition of the SDS-resistant c
11
aggregates
As described above, our c
11
ring preparations also con-
tained a distinct number of SDS-resistant complexes of

11
with one or more
c monomers attached. To investigate this possibility,
the homogeneous c
11
ring was isolated by electro-
elution of the c
11
band excised from the SDS gel
(Fig. 4). During storage for at least 1 month, no aggre-
gates or monomeric c units were formed from pure
c
11
ring preparations. However, after addition of isola-
ted c monomers and incubation overnight, the stable
aggregates were formed again. This suggests that the c
monomer assembled with other c subunits and rings to
form a ladder of higher aggregates. To test this hypo-
thesis, higher aggregates were specifically electroeluted
from the gel and subjected to SDS ⁄ PAGE without
heat treatment. The results showed that some aggre-
gates converted to c
11
and c
1
. It may therefore be con-
cluded that c
11
and c
1

11
band was cut out with a scalpel
and the protein was electroeluted from the gel pieces to obtain
pure c ring, as described in the Experimental procedures (lanes 2
and 5). As a control, c-ring bands migrating more slowly were cut
out and electroeluted (lane 3). Upon incubation of 2 lg of pure c
11
with 2 lgofc
1
purified in detergent, the slower migrating band
reappeared (lanes 4 and 8). Upon incubation of 2 lg of pure c
11
with 2 and 10 lgofc
1
purified in chloroform ⁄ methanol, the slower
migrating c ring aggregates did not reappear (lanes 6 and 7). Lane
9, c
1
purified by extraction with chloroform ⁄ methanol. Lane 10, c
1
purified by sucrose density gradient centrifugation with octylgluco-
side as the detergent. Lane 11, 2 lg of c ring after incubation with
5 lg of palmitoyl-oleyl-phosphatidylcholine. A molecular mass
standard is shown.
Fig. 3. Incubation of c ring with phospholipases and lipase. The
c ring samples isolated from Ilyobacter tartaricus wild-type cells
were incubated with phospholipase C (PLC), phospholipase A2
(PLA2) and lipase (Lip), as described in the Experimental proce-
dures, and 4 lg aliquots were loaded onto an SDS gel. The
enzymes alone were applied to separate lanes, as indicated. Also

cribed previously [20], and imaged by AFM. High-
resolution AFM topographs of c ring preparations
from I. tartaricus (Fig. 5) and P. modestum (Fig. 6)
showed surveys of crystalline (A) and densely packed
(B) regions of the reconstituted c subunits. The
undecameric subunit stoichiometry of the c rings was
more clearly visible in the densely packed regions of
the unprocessed topographs. Those c rings that were
assembled into a 2D crystal exhibited an upside-down
orientation, with one oligomer neighbored by three
oligomers showing an opposite orientation. In agree-
ment with previous results, the more elevated oligo-
mers (bright white areas) protruded from the lower
and wider c rings by about 1.1 ± 0.2 nm (n ¼ 50) and
thus partly prevented the AFM stylus from contouring
the wider rings [13]. However, for statistical analyses
we performed reference-free single particle analysis of
the densely packed c rings. All classes of complete c
rings exhibited 11 subunits forming the donut-like o ligo-
mer (first image of Figs 5D and 6D). However, some
rings were incomplete, missing one or more subunits.
Compared with AFM topographs of c rings isolated
from wild-type I. tartaricus ATP synthase [13,16], the
reconstituted samples investigated in the present study
showed more of these structural inconsistencies.
The presence of incompletely assembled c rings from
I. tartaricus and spinach chloroplast F-ATP synthases
was previously observed by AFM [16]. As the dia-
meter of the incomplete c rings did not change in any
Fig. 5. Atomic force microscopy (AFM) topographs of c subunit oligomers from Ilyobacter tartaricus F-ATP synthase overexpressed in

,c
9
and c
8
assem-
blies represented the most abundant species. These
defective rings could probably not be observed on the
SDS gel because the detergent dissociates the less
stable c
2
to c
10
assemblies into monomeric units.
Therefore, we assume that upon insertion of the last,
11th, c subunit, the assembly becomes resistant to SDS
or heat treatment. The observed accumulation of the
incomplete c
10
complex in the recombinant c ring
preparations suggests that the insertion of the last c
subunit forms the limiting step in the assembly process
of a functional oligomer.
Upon closer inspection, the occurrence of additional
protrusions in the cavity, and sometimes at the side of
some oligomers, became apparent (galleys of Figs 5
and 6). It may be assumed that these protrusions rep-
resent one or more c subunits attached to the ring-
shaped oligomer. Such a finding is in agreement with
the observation presented in Fig. 4, in which the com-
plete c subunit oligomers, hosting additional c sub-

k ring from the E. hirae V-ATPase [9]. As a result of
its extreme stability [22], the c
11
rotor ring from the
Na
+
-translocating F-ATP synthase from I. tartaricus
seems to be particularly suitable for structural investi-
gations. For a more detailed characterization of this
system, and to increase experimental options, we have
now investigated the aggregation behavior of the c
11
ring isolated from wild-type I. tartaricus cells, and we
have explored the subunit c assembly of the protein
expressed heterologously in E. coli. Under all investi-
gated conditions, these assemblies were found to con-
sist exclusively of rings of uniform size, allowing tight
packaging of 11 monomeric units. In accordance with
previous observations, some of these rings had gaps
indicative of the absence of, in most cases, one c sub-
unit [16]. As these rings had the same diameter as
the c
11
rings, they were regarded as incompletely
assembled. In preparations derived from recombinant
E. coli cells, the incomplete assemblies were more
abundant than in preparations derived from wild-type
I. tartaricus cells. In both wild-type and recombinant
preparations, the majority of the incomplete rings
lacked only one monomer. It can therefore be conclu-

formation of these supercomplexes is dependent on the
detergent because they are formed in octylglucoside,
but not in Triton X-100. These supercomplexes dis-
aggregate completely into the c
11
rings in the presence
of SDS. The second type of aggregate appears as a
ladder above the original c
11
band on SDS ⁄ PAGE and
consists of c
11
rings hosting varying amounts of the c
monomer. Aggregates are particularly abundant in
c ring preparations from E. coli expression clones
where the c monomer is present in high amounts. Once
these aggregates were formed they remained stable and
were minimally influenced by additives such as deter-
gents, organic solvents, salts or lipids (like 1-palmitoyl-
2-oleyl-sn-glycero-3-phosphocholine). AFM topographs
of these samples showed an exclusively undecameric
stoichiometry in the completely assembled rings. This
is also observed in the noncrystalline areas of the
reconstituted vesicles, demonstrating that it is not an
artifact from the 2D crystallization.
Slower migrating bands of c rings, as observed on
SDS gels, suggest that a certain fraction of c rings may
host additional subunits. The AFM topographs indi-
cate that these additional subunits may be hosted at the
outer sides and within the central cavities of the rings.

two restriction enzymes and ligated before transformation
into E. coli DH5a. Plasmid pt7c [26] was mutagenized with
the Quick Change Site Directed Mutagenesis Kit (Strata-
gene, La Jolla, CA, USA) to yield the single mutation,
T67C, in the P. modestum subunit c (plasmid pt7cT67C).
Synthesis and purification of c oligomers
from strain BL21(DE3) transformed with various
plasmids
E. coli BL21(DE3) (Novagen, Madison, WI, USA) was
transformed with plasmids pt7c, pt7cIT and pt7cT67C, as
described above. The transformed E. coli cells were grown
in 2 L of Luria–Bertani (LB) medium to reach an attenua-
nce (D) of 0.6 at 37 °C in the presence of 200 lgÆmL
)1
ampicillin. After cooling on ice for 5 min, the expression
was induced with 0.7 mm isopropyl thio-b-d-galactoside
and allowed to continue for 6 h at 30 °C, to yield typically
2.5 g of cells per L of medium. The cells (1 g wet weight)
were suspended in 8 mL of 50 mm potassium phosphate
buffer, pH 8.0, containing 1 mm 1,4-dithio-dl-threitol,
0.1 mm diisopropylfluorophosphate and a spatula tip of
DNaseI. Preparation of membranes was performed at 4 °C.
The cell suspension was passed twice through a French
pressure cell at 12 000 psi (8.3 · 10
4
kPa). After the
removal of cell debris by centrifugation at 15 000 g for
20 min, ultracentrifugation was performed at 200 000 g for
60 min. The membrane pellet was washed once with 4 mL
of 20 mm Tris, 5 mm EDTA, and then adjusted to pH 8.0

pooled and concentrated by ultracentrifugation (18 h,
200 000 g,4°C). The final protein concentration was typ-
ically between 1.5 and 3 mgÆmL
)1
. Fractions containing
the monomeric c subunit were also collected and used to
study the association with c
11
rings to stable c
11
(c
1
)
n
aggregates.
Reconstitution of densely packed and 2D
crystalline c ring samples
The c rings purified from these expression cultures were
crystallized in two dimensions by mixing octylglucoside-sol-
ubilized protein with 1 mgÆmL
)1
1-palmitoyl-2-oleyl-sn-gly-
cero-3-phosphocholine at a lipid : protein ratio of 0.8
(w ⁄ w) in a total volume of 50 lL, followed by dialysis for
24 h at 25 ° C against 200 mL of buffer (10 mm Tris ⁄ HCl,
pH 7.5, containing 200 mm NaCl and 0.02% NaN
3
), then
for another 24 h at 37 °C. The crystals were stored at 4 °C
for further analysis. Subunit c monomers solubilized in

Blue Native PAGE
Blue Native PAGE was carried out as described previously
[28]. Separation gels with a linear gradient of 5–17% acryl-
amide were prepared and overlayed with 4% sample gels.
Samples of 2–5 lg protein each were mixed with sample
buffer [50 mm Tris ⁄ HCl, pH 6.8, containing 12% (v ⁄ v) gly-
cerol and 0.01% (w ⁄ v) Serva blue G]. After running for 1 h
at 100 V with cathode buffer (50 mm Tricine, 15 mm Bis-
Tris ⁄ HCl, pH 7.0) containing 0.02% (w ⁄ v) Serva blue G,
T. Meier et al. Structural evidence for a constant c
11
ring stoichiometry
FEBS Journal 272 (2005) 5474–5483 ª 2005 FEBS 5481
the cathode buffer was replaced with buffer containing only
0.002% (w ⁄ v) Serva blue G and the run continued at
400 V. Native protein complexes were then analyzed by
SDS ⁄ PAGE, as described previously [27], with the lanes
from the Blue Native PAGE embedded into a 4% stacking
gel.
Atomic force microscopy
The samples were diluted to a concentration of 10
lgÆmL
)1
in 200 mm NaCl, 10 mm Tris ⁄ HCl, pH 7.5.
To allow adsorption of the membranes, a drop of 30 lL
was placed onto freshly cleaved mica. After an adsorption
time of 15 min, the sample was gently washed using the
above buffer solution containing no membrane proteins to
remove weakly attached material from the mica surface.
Contact mode AFM topographs were then recorded in the

shown). It appeared that all averaged classes showed a stoi-
chiometry of 11 subunits except for those of defect parti-
cles. The diameter of intact and defective c rings was
determined as described previously [16].
Other methods
Gels were stained with silver [29]. The protein concentra-
tion of samples was determined according to the bicinchon-
inic acid method [30] with bovine serum albumin as the
standard.
Acknowledgements
The authors thank Marijke Koppenol for critically
reading the manuscript. This work was supported by
the free state of Saxiona, the European community,
and the Deutsche Forschungsgemeinschaft (DFG).
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