Structural and functional relationship of ATP synthases (F1F0) from Escherichia coli and the thermophilic bacterium PS3.

Functional compatibility between the F1 and F0 parts of ATP synthases from Escherichia coli (EF1F0) and the thermophilic bacterium PS3 (TF1F0) was analyzed. F1-stripped everted membrane vesicles from both organisms bound the homologous or heterologous F1 part to the same extent. Titration of the reconstituted membrane vesicles with dicyclohexylcarbodiimide revealed a similar sensitivity of the homologous and hybrid F1F0 complexes towards the inhibitor. Furthermore, the heterologous enzymes exhibited ATP-dependent H+ translocation comparable to that of homologous F1F0. Antisera raised against EF1 or subunits a, b, and c of EF0 were analyzed for cross-reactivity with TF1 and TF0. Common antigenic sites have been detected with immunoblot analysis for subunit beta and subunit c of EF1F0 and the corresponding subunits from TF1F0. A weak binding of the anti-a and anti-b antisera with the TF0 part has been observed in an enzyme-linked immunosorbent assay. Based on these findings the structural and functional relationship between the mesophilic and thermophilic ATP synthase complexes is discussed.

Functional compatibility between the F, and Fo parts of ATP synthases from Escherichia coli (EFIFo) and the thermophilic bacterium PS3 (TFIFo) was analyzed. F,-stripped everted membrane vesicles from both organisms bound the homologous or heterologous F, part to the same extent. Titration of the reconstituted membrane vesicles with dicyclohexylcarbodiimide revealed a similar sensitivity of the homologous and hybrid FIFO complexes towards the inhibitor. Furthermore, the heterologous enzymes exhibited ATP-dependent H' translocation comparable to that of homologous FIFo. Antisera raised against EF, or subunits a, b, and c of EFo were analyzed for cross-reactivity with TF, and TFo. Common antigenic sites have been detected with immunoblot analysis for subunit fl and subunit c of EFIFo and the corresponding subunits from TFIFo. A weak binding of the anti-a and anti-b antisera with the TFo part has been observed in an enzyme-linked immunosorbent assay. Based on these findings the structural and functional relationship between the mesophilic and thermophilic ATP synthase complexes is discussed.
The ATP synthase (FIFO) is a key enzyme in energytransducing reactions. Driven by an electrochemical gradient of protons it catalyzes the phosphorylation of ADP (for review, see Ref. 1). This oligomeric complex, which is found in eukaryotic and prokaryotic organisms, is composed of two distinct entities: the F1 part, which carries the catalytic centers (2), is bound to the Fo part (3,4), an integral complex of the inner mitochondrial or thylakoid membrane of eukaryotes or the cytoplasmic membrane of bacteria. One of the most extensively studied ATP synthases is that of Escherichia coli (EFIFo) ( 5 ) . It is composed of eight different subunits, the primary structure of which has been deduced from the DNA sequence (for review, see Ref. 6). The F, part (EFJ which can be dissociated from Fo, is a soluble enzyme of 380 kDa with ATP-hydrolyzing activity (7, 8). The complex is composed of five different subunits which are present in a stoichiometry of nsP3y18,tl (9,10). The Fo part (EFo) in its native environment or reconstituted into phospholipid vesicles exhibits proton-translocating and F1-binding activity (11,12). The purified complex consists of the subunits a, b, and c with a * This work was supported in part by the Deutsche Forschungsgemeinschaft SFB 171, the Niedersachsische Ministerium fur Wissenschaft und Kunst, the Fonds der Chemischen Industrie, and the Studienstiftung des Deutschen Volkes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by a fellowship of the Italian Ministry of Education. § To whom correspondence should be addressed.
stoichiometry proposed to be 1:2:10fl (10). Analysis of deletion mutants (13) and reconstitution studies with isolated Fo subunits (14) revealed that all three subunits are necessary for the formation of an active Fo complex.
The ATP synthase of the thermophilic bacterium PS3 (TFIFo) is also well characterized. The enzyme exhibits an extraordinary stability against heat and denaturing reagents (15), thereby facilitating the biochemical characterization of the complex. Otherwise, this enzyme is very similar to the mesophilic counterpart from E. coli (16). In contrast to EF1, the ATP-hydrolyzing activity of TFl has a temperature optimum at 70 "C (17). The TFo complex is composed of three different subunits termed band 4 (19.0 kDa), band 6 (12.3 kDa), and band 8 protein' (8 kDa) (18). The latter exhibits extensive sequence homology to subunit c1 from E. coli (19).
In this article we have studied the function of F1-stripped membrane vesicles from both organisms reconstituted with homologous and heterologous F,. Immunological studies with antisera raised against components of the ATP synthase from E. coli were carried out to detect possible structural homologies in both complexes. After centrifugation (45 min at 180,000 X g) the incubation was repeated twice for 1 h at room temperature. Finally, the membranes, which exhibited less than 1% of the starting ATPase activity, were resuspended in 50 mM Tris/Cl, pH 8.0, 5 mM MgCl,, 1 mM dithiothreitol, 10% (v/v) glycerol, 6 mM p-aminobenzamidine, 0.1 mM phenylmethylsulfonyl fluoride (5 mg of protein/ml). Everted Flstripped membrane vesicles from PS3 were prepared by the same procedure. EF, (23), EF, (24), and TF, (17) were prepared as described. TFIFo was isolated as described in Ref. 16 with the following modification: the Triton X-100 extract (step 2) was dialyzed overnight at 4 "C against 10 volumes of 50 mM Tris/S04, pH 8.0, 0.5% (v/v) Triton X-100 and applied (400 ml/h) onto a DEAE-cellulose column (5 X 18 cm) previously equilibrated with the same buffer. After washing the column (600 ml/h) with 2 liters of 50 mM Tris/S04, pH 8.0, 0.05 M Na2S04, 0.5% (v/v) Triton X-100, the enzyme was eluted  (v/v) glycerol, 6 mM paminobenzamidine, 0.1 mM phenylmethylsulfonyl fluoride (0.5 mg/ ml). Samples of 2 ml were incubated with EF, (64 pmol X min" X mg-'; 0 ) or TF, (28 pmol X min" X mg"; 0) for 15 min at 37 "C. The membranes were centrifuged at 120,000 X g and washed twice in the same buffer (2 ml). The final pellets were resuspended in the same buffer (2 mg/ml) and analyzed for membrane-bound ATPase activity at the temperatures indicated. ATPase activity the enzyme was precipitated with ammonium sulfate and further purified by gel filtration according to Ref. 16. TF, was prepared from TFIFo by incubation with urea (18). The TF, pellet after the second urea treatment was resuspended to 8 mg/ml in 10 mM Tris/Cl, pH 8.0, 150 mM NaCI, 0.2 mM phenylmethylsulfonyl fluoride, 1% (w/v) cholate.
Immunological Procedures-For immunoblotting (25), antisera against SDS2-denatured subunits of EF, were raised in rabbits as described (25). The antiserum against EF, was prepared by the same immunization scheme as described in Ref. 25 using EF1 (1 mg of protein/injection) denatured in 2% (w/v) SDS. The ELISA was carried out as described in Ref. 26. For this assay antisera were raised against EFo subunits prepared under nondenaturing conditions as described in Ref. 14. Prior to immunization they were tested for reconstitution of an active Fo complex in liposomes (14).
Analytical Procedures-SDS-gel electrophoresis (7.5-17.5% acrylamide gradient gels) was carried out as described in Ref. lyzing activity of EF,. The phosphate liberated was determined by the continuous flow procedure described by Arnold et al. (29). One unit of ATPase activity is defined as the release of 1 pmol of Pi X min" at 37 "C for EF, and 60 "C for TF,.
Overath (Tubingen). All other chemicals were of analytical grade.

RESULTS
The F, part of E. coli or the thermophilic bacterium PS3 can be dissociated from everted membrane vesicles by treatment with buffer of low ionic strength in the presence of EDTA. Thus, these membranes lose their ATPase activity and become proton-permeable due to the protonophoric activity of the remaining membrane-embedded Fo complex. Incubation of F,-stripped membranes with F1 reconstitutes a membrane-bound ATPase activity which is coupled to proton translocation. This approach was used to reconstitute hybrid FIFo complexes starting from F, and F1-stripped membranes of E. coli and PS3.
F1-stripped membranes from E. coli were incubated with EF, or TF, (Fig. 1). Comparing the membrane-bound ATPase activity of the reconstituted vesicles at temperatures suited for the individual F, parts (37 "C for EF, and 60 "C for TF,),   Fig. 4. Incubation with DCCD was carried out as stated in the legend to Fig. 2. ATPase activity was determined at 37 "C for bound EF, and 60 "C for bound TF,.  binding was achieved after addition of 10 units of F1 to 1 mg of membrane protein (Fig. 1). In each case the membranebound ATPase activity is sensitive towards the energy transfer inhibitor DCCD (Fig. 2), indicating also a specific binding of TF, to EFo. This was confirmd by determining ATPdependent H' translocation (Fig. 3). The data indicate that the TF,EFo hybrid enzyme exhibits proton-translocating ac- tivity; however, the extent of fluorescence quenching was higher in vesicles reconstituted with homologous EF,. Since ATP-dependent proton translocation was assayed at different temperatures (37 "C for EF,, 50 "C for TF,) it could not be discriminated between a better fitting of EF, compared to TF, to the vesicles or a higher proton permeability of the mesophilic membranes at the elevated temperature.
Similar experiments were also carried out with F1-stripped membranes from PS3. With one exception, reconstitution of these vesicles with EFl or TF, resulted in membrane-bound ATPase activities (Fig. 4) comparable to those presented in Fig. 1: whereas E. coli membranes reconstituted with EF, exhibited a substantial ATPase activity at 60 "C ( Fig. 1) the hybrid enzyme EFITFo was completely inactive under the same conditions (Fig. 4). This observation might indicate a stabilizing effect of EFo on EF, ATPase activity at high temperature which could not be conferred by the thermophilic TFo part. The incubation of reconstituted membranes with DCCD revealed that at low inhibitor concentrations a higher sensitivity of TFITFo compared to EFITFo was observed (Fig.  5). In the ATP-dependent proton translocation assay, measured for both the homologous and heterologous reconstituted vesicles, TFITFo showed a higher initial quenching rate than the EFITFo hybrid enzyme (Fig. 6); however, the extent of fluorescence quenching was the same.
Structural similarities which might be the bases for the observed functional compatibility has been studied by immunological methods using F, and Fo from both organisms (Fig. 7A). In an immunoblot experiment the Fl and FO parts from both bacteria were probed with antibodies against EF, complex or SDS-denatured subunits of the EFo part. As also observed by others (30), the anti-EF1 antiserum reacted only with subunits (Y and p of EF,. Therefore, it was not possible to detect common antigenic sites for subunits y, 6, and t of EF, and TF,. A cross-reactivity of the antibodies against EF1 was observed for subunit p of TF, (Fig. 7B). Of the antibodies raised against the individual subunits from EFo only the anti-c antibodies exhibited cross-reactivity with band 8 protein (DCCD-binding protein of TFo). Interestingly, in this case the anti-c antibodies were most heavily associated with a 24-kDa protein band, which probably constitutes a trimer of the 8-kDa polypeptide (Fig. 7 E ) . The appearance of such oligomers in SDS-gel electrophoresis was also observed for subunit c (Fig. 7E) and might be related to the distinct hydrophobicity of DCCD-binding proteins. In addition, the TFo complex was also probed with antisera against nondenatured subunits a, b, and c in an ELISA. In this experiment the cross-reactivity between TFo and the anti-c antiserum could be confirmed (Fig. 8). Compared with EFo the binding avidity to TFo was significantly reduced. The antisera against nondenatured subunits a and b exhibited a weak cross-reactivity with TFo when probed in a competitive ELISA (Fig. 9). Based on these results it is difficult to judge whether structural similarities exist between subunits a and b of EFo and homologous subunits of TFo.

DISCUSSION
Reconstitution of hybrid enzymes with subunits from different species might be a tool to evaluate phylogenetic relationships as well as to obtain information about essential features of enzyme function. For the ATP synthase this approach was pioneered by the work of Futai et al. (31) who reconstituted hybrid ATP-hydrolyzing complexes from isolated subunits a , p, and y of E. coli and PS3. In this study we demonstrated that the F, and Fo parts from E. coli and the thermophilic bacterium PS3 are functionally compatible. Similar experiments were successfully carried out by Hsu et al. (32) working with F, and Fo from the closely related bacteria E. coli and Salmonella typhimurium. Recently, also the binding of TF, to thylakoid membranes from lettuce chloroplasts, which were depleted of CF,, has been demonstrated (33). In this case the rebinding of TF, was not coupled t o photophosphorylation, but the membranes became sealed against proton leakage. The authors concluded that "TF, appears to have only a structural role of sealing the membrane, but does not participate in the catalytic process itself." With E. coli and PS3 each combination of F, with homologous or heterologous F,-depleted membranes resulted in the formation of an ATP synthase complex which was sensitive towards DCCD and which exhibited ATP-dependent H'translocation. The better fitting of TF1 to E. coli than to thylakoid membranes is in agreement with the evolutionary tree based on amino acid sequence homologies of DCCD-binding proteins (4).
Immunological analyses indicate structural similarities only for subunit 0 of F, and the DCCD-binding protein of Fo. The data for subunit /3 are in agreement with DNA sequence analyses (34) revealing that this protein is more conserved than subunit a. This high conservation is also underlined by the reconstitution of a functional ATP synthase from 0-less chromatophores of Rhodospirillum rubrum with aubunit p from EF, (35) or CF, (36). These properties might reflect an intimate role of this subunit in the catalytic process (34). The cross-reactivity of band 8 protein of TFo with anti-c antibodies might be related to high sequence homology of both proteins. Positions of conserved amino acid residues are clustered at the polar loop in the center of the polypeptide chain, which is considered to be located at the F, binding site of the Fo complex (4, 37, 38). The poor cross-reactivity of TFo towards antisera against subunits a and b of EFo does not reflect the functional binding of the heterologous parts of both ATP synthases. Especially the hydrophilic and immunogenic (23,38) carboxyl-terminal region of subunit b, which plays a crucial role in EF1 binding, was expected to exhibit structural similarity to the hydrophilic band 6 protein of TFo, which also has TF, binding activity (18). On the other hand, experimental evidence indicates that also subunits c and 8 participate in the interaction of EF, and EFo: F1 binding to subunit c isolated with chloroform/methanol was detected by a solid phase radioimmune assay (39); after treating membranes of E. coli with urea and taurodeoxycholate, subunit @ exhibited a tight membrane binding property (40); subunit / 3 coulr' be cross-linked to subunit a by different bifunctional reag mts (41). Rc constitution studies with isolated subunits from EFo s tggests that the F, part only recognizes an oligomeric struct i r e conferred by all subunits of Fo (14). On the other hand, antibodies against single subunits might recognize those parts of the protein which are not essential for the interaction of F, and Fo and, therefore, could be quite different in both bacteria. Thus, it can be hypothesized that subunits with different amino acid sequences, which might have conserved amino acid residues at positions of strategical importance for enzyme function (e.g. polar loop of the DCCD-binding proteins; 37) form complexes with structures similar enough to allow a specific interaction between heterologous oligomers. In this context it is interesting to note that in immunoblot analysis an oligomeric form of band 8 protein exhibits a stronger cross-reactivity towards anti-c antibodies than its monomeric counterpart. A certain deviation in primary structure of TFIFo subunits is also expected to confer the thermostability to this ATP synthase. Further sequence data of TFIFo subunits will allow a deeper insight into the structural basis of functional compatibility between F, and Fo of E. coli and PS3.