Báo cáo khoa học: The transmembrane domain of subunitbof theEscherichia coli F1FOATP synthase is sufficient for H + -translocating activity together with subunitsaandc doc

The transmembrane domain of subunit
b
of the
Escherichia coli
F
1
F
O
ATP synthase is sufficient for H
+
-translocating activity
together with subunits
a
and
c
Jo¨ rg-Christian Greie, Thomas Heitkamp and Karlheinz Altendorf
Universita
¨
t Osnabru
¨
ck, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Osnabru
¨
ck, Germany
Subunit b is indispensable for the formation of a f unctional
H
+
-translocating F
O
complex both in vivo and in vitro.
Whereas t he very C-terminus of subunit b interacts w ith F
1

and p lays a crucial role in enzyme assembly, the C-terminal
region is also considered to be necessary for proper recon-
stitution of F
O
into liposomes. Here, we show that a syn-
thetic peptide, residues 1–34 of subunit b (b
1)34
) [Dmitriev,
O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) J. Biol.
Chem. 274, 15598–15604], corresponding to the membrane
domain of subunit b was sufficient in f orming an active F
O
complex when coreconstituted with purified ac subcomplex.
H
+
translocation w as shown to be sensitive to the specific
inhibitor N,N¢-dicyclohexylcarbodiimide, and the r esulting
F
O
complexes were deficient i n b inding of isolated F
1
.This
demonstrates that only the membrane part of subunit b is
sufficient,aswellasnecessary,forH
+
translocation across
the m embrane, whereas the binding of F
1
to F
O

is mainly
triggered by C-terminal residues beyond Glu34 in subunit b.
Comparison of the d ata with former r econstitution experi-
ments additionally indicated that parts of the hydrophilic
portion of the subunit b dimer are not involved in the p rocess
of ion t ranslocation itself, b u t might o rganize subunits a
and c in F
O
assembly. F urthermore, the d ata obtained
functionally support t he monomeric NMR structure of the
synthetic b
1)34
.
Keywords:F
1
F
O
ATP synthase; subunit b; reconstitution;
proton translocation; Escherichia coli.
Membrane-bound F-type A TPases (F
1
F
O
) occur ubiqui-
tously in mitochondria, c hloroplasts and Bacteria. They
reversibly catalyze the synthesis of ATP from ADP and
inorganic phosphate by use of an electrochemical ion
gradient, which is generated across the membrane by
respiration or photosynthesis. Although the distinct com-
position of this multisubunit enzyme complex varies some-

what between species, all F
1
F
O
complexes share high
homology with r espect to the mechanism of catalysis.
Although there is still som e controversy [ 1], it is g enerally
accepted that ion translocation t hrough the transmembrane
domain (F
O
) is coupled to ATP synthesis/hydrolysis in the
peripheral catalytic domain (F
1
) via a rotary mechanism [2].
Thus, the structural classification of the enzyme in F
1
(subunit composition a
3
b
3
cde in Escherichia coli)andF
O
(ab
2
c
10
) [3] is different compared to a functional division in
rotor and stator. During coupled catalysis, H
+
transloca-

tion through F
O
or ATP h ydrolysis in F
1
gene rate s a rotary
movement of the c entrally located ce subcomplex, which is
fixed to the ring-like subunit c oligomer [4,5]. Due to the
central rotor element, a second, peripheral stalk is necessary
for the stabilization of the F
1
F
O
complex, which is
composed at least o f the two copies of subunit b [6,7].
During catalysis, the subunit b dimer is supposed to
undergo t ransient elastic deformation in order to c ompen-
sate for t he torque, which i s built up by the p ropelling rotor
[4,8,9]. Finally, t orque is re leased by conformational c han-
ges leading to either H
+
pumping through F
O
or ATP
synthesis in F
1
. The molecular switch, by which one or the
other direction of catalysis is preferred, has recently been
attributed to the e subunit [10].
InbeingpartofthestatorelementoftheF
1

F
O
complex,
the subunit b dimer makes both m ultiple contacts with
subunits a, b an d d of the F
1
part [11] as well as with
subunit a of F
O
[12,13]. There a re several lines of evidence
that suggest t hat subunit b is absolutely essential f or the
binding of F
1
to F
O
[5,14], which is mainly attributed to its
C-terminal domain [15]. The multiple tasks performed by
subunit b have been attributed to different domains of the
polypeptide [11]. H owever, these domain s have been shown
not to function independently from each other. The binding
constant of the soluble C-terminal domain o f subunit b to
subunit d for example is much too low to withstand
the torque generated duri ng catalysis [2]. Deletion muta-
genesis of subunit b in assembled F
1
F
O
revealed tolerances
for segment gaps also affecting areas considered to be
crucial for dimerization of the cytoplasmic domain of

subunit b [16]. Thus, although spacially separated, a
balanced in terplay o f t he different domains of the subunit
Correspondence to J C.Greie,Universita
¨
t Osnabru
¨
ck,
Abteilung Mikrobiologie, D-49069 Osnabru
¨
ck, Germany.
Fax: + 49 541969 2870, Tel.: + 49 541969 2809,
E-mail:
Abbreviations:DCCD,N,N¢-dicyclohexylcarbodiimide; F
1
, peripheral
catalytic domain in F
1
F
O
ATP synthase; F
O
, transmembrane
domain in F
1
F
O
ATP synthase.
Enzyme:H
+
-transporting AT P synthase (EC 3.6.1.34).

(Received 3 1 March 2004, revised 2 5 May 2004,
accepted 28 May 2004)
Eur. J. Biochem. 271, 3036–3042 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04235.x
b dimer seems to be a prerequisite at least for a proper
assembly of the F
1
F
O
complex.
A few years ago, the monomeric structure of a synthetic
peptide corresponding to the membrane-spanning domain
of subunit b (b
1)34
) has been solved by NMR s pectroscopy
[17]. According to these data e ach of t he two b subunits is
predicted to f orm one transmembrane a helix, w hich, based
on chemically induced cysteine cross-linking experiments in
assembled F
1
F
O
complexes, are supposed to come together
to form a dimer. However, b
1)34
was n ot functional when
used for the coreconstitution of a H
+
-translocating F
O
complex in these studies, although it has previously been

shown that reconstitution of F
O
from single subunits is
possible [18]. T his lends support t o the notion that t he
C-terminal domain o f subunit b is also involve d in the
assembly of an a ctive F
O
complex [17]. Thus, a lthough i n
good accord with cross-linking studies and secondary
structure predictions, the NMR structure of the synthetic
b
1)34
has not yet been functionally validated, either in viv o
or in vitro.
Here, w e report t hat by t he use of preformed ac subcom-
plexes it was possible to coreconstitute b
1)34
into funct ional
F
O
complex capable of N,N¢-dicyclohexylcarbodiimide
(DCCD)-sensitive H
+
translocation. Hence, whereas the
membrane domain is sufficient to couple s ubunits a and c
during ion translocation, the soluble part of subunit b seems
to be neces sary for the p roper assembly of subunits a and c.
As expected, the resulting F
O
complexes were deficient in

the binding of F
1
, further restricting F
1
binding sites to the
C-terminal domain beyond residue Glu34 o f the s ubunit b
dimer.
Experimental procedures
Bacterial growth
Escherichia coli strain DK8 [19] lacking the at p operon was
transformed with plasmid pBWU13 [20] carrying the atp
operon except for atpI. Cells were grown o n minimal
medium supplemented with thiamine (2 lgÆmL
)1
), thymine,
asparagine, isoleucine and valine (50 lgÆmL
)1
each)
together with 75 m
M
glycerol as carbon source, harvested
at late exponential phase and stored at )80 °C.
Preparative procedures
F
O
and F
1
complexes as well as sub unit b and ac
subcomplex isolated from dissociated F
O

complexes were
prepared as described [14,15,21]. Synthetic peptide b
1)34
(2 m
M
solution in chloroform/methanol/H
2
O4:4:1,
v/v/v) was a kind gift of O. Y. Dmitriev and R. H.
Fillingame (University of Wisconsin Medical School,
Madison, WI, U SA), the synthesis of which was described
previously by Dmitriev et al . [17].
Reconstitution into proteoliposomes
Proteoliposomes were prepared as described [22] with the
following modifications. E. coli lipids present in chloroform
at 20 mg ÆmL
)1
(Avanti Pro Lipids) were dried under a
gentle stream of argon and redissolved to 40 mg ÆmL
)1
in
detergent buffer before the addition of protein. The w eight
ratio o f F
O
to phospholipid was 1 : 50. In the case of
subcomplexes and s ingle subunits except b
1)34
, the corres-
ponding amount of protein was initially calculated using the
particular stoichiometric abundance with respect to a

stoichiometry of ab
2
c
10
for F
O
. In either case, proper
stoichiometric amounts of particular F
O
subunits were
finally confirmed by SDS/PAGE. For samples containing
b
1)34
present in c hloroform/methanol/H
2
O4:4:1(v/v/v),
aliquots of the latter were added to the lipid solution prior to
the removal of the organic solvent. Proper stoichiometric
amounts were calcu lated based on t he amino a cid analysis
performed during the synthesis of b
1)34
and c alibrated w ith
the F
O
sample assuming a stoichiometry of ab
2
c
10
. Dialysis
wascarriedoutfor40hat4°C changing the buffer once.

Loading of proteoliposomes with K
+
was carried out
as described [13]. For the inhibition of passive H
+
translocation, samples were t reated with 50 l
M
DCCD
for 5 min directly in the assay medium according to
Dmitriev et al . [18].
Assays
Rates of passive H
+
translocation were measured as
described [ 13] by use o f 2 l
M
valinomycin for induction
of the K
+
diffusion potential. After rebinding of F
1
,
reconstituted DCCD-sensitive ATPase activities were
measured acco rding to Steffe ns et al. [ 23]. Protein concen-
trations were determined with the bicinchoninic a cid assay
(Pierce) used as recommended by t he supplier. Proteins
were separated by SDS/PAGE [24] and detected by silver
staining [25].
Results
Stoichiometric mixing of subcomplexes

for reconstitution
Previous studies revealed that the rate of H
+
transloca-
tion through F
O
reconstituted from subcomplexes was
sensitive t o t heir particular stoichiometric am ount in the
reconstitution assay [ 13,14]. Hence, in order to compare
the effect of b
1)34
with intact subunit b,itwasimportant
to determine and exactly adjust stoichiometric propor-
tions of b oth b and b
1)34
with respect to the preformed ac
subcomplexes. Whereas the concentration of the b
1)34
sample was determined b y a mino acid analysis [17], the
determination of protein concentrations of F
O
, ac
subcomplex and s ubunit b by conventional c olourimetric
assays revealed to be biased by partial i mpurities of t he
preparation and by the particular buffer c omposition a s
well as by the specific biochemical properties of each
polypetide (data not shown; also compare [13]). H ence,
the stoichiometric ratios of subcomplexes (except for
b
1)34

) m ixed for r econstitution were only initially judged
by the colourimetric bicin choninic acid assays of protein
samples, but were finally adjusted by the d ensitometric
comparison of silver stained protein bands in SDS/PAGE
(Fig. 1 ). Aliquots we re taken directly f rom t he samples
before the addition of lipid or lipid plus b
1)34
.The
comparison of corresponding band intensities revealed a
proper stoichiometric relationship of F
O
subunits in each
of the s amples taken for reconstitution.
Ó FEBS 2004 Functional reconstitution of b
1)34
(Eur. J. Biochem. 271) 3037
Reconstitution of F
O
from
b
1)34
and preformed
ac
subcomplexes
Previous studies dealing with the reconstitution of chloro-
form/methanol extracted subunit c revealed the necessity for
the addition of detergent t o the sample prior to the removal
of the s olvent by e vaporation in o rder to facilitate resolu-
bilization and to prevent partial denaturation of the
polypeptide [18]. This c ould be o vercome b y the dire ct

addition of the protein to the lipid solution, also present in
organic solvent, prior to the e vaporation [13], thereby
transferring the polypeptide from the solvent immediately
into the lipid environment without the n eed for additional
detergents. Thus, the same technique was successfully used
for the reconstitution of b
1)34
present in chloroform/
methanol/H
2
O 4 : 4 : 1 ( v/v/v ).
Reconstitution of preformed ac subcomplexes with intact
subunit b as well as with the subunit b t ransmembrane
domain b
1)34
resulted in the formation of functional F
O
complexes as demonstrated by rapid K
+
/valinomycin-
triggered H
+
uptake into p roteoliposomes (Fig. 2 ). Traces
of passive H
+
translocation were in g ood accordance with
those obtained for the reconstituted F
O
complex from single
subunits a, b and c [18]. Whereas significant initial rates of

H
+
translocation could already be observed with a
stoichiometric ratio o f intact s ubunit b and ac subco mplex,
a 6 .6-fold molar excess of b
1)34
was n ecessary to obtain
similar results. Reconstituted ac subcomplexes without
added b subunits, a s well as the control c ontaining subunit
b only, revealed slightly higher rates of passive H
+
translocation than control liposomes. This is due to residual
amounts of subunit b or subunits a and c, respectively, in the
corresponding protein preparations (compare Fig. 1, lanes 3
and 6). These findings are also reflected by a higher
background rate of reconstituted A TPase activity i n these
samples with respect to the control (see below).
Quantitative titration of
b
1)34
in reconstitution
Comparable rates of H
+
translocation for intact subunit b
and b
1)34
were only obtained with a stoichiometric surplus
of the latter. Recent studies dealing w ith the reconstitution
of chloroform/methanol-extracted subunit c also revealed
the necessity of an excess of the polypetide, which is also

present i n c hloroform/methano l/H
2
O p rior to reconstitu-
tion [13]. T his points t o a more general t han specific effect
due to the use of organic solvent in protein preparation.
Furthermore, the amount of b
1)34
taken for coreconstitu-
tion was stoichiometrically calibrated with the protein
concentration of the F
O
sample, w hich was f ound to be
biased by several factors.
However, in order to f urther elucidate s aturating condi-
tions of passive H
+
translocation against t he stoichiometric
abundance of b
1)34
, preformed ac subcomplexes were
titrated with increasing amounts of b
1)34
in the reconstitu-
tion assay (Fig. 3). Again, low basal H
+
translocation
activity could be observed in c ase of ac subcomplex by itself
(2.2 lmol H
+
Æmin

)1
Æmg
)1
), whereas the control containing
a 1 3.3-fold molar excess of b
1)34
only s howed unspecific
linear H
+
drift ( 0.2 lmol H
+
Æmi n
)1
Æmg
)1
) i nstead of a
corresponding exponential rise in translocation activity
following the potential jump. T his unspecific H
+
drift is
Fig. 1. Quantitative c omparison of F
O
subunits in s ubcomplexes mixed
for reconstitution. Silver stained SDS/PAGE of samples taken directly
for reconstitution. Aliquots of 2 lL were taken for electrophoresis
prior t o the addition of lipid in the reconstitution p rocedure. Lane 1,
buffer control; lane 2, F
O
(7.2 lg); lane 3, ac subcomplex; lane 4,
ac + b; lane 5 , as l ane 3, p rio r to ad dition of lipid plus b

1)34
;lane6,
subunit b. MW, molecular mass marker.
Fig. 2. Passive H
+
translocation o f F
O
obtained by coreconstitution of
b
1)34
into proteoliposomes. F
O
, ac su bcomplex and intact subunit b
were reconstituted in stoichiometric amounts. In the case o f b
1)34
,a
6.7-fold stoichiometric excess was used for corecon stitution with ac,
whereas a 13.3-fold stoichiometric excess was used a s a control. Passive
H
+
uptake wa s measured b y use of a K
+
/valinomycin diffusion
potential. Traces are correspondingly labelled. Control, plain lipo-
somes without protein. The addition of valinomycin is indicated by the
arrow.
3038 J C. Greie et al.(Eur. J. Biochem. 271) Ó FEBS 2004
most likely due to the high amount of membrane pro-
tein present in the proteoliposome, as the 13.3-fold
stoichiometric amount of b

1)34
was used as control.
Generally, u nspecific H
+
drift c an be clearly s eparated
from specific potenti al-driven H
+
translocation because t he
former results in a linear curve whereas the latter leads to an
initial e xponential rise on top of the drift. However, the use
of a 3.3-fold molar excess of b
1)34
revealed an only slight
increase in passive H
+
translocation a ctivity w hen c ore-
constituted with ac subcomplex (4.2 lmol H
+
Æmin
)1
Æmg
)1
).
In contrast, a strong effect was observed in the case of the
6.7-fold stoichiometric amount (6.8 lmol H
+
Æmin
)1
Æmg
)1

),
whereas no further increase was obtained, even with a 13.3-
fold molar excess of b
1)34
(4.5 lmol H
+
Æmin
)1
Æmg
)1
).
Instead, a d ecrease in the initial H
+
uptake rate c ould be
observed, which is due to the already described negative
effect of unspecific H
+
drift on t he driving force reflecting
the large amount of protein present in the membrane. In
summary, the titration experiments revealed that an
approximately 6-fold molar excess of b
1)34
was necessary
to obtain saturated H
+
translocation activities, whereas
the use of higher molar ratios had no further stimulating
effect.
Reconstituted ATPase activity after rebinding of F
1

From previous studies it is known that t he C-terminal
hydrophilic domain of the subunit b dimer is involved in the
binding of F
1
[5,14,15]. Deletion mutagenesis of hydrophilic
segments of subunit b more proximal to F
O
also revealed
defects in F
1
F
O
assembly [16]. Interactions in cou pling
between F
1
and F
O
have also been shown t o occur via the
subunit c ring, a lthough these are not sufficient for the tight
binding of F
1
to ac subcomplexes [8]. It is still unknown
whether the N-terminal domain o f subunit b is involved in
F
1
interaction, either in a direct or indirect way, the latter of
which could occur via a possible stabilizing e ffect of the
subunit b transmembrane domains on the ac subco mplex.
Therefore, F
O

complexes reconstituted from ac subcom-
plexes and b
1)34
were tested for their F
1
binding ability
(Table 1). Significant rates of reconstituted ATPase activity
were obtained in the c ase of p roteoliposomes containing F
O
and ac + b , w hich is in accordance with the rates obtained
from the passive H
+
translocation measurements. In
contrast, even by the use of a 13.3-fold stoichiometric
excess o f b
1)34
, t here was n o corresponding i ncrease in
activity when coreconstituted with ac subcomplex. As
already mentioned, the very minor background activity in
control samples only containing ac subcomplex or intact
subunit b is again due to residual impurities of other
corresponding F
O
subunits, which can thus far not be
avoided during the preparatio n (compare Figs 1 and 2). In
conclusion, F
O
complexes assembled f rom ac subcomplexes
and b
1)34

are not competent in F
1
binding due to the lack of
corresponding sites o f interaction. Thus, the N-terminal
stretch o f residues of subunit b up to Glu34 is n ot s ufficient
to trig ger F
1
binding even in as sembled F
O
complexes
capable of H
+
translocation.
Inhibition of reconstituted H
+
translocation by DCCD
In order t o demonstrate that th e passive H
+
translocation
observed for F
O
complexes reconstituted from ac + b
1)34
is
specific, both ac + b and ac + b
1)34
were incubated with
and without 50 l
M
DCCD prior to the measurements

(Fig. 4 ). Both resulting F
O
complexes s howed comparable
rates o f inhibition, whereas the addition of a corresponding
amount of e than ol to the non inhibited samples had no
inhibitory effect in either case. The corresponding behaviour
Fig. 3. Saturating titration of ac subcomplexes with b
1)34
in reconsti-
tution. Increasing stoichiometric amounts o f b
1)34
were used to
reconstitute ac subcomplexes. Passive H
+
uptake w as m e asu red by u se
of a K
+
/valinomycin d iffusion potential. Trace s are correspondingly
labelled. The values in parentheses in the c ase of b
1)34
indicate the
corresponding stoichiometric amount, for e xample 3.3· means a 3.3-
fold stoic hiometric excess of the polype ptide with respect t o a stoi-
chiometry of ab
2
c
10
for F
O
. The ad dition of valinomycin is indicated by

the arrow.
Table 1. Reconstituted coupled ATPase a ctivities after rebinding of F
1
.
DCCD-sensitive ATPase activities were measured after the binding of
isolated F
1
complexes to proteoliposomes. According to the assays of
passive H
+
translocation, an increasing amount of b
1)34
was used
in the reconstitution. The values in parenthese s indicate the
corresponding stoich iometric amo unt p resen t, for e xample 3.3 · means
a 3 .3-fold stoichiometric excess of the p olypeptid e with resp ect to a
stoichiometry of ab
2
c
10
for F
O
.
Proteoliposome sample
taken for the rebinding
of isolated F
1
DCCD-sensitive
ATPase activity
(lmol P

i
Æmin
)1
Æmg
)1
)
Plain liposomes 0.8
F
O
14.6
ac 4.7
b 2.4
b
1)34
(13.3·) 0.2
ac + b 11.2
ac + b
1)34
(3.3·) 5.4
ac + b
1)34
(6.7·) 4.6
ac + b
1)34
(13.3·) 3.4
Ó FEBS 2004 Functional reconstitution of b
1)34
(Eur. J. Biochem. 271) 3039
of b
1)34

and intact subunit b clearly a rgues in favour of a
homologous function in the H
+
translocation process a nd,
hence, reve ale d that b
1)34
is capable of forming functional
F
O
complexes in vitro.
Discussion
Due to the rotary mechanism of the enzyme, the subunit b
dimer accomplishes multiple tasks in assembled ATP
synthase, the most obvious one of which is the structural
linkage between F
1
and F
O
[6]. This physical linkage
between the site o f catalysis and ion translocation is further
associated with functional needs of coupling by means of
elasticity. The axial deformation of the intertwined helices of
subunit c are supposed to be counteracted by the parallel
paired helices of the subunit b dimer, thus formin g a
parallelogram-like s pring transiently loaded with elastic
torque [4]. It is tempting to independently allocate different
functions of subunit b to different domains of the polypep-
tide. Hence, stator interactions with F
O
and F

1
subunits are
supposed to occur mainly in the N- and C-terminal regions,
respectively [11,13], whereas the middle p art of the poly-
peptide was shown t o adopt a r ight-handed coiled-coil
structure essential for dimerization and presumably
involved in the transient storage of energy [26]. Due t o the
tension, which is built up during catalysis, stator resistance
was shown to be at least balanced with the torque produced
by the rotor [27]. Although a strong binding has been
observed between the cytoplasmic domain of the subunit b
dimer and F
1
in solution [28], the interplay o f all three F
O
subunits is necessary for the reconstitution of F
1
ATPase
activity on the membrane l evel. Neither the subunit b dimer
[8] or t he ab
2
stator subcomplex [13], nor subunit a together
with the ring of c su bunits [14] or the s ubunit c ring alone
[13], can be held responsible for F
1
binding. Thus, s ubunit
interactions occurring solely within the central or the second
stalk a re not sufficient to couple F
1
to F

O
on the functional
level of the membrane.
When se parated in vitro, both F
1
and F
O
act independ-
ently according to their f unction in viv o, i.e. ATP hydrolys is
or H
+
translocation, respectively. Thus, it should be
possible to discriminate between residues in subunit b
which are essential for the function of F
O
or the c oupling
to F
1
when reconstituted together w ith o ther F
O
subunits.
Whereas the soluble hydrophilic domain of subunit b has
already been extensively characterized with respect to both
structure and function [11], the membrane part of the
polypeptide has received comparatively little attention. The
monomeric structure o f the synthetic peptide b
1)34
corres-
ponding to the transmembrane domain of s ubunit b has
been determined at high resolution with two-dimensional

1
H NMR in organic solvent [17]. Although i n g ood accord
with cross-linking studies and secondary structure predic-
tions, t his N MR structure has n ot y et been functionally
validated, either in vivo or in vitro. Although the reconsti-
tution of functional F
O
complexes from single a, b and c
subunits has already been reported [ 18], the same approach
initially failed in the case of b
1)34
,fromwhichitwas
deduced that the C-terminal segment of subunit b is
essential for the reconstitution and functional assembly of
an active F
O
complex [17]. However, in this case
coreconstitution of b
1)34
was p erformed by use of s ingle
subunits a and c.
In contrast, our data clearly demonstrate, that by use of
preformed ac subcomplexes, only t he membrane part of
subunit b is sufficient, as well as necessary, for H
+
translocation across t he membrane, w hereas the b inding
of F
1
to F
O

is triggered by C-terminal residues in s ubunit b.
This clearly attributes two distinct functions to the subunit b
dimer, which are spatially separated.
An excess of b
1)34
with respect to isolated s ubunit b was
necessary to obtain comparable rates of passive H
+
translocation w hen coreconstituted with ac subcomplex.
The necessity of an excess of free F
O
subunits, which are
present in chloroform/methanol/H
2
O prior to the reconsti-
tution, is already known from other recent e xperiments [13]
and m igh t in part result from potential damage of the
polypeptides during t he extraction in organic solvent.
Furthermore, the protein is likely to integrate in different
orientations with respect to the c oreconstituted subcom-
plexes in general, which decreases the fraction of properly
assembled p rotein complexes. In addition, b
1)34
was s hown
to be a monomer in chloroform/methanol/H
2
O [17], which
might produce nonfunctional antiparallel orientations of
the r esulting dimer during r econstitution. H
+

translocation
rates were generally lowe r in t he case of b
1)34
than in the
case of intact s ubunit b. This is d ue to the n eed for a
relatively high protein content in the membrane due to the
different possible o rientations of b
1)34
,whichleadstoa
decreased driving force caused b y unspecific H
+
leakage
following the potential jump. In a ddition, the c hemically
Fig. 4. DCCD-inhibited H
+
translocat ion o f ac subcomplexes recon-
stituted with subunit b or b
1)34
. Th e ac subcomplexes were recon stitute d
either with stoichiome tric amounts of subunit b or with a 6.7-fold
stoichiometric excess of b
1)34
. Samples were trea ted with 50 l
M
DCCD for 5 min in t he assay medium prior to the measure ments.
Passive H
+
uptake was measured by use of a K
+
/valinomycin di ffu-

sion po ten tial. Traces are correspondingly labe lled . As a co ntrol,
ac + b plus the c orresp onding amount of ethanol as in the DCCD
inhibition assays was used (top). T he addition of valin omycin is indi-
cated by t he arrow.
3040 J C. Greie et al.(Eur. J. Biochem. 271) Ó FEBS 2004
synthesized b
1)34
certainly r epresents a more artificial
population of the polypeptide than a subunit b dimer
purified by dissociation from already functional F
O
com-
plexes, thereby exhibiti ng a g enerally lower activity. This
view is supported b y an analogous set o f experiments with
intact subunit b prepared by denaturation with SDS and
refolding according to Gr eie et al.[8].Thisb subunit also
showed a r educed rate of H
+
conductivity in the corecon-
stitution assay with respect to intact subunit b prepared by
dissociation of F
O
complexes, with rates more comparable
to that of b
1)34
(data not shown). T he traces of DCCD
inhibition were again comparable to those obtained by
Dmitriev et al.forF
O
complexes reconstituted f rom single

subunits a, b and c [18].
However, isolated ac subcomplexes were s h own t o b e
deficient in H
+
conduction, although both subunits directly
involved in ion t ranslocation are present [8]. Our r esults
demonstrate that t he presence of the transmembrane spans
of subunit b are both sufficient as well as necessary to build
up a functional H
+
-translocating F
O
complex. Therefore,
an essential function of the membrane part of subunit b may
be that of keeping the rotor and stator in a proper
configuration while the subunit c ring slides along the
surface of subunit a. Thus, a tight interaction with subunit a
seems reasonable and was r ecently d emonstrated by the
purification of a stable ab
2
subcomplex [13]. L ess extensive
contact w ith t he rotating subunit c oligomer can be derived
from cross-linking data [29].
As already mentioned, functional coreconstitution of
b
1)34
failed when mixed with single subunits a and c,
although the membrane part of subunit b should be
sufficient for stabilizing subunits a and c during H
+

translocation. Hence, the C-terminal domain seems to be
involved in the a ssembly or education o f subunits a and c.
This view is s upported by t he fact that F
O
complexes
containing subunit b were shown to assemble unidirection-
ally into the outer shell of the multilamellar proteoliposome
during reconstitution [14,15], which is m ost likely due to the
large h ydrophilic domain of the subunit b dimer. Thus, this
would imply th at the C -terminal domain o f b might be
important not for the insertio n of F
O
subunits into the
membrane itself but for the proper alignment of F
O
subunits
during assembly. As a cons equence, subcom plexes lack ing
this domain, as in the case of ac, ac + b
1)34
and b
1)34
alone,
would t end to a ssemble rather randomly with respect to
their topological orientation, thus leading to a significant
decrease of function al F
O
complexes compared to s amples
containing intact subunit b. T his is e xactly what was found
in the reconstitution of b
1)34

.
That distinct parts of the hydrophilic portion of subunit b
are involved in F
1
F
O
assembly can also be derived from
deletion mutagenesis e xperiments [16]. Several deletions
with increasing sizes affecting r esidues 50–60 were shown to
be impaired in the assembly process, but were not affected in
activity. Thus, t his stretch of residues is probably important
for assembly but not directly involved in catalytic function.
The d isruption of interactions with subunit a during
assembly has been discussed. Recent cross-linking experi-
ments demonstrated a close proximity of a putative a-helic al
face of subunit b between residues Ala32 and Arg36 and
hydrophilic loops of subunit a [30,31]. In combination
with our r esults these data suggest that residues between
positions 35 and 60 might be important for t he assembly of
subunits a and c.
The determination of high resolution three-dimensional
protein structures from F
O
subunits has only been accom-
plished in case o f subunits c and b
1)34
by use of s ingle
monomeric polypeptides prepared in organic solvent
[17,32,33]. Although t he mixture of c hloroform/methanol/
water i s r egarded a s membrane mimetic, c orresponding

protein samples can only be validated for their physiological
relevance by subsequent functional reconstitution. Protein
structure i s strongly supported to be retained during t he
transfer of the polypeptide from organic solvent to the lipid
environment as was shown f or subunit c [32]. Whereas the
coreconstitution of isolated subunit c has therefore already
been achieved in several cases [13,18], similar experiments
with b
1)34
initially failed [ 17]. Our data clearly de monstrate
that the synthetic peptide b
1)34
reflects functional properties
of intact subunit b in H
+
translocation a nd st rongly argues
in favour of the corresponding NMR structure.
Acknowledgements
Drs O . Y. Dmitriev and R. H. Fillingame (University of Wisconsin
Medical School, Madison, WI, USA) are kindly acknowledged for
generously providing p eptide b
1)34
. This w ork w as supported b y t he
Deutsche Forschungsgemeinschaft (SFB 431-P2) and by the Fonds der
Chemischen Industrie.
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