This demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H+translocation across the membrane, whereas the binding of F1to FOis mainly triggered b
Trang 1The transmembrane domain of subunit b of the Escherichia coli
F1FO ATP synthase is sufficient for H+-translocating activity
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 functional
H+-translocating FO complex both in vivo and in vitro
Whereas the very C-terminus of subunit b interacts with F1
and plays a crucial role in enzyme assembly, the C-terminal
region is also considered to be necessary for proper
recon-stitution of FOinto liposomes Here, we show that a
syn-thetic peptide, residues 1–34 of subunit b (b1)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 forming an active FO
complex when coreconstituted with purified ac subcomplex
H+translocation was shown to be sensitive to the specific
inhibitor N,N¢-dicyclohexylcarbodiimide, and the resulting
FOcomplexes were deficient in binding of isolated F1 This
demonstrates that only the membrane part of subunit b is sufficient, as well as necessary, for H+translocation across the membrane, whereas the binding of F1to FOis mainly triggered by C-terminal residues beyond Glu34 in subunit b Comparison of the data with former reconstitution experi-ments additionally indicated that parts of the hydrophilic portion of the subunit b dimer are not involved in the process
of ion translocation itself, but might organize subunits a and c in FO assembly Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic b1)34
Keywords: F1FO ATP synthase; subunit b; reconstitution; proton translocation; Escherichia coli
Membrane-bound F-type ATPases (F1FO) occur
ubiqui-tously in mitochondria, chloroplasts 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 F1FO complexes share high
homology with respect to the mechanism of catalysis
Although there is still some controversy [1], it is generally
accepted that ion translocation through the transmembrane
domain (FO) is coupled to ATP synthesis/hydrolysis in the
peripheral catalytic domain (F1) via a rotary mechanism [2]
Thus, the structural classification of the enzyme in F1
(subunit composition a3b3cde in Escherichia coli) and FO
(ab2c10) [3] is different compared to a functional division in
rotor and stator During coupled catalysis, H+
transloca-tion through FOor ATP hydrolysis in F1generates a rotary
movement of the centrally 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 F1FO complex, which is composed at least of the two copies of subunit b [6,7] During catalysis, the subunit b dimer is supposed to undergo transient elastic deformation in order to compen-sate for the torque, which is built up by the propelling rotor [4,8,9] Finally, torque is released by conformational chan-ges leading to either H+ pumping through FO or ATP synthesis in F1 The molecular switch, by which one or the other direction of catalysis is preferred, has recently been attributed to the e subunit [10]
In being part of the stator element of the F1FOcomplex, the subunit b dimer makes both multiple contacts with subunits a, b and d of the F1 part [11] as well as with subunit a of FO[12,13] There are several lines of evidence that suggest that subunit b is absolutely essential for the binding of F1to FO[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] However, these domains have been shown not to function independently from each other The binding constant of the soluble C-terminal domain of subunit b to subunit d for example is much too low to withstand the torque generated during catalysis [2] Deletion muta-genesis of subunit b in assembled F1FOrevealed 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 interplay of the 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: joerg.greie@biologie.uni-osnabrueck.de
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 ATP synthase (EC 3.6.1.34).
(Received 31 March 2004, revised 25 May 2004,
accepted 28 May 2004)
Trang 2b dimer seems to be a prerequisite at least for a proper
assembly of the F1FOcomplex
A few years ago, the monomeric structure of a synthetic
peptide corresponding to the membrane-spanning domain
of subunit b (b1)34) has been solved by NMR spectroscopy
[17] According to these data each of the two b subunits is
predicted to form one transmembrane a helix, which, based
on chemically induced cysteine cross-linking experiments in
assembled F1FOcomplexes, are supposed to come together
to form a dimer However, b1)34was not functional when
used for the coreconstitution of a H+-translocating FO
complex in these studies, although it has previously been
shown that reconstitution of FO from single subunits is
possible [18] This lends support to the notion that the
C-terminal domain of subunit b is also involved in the
assembly of an active FOcomplex [17] Thus, although in
good accord with cross-linking studies and secondary
structure predictions, the NMR structure of the synthetic
b1)34has not yet been functionally validated, either in vivo
or in vitro
Here, we report that by the use of preformed ac
subcom-plexes it was possible to coreconstitute b1)34into functional
FO complex capable of N,N¢-dicyclohexylcarbodiimide
(DCCD)-sensitive H+ translocation Hence, whereas the
membrane domain is sufficient to couple subunits a and c
during ion translocation, the soluble part of subunit b seems
to be necessary for the proper assembly of subunits a and c
As expected, the resulting FOcomplexes were deficient in
the binding of F1, further restricting F1binding sites to the
C-terminal domain beyond residue Glu34 of the subunit b
dimer
Experimental procedures
Bacterial growth
Escherichia colistrain DK8 [19] lacking the atp operon was
transformed with plasmid pBWU13 [20] carrying the atp
operon except for atpI Cells were grown on minimal
medium supplemented with thiamine (2 lgÆmL)1), thymine,
asparagine, isoleucine and valine (50 lgÆmL)1 each)
together with 75 mMglycerol as carbon source, harvested
at late exponential phase and stored at)80 C
Preparative procedures
FO and F1 complexes as well as subunit b and ac
subcomplex isolated from dissociated FOcomplexes were
prepared as described [14,15,21] Synthetic peptide b1)34
(2 mM solution in chloroform/methanol/H2O 4 : 4 : 1,
v/v/v) was a kind gift of O Y Dmitriev and R H
Fillingame (University of Wisconsin Medical School,
Madison, WI, USA), 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 mgÆmL)1 (Avanti Pro Lipids) were dried under a
gentle stream of argon and redissolved to 40 mgÆmL)1in
detergent buffer before the addition of protein The weight
ratio of FO to phospholipid was 1 : 50 In the case of subcomplexes and single subunits except b1)34, the corres-ponding amount of protein was initially calculated using the particular stoichiometric abundance with respect to a stoichiometry of ab2c10 for FO In either case, proper stoichiometric amounts of particular FO subunits were finally confirmed by SDS/PAGE For samples containing
b1)34present in chloroform/methanol/H2O 4 : 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 calculated based on the amino acid analysis performed during the synthesis of b1)34and calibrated with the FOsample assuming a stoichiometry of ab2c10 Dialysis was carried out for 40 h at 4C changing the buffer once Loading of proteoliposomes with K+ was carried out
as described [13] For the inhibition of passive H+ translocation, samples were treated with 50 lM 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 of 2 lM valinomycin for induction
of the K+ diffusion potential After rebinding of F1, reconstituted DCCD-sensitive ATPase activities were measured according to Steffens et al [23] Protein concen-trations were determined with the bicinchoninic acid assay (Pierce) used as recommended by the 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 FO reconstituted from subcomplexes was sensitive to their particular stoichiometric amount in the reconstitution assay [13,14] Hence, in order to compare the effect of b1)34with intact subunit b, it was important
to determine and exactly adjust stoichiometric propor-tions of both b and b1)34with respect to the preformed ac subcomplexes Whereas the concentration of the b1)34 sample was determined by amino acid analysis [17], the determination of protein concentrations of FO, ac subcomplex and subunit b by conventional colourimetric assays revealed to be biased by partial impurities of the preparation and by the particular buffer composition as well as by the specific biochemical properties of each polypetide (data not shown; also compare [13]) Hence, the stoichiometric ratios of subcomplexes (except for
b1)34) mixed for reconstitution were only initially judged
by the colourimetric bicinchoninic acid assays of protein samples, but were finally adjusted by the densitometric comparison of silver stained protein bands in SDS/PAGE (Fig 1) Aliquots were taken directly from the samples before the addition of lipid or lipid plus b1)34 The comparison of corresponding band intensities revealed a proper stoichiometric relationship of FOsubunits in each
of the samples taken for reconstitution
Trang 3Reconstitution of FOfromb1)34and preformed
ac subcomplexes
Previous studies dealing with the reconstitution of
chloro-form/methanol extracted subunit c revealed the necessity for
the addition of detergent to the sample prior to the removal
of the solvent by evaporation in order to facilitate
resolu-bilization and to prevent partial denaturation of the
polypeptide [18] This could be overcome by the direct
addition of the protein to the lipid solution, also present in
organic solvent, prior to the evaporation [13], thereby
transferring the polypeptide from the solvent immediately
into the lipid environment without the need for additional
detergents Thus, the same technique was successfully used
for the reconstitution of b1)34 present in chloroform/
methanol/H2O 4 : 4 : 1 (v/v/v)
Reconstitution of preformed ac subcomplexes with intact
subunit b as well as with the subunit b transmembrane
domain b1)34resulted in the formation of functional FO
complexes as demonstrated by rapid K+
/valinomycin-triggered H+uptake into proteoliposomes (Fig 2) Traces
of passive H+translocation were in good accordance with
those obtained for the reconstituted FOcomplex from single
subunits a, b and c [18] Whereas significant initial rates of
H+ translocation could already be observed with a
stoichiometric ratio of intact subunit b and ac subcomplex,
a 6.6-fold molar excess of b1)34 was necessary to obtain
similar results Reconstituted ac subcomplexes without
added b subunits, as well as the control containing 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 ATPase activity in these
samples with respect to the control (see below)
Quantitative titration ofb1)34in reconstitution Comparable rates of H+translocation for intact subunit b and b1)34were only obtained with a stoichiometric surplus
of the latter Recent studies dealing with the reconstitution
of chloroform/methanol-extracted subunit c also revealed the necessity of an excess of the polypetide, which is also present in chloroform/methanol/H2O prior to reconstitu-tion [13] This points to a more general than specific effect due to the use of organic solvent in protein preparation Furthermore, the amount of b1)34taken for coreconstitu-tion was stoichiometrically calibrated with the protein concentration of the FO sample, which was found to be biased by several factors
However, in order to further elucidate saturating condi-tions of passive H+translocation against the stoichiometric abundance of b1)34, preformed ac subcomplexes were titrated with increasing amounts of b1)34in the reconstitu-tion assay (Fig 3) Again, low basal H+ translocation activity could be observed in case of ac subcomplex by itself (2.2 lmol H+Æmin)1Æmg)1), whereas the control containing
a 13.3-fold molar excess of b1)34 only showed unspecific linear H+ drift (0.2 lmol H+Æmin)1Æmg)1) instead of a corresponding exponential rise in translocation activity following the potential jump This unspecific H+drift is
Fig 1 Quantitative comparison of F O subunits in subcomplexes mixed
for reconstitution Silver stained SDS/PAGE of samples taken directly
for reconstitution Aliquots of 2 lL were taken for electrophoresis
prior to the addition of lipid in the reconstitution procedure Lane 1,
buffer control; lane 2, F O (7.2 lg); lane 3, ac subcomplex; lane 4,
ac + b; lane 5, as lane 3, prior to addition of lipid plus b1)34; lane 6,
subunit b MW, molecular mass marker.
Fig 2 Passive H+translocation of F O obtained by coreconstitution of
b1)34into proteoliposomes F O , ac subcomplex and intact subunit b were reconstituted in stoichiometric amounts In the case of b1)34, a 6.7-fold stoichiometric excess was used for coreconstitution with ac, whereas a 13.3-fold stoichiometric excess was used as a control Passive
H + uptake was measured by 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.
Trang 4most likely due to the high amount of membrane
pro-tein present in the proteoliposome, as the 13.3-fold
stoichiometric amount of b1)34 was used as control
Generally, unspecific H+ drift can be clearly separated
from specific potential-driven H+translocation because the
former results in a linear curve whereas the latter leads to an
initial exponential rise on top of the drift However, the use
of a 3.3-fold molar excess of b1)34revealed an only slight
increase in passive H+ translocation activity when
core-constituted with ac subcomplex (4.2 lmol H+Æmin)1Æmg)1)
In contrast, a strong effect was observed in the case of the
6.7-fold stoichiometric amount (6.8 lmol H+Æmin)1Æmg)1),
whereas no further increase was obtained, even with a
13.3-fold molar excess of b1)34 (4.5 lmol H+Æmin)1Æmg)1)
Instead, a decrease in the initial H+uptake rate could be
observed, which is due to the already described negative
effect of unspecific H+drift on the 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 b1)34was 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 F1
From previous studies it is known that the C-terminal
hydrophilic domain of the subunit b dimer is involved in the
binding of F1[5,14,15] Deletion mutagenesis of hydrophilic
segments of subunit b more proximal to F also revealed
defects in F1FO assembly [16] Interactions in coupling between F1and FOhave also been shown to occur via the subunit c ring, although these are not sufficient for the tight binding of F1 to ac subcomplexes [8] It is still unknown whether the N-terminal domain of subunit b is involved in
F1interaction, either in a direct or indirect way, the latter of which could occur via a possible stabilizing effect of the subunit b transmembrane domains on the ac subcomplex Therefore, FO complexes reconstituted from ac subcom-plexes and b1)34 were tested for their F1 binding ability (Table 1) Significant rates of reconstituted ATPase activity were obtained in the case of proteoliposomes containing FO
and ac + b, which 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 of b1)34, there was no corresponding increase 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 FO subunits, which can thus far not be avoided during the preparation (compare Figs 1 and 2) In conclusion, FOcomplexes assembled from ac subcomplexes and b1)34are not competent in F1binding due to the lack of corresponding sites of interaction Thus, the N-terminal stretch of residues of subunit b up to Glu34 is not sufficient
to trigger F1 binding even in assembled FO complexes capable of H+translocation
Inhibition of reconstituted H+translocation by DCCD
In order to demonstrate that the passive H+translocation observed for FOcomplexes reconstituted from ac + b1)34is specific, both ac + b and ac + b1)34were incubated with and without 50 lM DCCD prior to the measurements (Fig 4) Both resulting FOcomplexes showed comparable rates of inhibition, whereas the addition of a corresponding amount of ethanol to the noninhibited 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 of b1)34 were used to
reconstitute ac subcomplexes Passive H+uptake was measured by use
of a K + /valinomycin diffusion potential Traces are correspondingly
labelled The values in parentheses in the case of b1)34indicate the
corresponding stoichiometric amount, for example 3.3· means a
3.3-fold stoichiometric excess of the polypeptide with respect to a
stoi-chiometry of ab 2 c 10 for F O The addition of valinomycin is indicated by
the arrow.
Table 1 Reconstituted coupled ATPase activities 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 b1)34was used
in the reconstitution The values in parentheses indicate the corresponding stoichiometric amount present, for example 3.3· means
a 3.3-fold stoichiometric excess of the polypeptide with respect 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)1Æmg)1)
Trang 5of b1)34and intact subunit b clearly argues in favour of a
homologous function in the H+translocation process and,
hence, revealed that b1)34is capable of forming functional
FOcomplexes 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 F1 and FO [6] This physical linkage
between the site of 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 forming a
parallelogram-like spring 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 FOand F1subunits are
supposed to occur mainly in the N- and C-terminal regions,
respectively [11,13], whereas the middle part of the
poly-peptide was shown to adopt a right-handed coiled-coil
structure essential for dimerization and presumably
involved in the transient storage of energy [26] Due to 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 F1in solution [28], the interplay of all three FO
subunits is necessary for the reconstitution of F1ATPase activity on the membrane level Neither the subunit b dimer [8] or the ab2stator subcomplex [13], nor subunit a together with the ring of c subunits [14] or the subunit c ring alone [13], can be held responsible for F1binding Thus, subunit interactions occurring solely within the central or the second stalk are not sufficient to couple F1to FOon the functional level of the membrane
When separated in vitro, both F1and FOact independ-ently according to their function in vivo, i.e ATP hydrolysis
or H+ translocation, respectively Thus, it should be possible to discriminate between residues in subunit b which are essential for the function of FOor the coupling
to F1when reconstituted together with other FOsubunits 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 of the synthetic peptide b1)34 corres-ponding to the transmembrane domain of subunit b has been determined at high resolution with two-dimensional
1H NMR in organic solvent [17] Although in good accord with cross-linking studies and secondary structure predic-tions, this NMR structure has not yet been functionally validated, either in vivo or in vitro Although the reconsti-tution of functional FOcomplexes from single a, b and c subunits has already been reported [18], the same approach initially failed in the case of b1)34, from which it was deduced that the C-terminal segment of subunit b is essential for the reconstitution and functional assembly of
an active FO complex [17] However, in this case coreconstitution of b1)34 was performed by use of single subunits a and c
In contrast, our data clearly demonstrate, that by use of preformed ac subcomplexes, only the membrane part of subunit b is sufficient, as well as necessary, for H+ translocation across the membrane, whereas the binding
of F1to FOis triggered by C-terminal residues in subunit b This clearly attributes two distinct functions to the subunit b dimer, which are spatially separated
An excess of b1)34with respect to isolated subunit b was necessary to obtain comparable rates of passive H+ translocation when coreconstituted with ac subcomplex The necessity of an excess of free FOsubunits, which are present in chloroform/methanol/H2O prior to the reconsti-tution, is already known from other recent experiments [13] and might in part result from potential damage of the polypeptides during the extraction in organic solvent Furthermore, the protein is likely to integrate in different orientations with respect to the coreconstituted subcom-plexes in general, which decreases the fraction of properly assembled protein complexes In addition, b1)34was shown
to be a monomer in chloroform/methanol/H2O [17], which might produce nonfunctional antiparallel orientations of the resulting dimer during reconstitution H+translocation rates were generally lower in the case of b1)34than in the case of intact subunit b This is due to the need for a relatively high protein content in the membrane due to the different possible orientations of b1)34, which leads to a decreased driving force caused by unspecific H+leakage following the potential jump In addition, the chemically
Fig 4 DCCD-inhibited H+translocation of ac subcomplexes
recon-stituted with subunit b or b 1)34 The ac subcomplexes were reconstituted
either with stoichiometric amounts of subunit b or with a 6.7-fold
stoichiometric excess of b 1)34 Samples were treated with 50 l M
DCCD for 5 min in the assay medium prior to the measurements.
Passive H+uptake was measured by use of a K+/valinomycin
diffu-sion potential Traces are correspondingly labelled As a control,
ac + b plus the corresponding amount of ethanol as in the DCCD
inhibition assays was used (top) The addition of valinomycin is
indi-cated by the arrow.
Trang 6synthesized b1)34 certainly represents a more artificial
population of the polypeptide than a subunit b dimer
purified by dissociation from already functional FO
com-plexes, thereby exhibiting a generally lower activity This
view is supported by an analogous set of experiments with
intact subunit b prepared by denaturation with SDS and
refolding according to Greie et al [8] This b subunit also
showed a reduced rate of H+conductivity in the
corecon-stitution assay with respect to intact subunit b prepared by
dissociation of FOcomplexes, with rates more comparable
to that of b1)34 (data not shown) The traces of DCCD
inhibition were again comparable to those obtained by
Dmitriev et al for FOcomplexes reconstituted from single
subunits a, b and c [18]
However, isolated ac subcomplexes were shown to be
deficient in H+conduction, although both subunits directly
involved in ion translocation are present [8] Our results
demonstrate that the presence of the transmembrane spans
of subunit b are both sufficient as well as necessary to build
up a functional H+-translocating FOcomplex 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 recently demonstrated by the
purification of a stable ab2subcomplex [13] Less extensive
contact with the rotating subunit c oligomer can be derived
from cross-linking data [29]
As already mentioned, functional coreconstitution of
b1)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 assembly or education of subunits a and c
This view is supported by the fact that FO complexes
containing subunit b were shown to assemble
unidirection-ally into the outer shell of the multilamellar proteoliposome
during reconstitution [14,15], which is most likely due to the
large hydrophilic domain of the subunit b dimer Thus, this
would imply that the C-terminal domain of b might be
important not for the insertion of FO subunits into the
membrane itself but for the proper alignment of FOsubunits
during assembly As a consequence, subcomplexes lacking
this domain, as in the case of ac, ac + b1)34and b1)34alone,
would tend to assemble rather randomly with respect to
their topological orientation, thus leading to a significant
decrease of functional FOcomplexes compared to samples
containing intact subunit b This is exactly what was found
in the reconstitution of b1)34
That distinct parts of the hydrophilic portion of subunit b
are involved in F1FO assembly can also be derived from
deletion mutagenesis experiments [16] Several deletions
with increasing sizes affecting residues 50–60 were shown to
be impaired in the assembly process, but were not affected in
activity Thus, this stretch of residues is probably important
for assembly but not directly involved in catalytic function
The disruption of interactions with subunit a during
assembly has been discussed Recent cross-linking
experi-ments demonstrated a close proximity of a putative a-helical
face of subunit b between residues Ala32 and Arg36 and
hydrophilic loops of subunit a [30,31] In combination
with our results these data suggest that residues between
positions 35 and 60 might be important for the assembly of subunits a and c
The determination of high resolution three-dimensional protein structures from FOsubunits has only been accom-plished in case of subunits c and b1)34 by use of single monomeric polypeptides prepared in organic solvent [17,32,33] Although the mixture of chloroform/methanol/ water is regarded as membrane mimetic, corresponding protein samples can only be validated for their physiological relevance by subsequent functional reconstitution Protein structure is strongly supported to be retained during the transfer of the polypeptide from organic solvent to the lipid environment as was shown for subunit c [32] Whereas the coreconstitution of isolated subunit c has therefore already been achieved in several cases [13,18], similar experiments with b1)34initially failed [17] Our data clearly demonstrate that the synthetic peptide b1)34reflects functional properties
of intact subunit b in H+translocation and strongly 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 peptide b 1 )34 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 431-P2) and by the Fonds der Chemischen Industrie.
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