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: 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 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. References 1. Berden, J.A. & Hartog, A .F. (2000) A nalysis of t he nucleotide binding sites of mitochondrial A TP synth ase p rovides evide nce for a two-site catalytic mechanism. Biochim. Biophys. Acta 1458, 234– 251. 2. Weber, J. & S enior, A.E. (2003) ATP synthesis driven by proton transport in F 1 F O -ATP synthase. FEB S Lett. 545 , 61–70. 3. Fillingame, R.H. & Dmitriev, O.Y. 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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¨. 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