The b and delta  Subunits of the Escherichia coli ATP Synthase Interact via Residues in their C-terminal Regions*

Derek T. McLachlinDagger , Jennifer A. Bestard, and Stanley D. Dunn§

From the Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5C1

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

An affinity resin for the F1 sector of the Escherichia coli ATP synthase was prepared by coupling the b subunit to a solid support through a unique cysteine residue in the N-terminal leader. b24-156, a form of b lacking the N-terminal transmembrane domain, was able to compete with the affinity resin for binding of F1. Truncated forms of b24-156, in which one or four residues from the C terminus were removed, competed poorly for F1 binding, suggesting that these residues play an important role in b-F1 interactions. Sedimentation velocity analytical ultracentrifugation revealed that removal of these C-terminal residues from b24-156 resulted in a disruption of its association with the purified delta  subunit of the enzyme. To determine whether these residues interact directly with delta , cysteine residues were introduced at various C-terminal positions of b and modified with the heterobifunctional cross-linker benzophenone-4-maleimide. Cross-links between b and delta  were obtained when the reagent was incorporated at positions 155 and 158 (two residues beyond the normal C terminus) in both the reconstituted b24-156-F1 complex and the membrane-bound F1F0 complex. CNBr digestion followed by peptide sequencing showed the site of cross-linking within the 177-residue delta  subunit to be C-terminal to residue 148, possibly at Met-158. These results indicate that the b and delta  subunits interact via their C-terminal regions and that this interaction is instrumental in the binding of the F1 sector to the b subunit of F0.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the process of oxidative phosphorylation or photophosphorylation, the electron transport chain generates a transmembrane proton gradient. The ATP synthase, or F1F0-ATPase, allows protons to flow down this electrochemical gradient and uses the energy obtained to synthesize ATP (for reviews, see Refs. 1-4). Under appropriate conditions ATP synthase can hydrolyze ATP to pump protons. The enzyme is composed of two sectors; the F0 sector is membrane-integral and is responsible for proton translocation, and the F1 sector is attached to the membrane via F0 and houses the catalytic sites for ATP synthesis. F1 is easily detached from the membrane and can be purified as a soluble protein with ATPase activity.

ATP synthases contain at least eight types of subunits. In the relatively simple enzyme from Escherichia coli, the F1 sector has the stoichiometry alpha 3beta 3gamma 1delta 1epsilon 1, whereas F0 is composed of three subunits of stoichiometry a1b2c9-12. The a and c subunits, but not b, contain residues essential for the translocation of protons across the membrane (3). The 156-residue b subunit is believed to span the membrane once at its hydrophobic N terminus, whereas the remainder of the protein is very hydrophilic. b is thought to exist as a dimer in the complex (5-7), and proteolysis studies have shown that the hydrophilic region of b is required for the association of F1 with the membrane (7-9). Removal of two residues from the C terminus of b disrupts normal assembly of the complex (10), as does mutation of Gly-131 to aspartate (11). Thus the b subunit is essential for linking the F1 and F0 sectors and likely plays a key role in the coupling of energy from proton translocation to ATP synthesis.

The crystal structure of the mitochondrial F1 has shown that the alpha  and beta  subunits alternate in a hexagonal ring structure, with two long alpha -helices from gamma  extending into a hole in the center of the ring (12). Recent evidence strongly suggests that gamma  and probably epsilon  rotate relative to alpha  and beta  during catalysis (13-16). Several studies have implied that the delta  subunit is located near the top of the alpha beta cluster (17-20). Like b, delta  is required for the binding of F1 to F0. Membranes of mutant E. coli strains expressing truncated forms of delta  showed little ATPase activity (21), suggesting that F1 cannot bind to the membrane in the absence of delta . In truncation and mutagenesis studies using the mitochondrial (22, 23) and yeast (24) homologues of delta , called OSCP1 for oligomycin sensitivity conferring protein, the C-terminal region of OSCP was implicated in F0 binding.

The only subunit of F0 able to span the distance from the membrane to the top of F1 is b. The hydrophilic portion of b is dimeric, highly alpha -helical, has an elongated shape, and binds to F1 (25). Although chemical cross-linking of E. coli ATP synthase has failed to reveal b-delta cross-links, such products have been obtained with the chloroplast (26) and mitochondrial (27) enzymes. In the chloroplast work, the cross-link produced by 1-ethyl-3,3-(dimethylaminopropyl)-carbodiimide was mapped to the C-terminal part of b. The site in delta  was determined to be within the cyanogen bromide fragment encompassing residues Val-1 to Met-165 of the 187-residue polypeptide. Recent studies (28-31) have demonstrated the interaction of E. coli b and delta  in the absence of other subunits.

In the present work we examine the interaction between E. coli b and F1. An F1 affinity resin has been generated by linking the hydrophilic portion of b to a solid support. By using binding assays based on this resin, analytical ultracentrifugation, and chemical cross-linking, we have gathered evidence to demonstrate an interaction between residues at the C terminus of b and the C-terminal region of delta .

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Construction of Plasmids-- Molecular biological procedures were carried out as described by Sambrook et al. (32). Plasmid pMR2, which was used as an intermediate during the construction of pJB2, was generated from pSD80 (33) by elimination of an NdeI site outside of the multiple cloning region, followed by insertion of the PCR-amplified uncF gene from pSD51 (25) into the EcoRI and HindIII sites. The PCR primer was designed such that the initiating ATG codon is part of an NdeI site.

Plasmid pJB2, encoding the bMERC protein, was constructed as follows. pDM3 (34) was used as the template for PCR, using the mutagenic primer 5'-GCGCATATGGAACGTTGCTCGAATTCCCACTACG-3', the 5' end contains an NdeI site and the 3' end is complementary to the beginning of the gene encoding b24-156. The initial ATG codon is underlined. The second primer for PCR was the M13 forward sequencing primer. The resulting PCR product was inserted into the NdeI and HindIII sites of pMR2 to encode a protein beginning with the amino acid sequence MERCSNSH followed by residues Tyr-24 through Leu-156 of the b sequence. The entire open reading frame was sequenced to ensure that no mutation had arisen during the PCR.

Plasmids expressing other variants of b24-156 (b24-155, b24-152, and the mutations D150C, K151C, E155C, and 158C) were based on pDM3 (34). In general, mutagenic PCR primers were designed to encode the desired mutation and were cloned into pDM3 using unique restriction endonuclease sites. Correct incorporation of the desired mutations into all of the above plasmids was determined by DNA sequencing. To introduce mutations into the full-length b protein, appropriate restriction endonucleases were used to cut the desired fragment from the pDM3-based plasmid and transfer it to pDM8 (34). The construction of plasmid pSD114 encoding b34-156 has been described previously (34).

Expression and Purification of Proteins-- b24-156 and b34-156 were expressed and purified as described (34). Cysteine-containing and truncated forms of b24-156 as well as bMERC were expressed and purified in the same manner as b24-156, except that proteins containing cysteine residues were purified in the presence of 1 mM dithiothreitol (DTT). Plasmids encoding wild type or mutated full-length b were transformed into the uncF E. coli strain KM2 (35). Expression of the proteins and preparation of membranes were performed as described (34). The delta  subunit was expressed and purified as described previously (31). F1 was purified by standard methods (36).

Production of the F1 Affinity Resin-- DTT was removed from bMERC by passing it through a Sephadex G-25 size exclusion column equilibrated with 50 mM triethanolamine HCl (TEA-HCl), pH 7.5, 1 mM EDTA. Sulfo-link Coupling gel, obtained from Pierce, was washed with 8 volumes of 50 mM TEA-HCl, pH 7.5, 5 mM EDTA before addition of 2.5 mg of bMERC per ml of resin. The mixture was incubated at room temperature with agitation for 1 h, and then excess buffer was removed and assayed for protein. Based on the amount of protein remaining in the solution, the amount of bMERC bound to the resin was inferred to be 2.2 mg per ml of resin. The resin was then incubated at room temperature in 50 mM Tris-HCl, pH 8.5, 1 mM EDTA containing 50 mM cysteine to block any unreacted thiol-binding sites. After 1 h, excess buffer was removed; the resin was washed with 1 M NaCl, and it was resuspended to its original volume in buffer at pH 7.4. The resin was stored at 4 °C. A control resin was prepared by incubating the Sulfo-link Coupling gel with 50 mM Tris-HCl, pH 8.5, 1 mM EDTA containing 50 mM cysteine instead of the bMERC protein.

Assays for F1 Binding by the Affinity Resin-- Two assays for F1 binding were used. In the "soluble ATPase activity" assay, 31.3 µg of F1 supplemented with 0.8 µg of the delta  subunit was incubated with 20 µl of the affinity resin in a final volume of 50 µl of buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2 mM ATP, 10% glycerol, and 0.15 mg/ml BSA (binding buffer). The delta  subunit was added to the assay to make up for any deficiency of that subunit in the F1-ATPase, since a fraction of delta  is easily lost during purification of the complex (37). After gentle agitation for 1 h at room temperature, the mixture was centrifuged to pellet the resin. The ATPase activity of the supernatant was assayed as described (37).

In the "SDS-PAGE" assay, F1, delta , and the affinity resin in the amounts described above were mixed in 250 µl of the binding buffer. After the 1-h incubation and centrifugation, the supernatant solution was discarded, and the pellet was resuspended in 50 µl of SDS-PAGE sample buffer containing DTT. After heating at 100 °C for 10 min, the mixture was centrifuged to sediment the resin, and 10 µl of the supernatant solution were analyzed by SDS-PAGE.

Analytical Ultracentrifugation-- Analytical ultracentrifugation was carried out using a Beckman XL-A ultracentrifuge at 20 °C. Buffer containing 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA (centrifugation buffer) was used. In sedimentation velocity experiments the rotor speed was 60,000 rpm, and scans were taken at 10-min intervals. During analysis of delta , 1 mM DTT was added to the centrifugation buffer; the buffer used during experiments in which delta  and forms of b were mixed contained 0.5 mM DTT. The data were analyzed with the Beckman software using the time derivative method of Stafford (38). The values of Cohn and Edsall (39) were used to calculate partial specific volumes. Sedimentation equilibrium experiments were performed at 20 °C and 20,000 rpm using b24-152 at concentrations of 0.5, 1.0, or 2.0 mg of protein per ml in centrifugation buffer. Three molecular weight determinations were done at each concentration.

Cross-linking of Membranes-- Membranes containing F1F0 complexes including either wild type or mutated b were diluted to 0.25 mg of total protein per ml with buffer containing 50 mM Tris-HCl, pH 8.0, 5 mM MgCl2, and 10% glycerol. Benzophenone-4-maleimide (BPM) (Molecular Probes, Eugene, OR) dissolved in dimethylformamide (DMF) was added to a final concentration of 1 mM, and the mixture was allowed to stand at room temperature for 30 min. Controls were performed in which only DMF was added to the membranes. The treated membranes were exposed to long wave ultraviolet light from an Ultra-Violet Products model TM-36 transilluminator for 5 min. As a control some BPM-modified samples were placed on the transilluminator but were removed before it was turned on. After illumination, SDS-PAGE sample buffer was added to the samples, which were then heated at 100 °C for 5 min and analyzed by SDS-PAGE followed by Western blotting.

Cross-linking of Reconstituted F1b-- Purified F1 and wild type or cysteine-containing b24-156 were passed separately through 1-ml centrifuge columns (40) containing Bio-Gel P-10 resin (Bio-Rad) equilibrated with 50 mM sodium phosphate, pH 7.5, and 1 mM EDTA. Tris-(2-carboxyethyl)phosphine (Molecular Probes, Eugene, OR) was added to all b24-156 samples in a 1.1-fold molar excess to ensure the reduction of any disulfide bonds. BPM in DMF was added in a 5-fold molar excess over b24-156; in control samples DMF alone was added. After incubation for 15 min at room temperature, an excess of DTT was added to consume unreacted BPM. The b24-156 was then mixed with the column-centrifuged F1 at a molar ratio of 1.2 F1 per b24-156 dimer, in the presence of 5 mM MgCl2. Cross-linking and analysis was carried out as described above for the membrane samples.

Cyanogen Bromide Cleavage and Analysis of Cross-linked Products-- Cross-linked b24-156E155C-F1 and b24-158158C-F1 were analyzed by SDS-PAGE and stained briefly with Coomassie Blue. After destaining for 10 min with 10% acetic acid, gel slices containing bands to be cleaved were excised from the gel and were dried by lyophilization. The dried gel slices were then treated with 2% CNBr in 70% formic acid. After 30 min the slices had swollen back to their original sizes. At this time the excess CNBr solution was removed, and the tubes containing the gel slices were sealed and incubated at 37 °C overnight. The pH of the slices was then adjusted by three successive 15-min incubations in 150 µl of 1.0 M Tris-HCl, pH 8.0, followed by 15-min incubations in 150 µl of 1.0 M Tris-HCl, pH 6.8, 150 µl of 67 mM Tris-HCl, pH 6.8, and 150 µl of SDS-PAGE sample buffer containing DTT. The slices were placed in the wells of a second SDS-polyacrylamide gel that had been pre-electrophoresed for 1 h at 100 V in the presence of 50 mM Tris-HCl, pH 8.0, 0.1% SDS, containing 0.1 mM sodium thioglycolate to scavenge free radicals in the gel. Electrophoresis was carried out in the presence of 0.1 mM thioglycolate. The gel was either stained with Coomassie Blue or Western blotted onto a polyvinylidene difluoride (PVDF) membrane. After blotting, the membrane was stained briefly with Coomassie Blue and destained with 30% methanol before the bands of interest were excised and analyzed by peptide sequencing.

Other Methods-- SDS-PAGE was performed by the method of Laemmli (41) using 15% separating gels. The proteins were stained with Coomassie Brilliant Blue R-250. Protein blotting onto PVDF membranes was carried out using carbonate blot buffer (42). The anti-b monoclonal antibodies 10-1A4 and 10-6D1 were generous gifts of Drs. Karlheinz Altendorf and Gabriele Deckers-Hebestreit of Universität Osnabrück, Germany. Anti-delta polyclonal antiserum was raised against purified delta  subunit (43), and the anti-delta antibodies were affinity purified on a column containing immobilized recombinant delta  (31). Antibodies were labeled with 125I by the IODO-GEN method (44). Protein concentrations were determined by the method of Bradford (45) or Lowry et al. (46). Peptide sequencing was performed at the Laboratory for Macromolecular Structure at Purdue University (West Lafayette, IN) using an Applied Biosystems 470A sequencer.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

F1 Affinity Resin-- To characterize better the binding between the F1 sector and the hydrophilic portion of b, we coupled this region of b to a solid matrix to form an affinity resin for F1. The Sulfo-link coupling gel from Pierce, to which proteins can be coupled specifically via thiol groups, was chosen as a matrix. Since the hydrophilic region of b has no cysteine residues, the site of coupling to the resin can be specified by site-directed mutagenesis of b. Because the C-terminal region was thought most likely to be involved in b-F1 contacts, we introduced a cysteine residue near the N terminus of the hydrophilic region. A construct encoding the polypeptide sequence MERCSNSHY24-L156 (bMERC) was produced as described under "Experimental Procedures." The first four amino acids of this sequence were taken from the E. coli enzyme 3-methyladenine-DNA glycosylase I, because of the polar nature of the sequence and the high expression of this enzyme in a recombinant system (47).

The purified bMERC was coupled to the Sulfo-link resin as described under "Experimental Procedures," at a final concentration of 2.2 mg per ml of resin. Upon incubation of the modified resin with the F1 complex followed by centrifugation, a significant amount of F1 sedimented with the resin, as determined by SDS-PAGE (Fig. 1A, first lane). Only a small amount of F1 co-sedimented with the resin that had been modified with cysteine (Fig. 1A, last lane). The F1 present in this pellet was probably not bound specifically to the resin but rather was trapped between and within the resin particles. As a control for the volume of trapped liquid, BSA was included in all incubations; similar amounts of BSA were observed to co-sediment with both resins under all conditions used (Fig. 1). Thus the bMERC-modified resin is able to bind F1 specifically. The faint band migrating at the position of b24-156 in the first lane represents a trace of bMERC eluted from the resin during the incubation in SDS sample buffer. Most likely this arose from instances in which only one subunit of the dimer became covalently coupled to the resin.


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Fig. 1.   Binding of F1 to affinity resin and competition by soluble forms of b, as analyzed by SDS-PAGE. The F1 affinity resin, prepared from bMERC as described under "Experimental Procedures," was incubated for 1 h in the presence of purified F1 and the indicated soluble forms of b, expressed as concentration of the dimer. BSA was included as a control for trapping (see text). The resin was then sedimented by centrifugation, and the pellet was resuspended in SDS-PAGE sample buffer and analyzed by SDS-PAGE. The asterisks indicate experiments in which the control resin, bearing cysteine instead of bMERC, was used. A, a wide variety of b24-156 concentrations was used to determine the range in which substantial effects would be observed. B, b24-156, b24-155, and b24-152 were each tested at dimer concentrations of 1 and 10 µM to determine their relative strengths of binding.

Competition for F1 Binding by the SDS-PAGE Assay-- To test the ability of the hydrophilic portion of b to compete with the resin for binding to F1, the resin was incubated with F1 in the presence of increasing amounts of b24-156 (formerly known as bsyn; Ref. 33). After centrifugation and analysis of the pellet by SDS-PAGE, the amount of F1 bound to the resin was observed to decrease as the concentration of b24-156 increased, until essentially no F1 was bound by the resin at 6.5 µM of b24-156 dimer (Fig. 1A). Note that the amounts of soluble b24-156 trapped within the pelleted resin provide an internal representation of the amount of competitor added. These experiments demonstrate that b24-156 is able to compete with the bMERC-modified resin for binding to F1, establishing a simple competition assay for determining the relative affinity of any mutant form of b for F1.

Such experiments were carried out using b24-155 and b24-152, which lack one and four residues from the C terminus, respectively. It was found that these forms of b competed very poorly, relative to b24-156, with the bMERC-modified resin (Fig. 1B). At a dimer concentration of 10 µM, b24-155 showed a small amount of competition, whereas at the same concentration b24-152 showed no detectable competition by the SDS-PAGE assay. It is evident, however, that very weak competition is difficult to detect in this manner, as it requires seeing a small difference in band intensity.

Competition for F1 Binding by the Soluble ATPase Activity Assay-- To determine weak competition more reliably and in a quantifiable way, the assay was modified such that soluble ATPase activity, rather than bound ATPase protein, was measured. Under the conditions used in these assays, more than 90% of the added enzyme was bound by the resin. Competition for F1 binding by b24-156, b24-155, and b24-152 was determined by the increase in ATPase activity remaining in the supernatant solution when these forms of b were added to the incubations. As expected, the amount of F1 in the supernatant solution increased sharply with increasing concentration of b24-156, whereas the ability of b24-155 and b24-152 to compete with the bMERC-modified resin was far weaker, although still detectable (Fig. 2). These results show that the C-terminal residues of b are essential for its proper interaction with F1-ATPase. The preparations of soluble b were tested directly for ATP hydrolysis activity to make certain that trace contamination with an enzyme such as alkaline phosphatase could not account for the increase in soluble ATPase activity. In no case could such a contaminant account for more than 2% of the observed soluble activity.


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Fig. 2.   F1 binding competition between forms of b and the affinity resin as analyzed by soluble ATPase activity. The F1 affinity resin was incubated for 1 h in the presence of F1 and various amounts of b24-156 (black-square), b24-155 (black-triangle), or b24-152 (bullet ). The resin was then sedimented by centrifugation, and the supernatant solution was assayed for ATPase activity as described under "Experimental Procedures." The abscissa represents concentration of b dimer. The activities are expressed as percentages of the activity present in the supernatant solution when the control resin, bearing cysteine instead of bMERC, was used.

Sedimentation Velocity Analysis-- Recent ultracentrifugation results from this laboratory have provided evidence for the formation of an elongated complex by two molecules of the hydrophilic region of b and one molecule of the delta  subunit (31). We performed further centrifugation experiments to determine whether the reduced binding of the C-terminal truncations of b to F1 could be due to loss of contacts with delta . The isolated b24-156 and delta  subunits showed sedimentation coefficients (Table I) which were similar to those previously reported for bsol (25) and delta  (31). The increase in the s20,w upon mixture of equimolar amounts of dimeric b24-156 and delta  is confirmation that these proteins form a complex in solution. Mixtures of delta  with b24-155 or with b24-152 resulted in s values only slightly above the weighted averages of the two components, indicating that binding of b to delta  is significantly disrupted by removal of one or four residues from the C terminus. Thus the essential nature of these residues for binding F1 derives from their role in binding delta . The molecular mass of b24-152 was determined by sedimentation equilibrium, as described under "Experimental Procedures," to be 28,100 ± 1000 Da confirming that the dimeric nature of b was unaffected by the C-terminal truncation. Unlike b24-156 (34), no aggregation of b24-152 was observed at high concentrations.

                              
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Table I
Sedimentation velocity analysis of delta  with soluble b domains
Proteins as indicated were analyzed by ultracentrifugation at 60,000 rpm as described under "Experimental Procedures." Where applicable, b was present at a dimer concentration of 53.5 µM, and delta  was present at 53 µM.

The protein b34-156, which lacks 10 residues of the b sequence relative to b24-156, was found to have a sedimentation coefficient comparable with b24-156 (Table I). Interestingly, the complex formed when b34-156 was mixed with delta  had a significantly lower sedimentation coefficient than that of b24-156 and delta . This lower sedimentation coefficient was much more in line with the value of 2.07-2.16 recently reported for the b2delta complex found with the bST34-156 construct that also contained residues 34-156 of b (31). It seems unlikely that removal of 10 residues near the N terminus of b would have a significant effect on its binding to delta , and the molecular masses of b24-156 and b34-156 differ by only about 1.5 kDa. It therefore appears that the presence of residues 24-33, which are mostly hydrophobic, in the soluble b alters the conformation of the b2delta complex to make it less asymmetric, thereby increasing its sedimentation coefficient.

It is also of note that the sedimentation coefficients of b24-153 and b24-155 alone are substantially lower than that of b24-156, which is only a few residues greater in length. This finding implies that the truncated forms have greater frictional coefficients than b24-156, probably due to a conformational change in the C-terminal region upon deletion of the C-terminal residues. Such a conformational change could be responsible for the reduced binding of these forms of b to delta . To distinguish between direct and conformational roles of the C terminus of b in binding delta , we attempted to generate chemical cross-links to delta  from sites within this region.

Cross-linking of b and delta -- Individual cysteine residues were introduced into full-length b at positions 150, 151, and 155. A fourth construct was made that encoded a protein, referred to as b158C, having two residues, glycine and cysteine, attached to the C terminus of b. It was anticipated that the glycine would provide conformational flexibility to the C-terminal cysteine, increasing the likelihood of obtaining a cross-link. Plasmids bearing these mutated forms of b were all able to complement the uncF strain KM2 for growth on minimal media with succinate as the sole carbon/energy source, indicating that the b-F1 interaction was not disrupted in the mutants.

Membrane preparations bearing F1F0 complexes containing the mutated b subunits were incubated with the photoreactive cross-linker benzophenone-4-maleimide (BPM) and then exposed to ultraviolet light. The samples were analyzed by Western blotting, using 125I-radiolabeled monoclonal antibodies raised against b as probes. No cross-linking was observed with either bD150C or bK151C (data not shown). However, cross-linked products of about the same size were observed with both bE155C and b158C (Fig. 3). The new bands were approximately the size expected for a b-delta cross-link and showed reactivity with anti-delta polyclonal antibodies (Fig. 3), indicating that cross-links had been formed between b and delta . Membranes containing cross-linked F1F0 showed no apparent loss of activity compared with control membranes treated with either BPM or UV light (data not shown).


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Fig. 3.   Cross-linking of b to delta  in the F1F0 complex. Membranes were prepared from uncF E. coli cells complemented by a plasmid expressing either b containing the E155C mutation (A), b containing the 158C mutation (B), or the wild type b subunit (both panels). The membranes were treated with BPM and/or UV light as shown and were then analyzed by Western blotting. The blots were probed with antibodies raised against either b or delta .

To characterize further the cross-linked products, the E155C and 158C mutations were incorporated into b24-156. After modification of each of these proteins with BPM, reconstitution with F1, and exposure to ultraviolet light, new bands of an appropriate size were observed on SDS-PAGE (Figs. 4A and 5A). The new bands were recognized by antibodies directed against b and delta  (Figs. 4B and 5B), confirming the identity of a b24-156-delta cross-link. Exposure of the BPM-modified E155C and 158C proteins to UV light in the absence of F1 caused an apparent reduction in the total amount of protein on the stained gels (Figs. 4A and 5A). Western blotting of similar samples revealed a series of dimeric and higher order aggregates (not shown), which were probably not visible on the stained gels because of their heterogeneous nature.


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Fig. 4.   Cross-linking of b24-156 via position 155 to purified F1. b24-156 containing the E155C mutation, as well as wild type b24-156, was modified with BPM and reconstituted with purified F1. After exposure to UV light, the samples were analyzed by SDS-PAGE (A) and Western blotting (B), probing with 125I-labeled antibodies raised against either b or delta . Controls were performed in which the reconstituted b24-156-F1 complex was exposed only to BPM or to UV light, and in which the b24-156, with or without the E155C mutation, was treated in the absence of F1.


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Fig. 5.   Cross-linking of b24-158 via position 158 to purified F1. An experiment identical to that described in the legend to Fig. 4 was performed, except that the 158C mutation was used instead of the E155C mutation.

Cross-linking of the soluble forms of b gave rise to a second cross-link in each case that had a slightly greater mobility than b24-156 on SDS-PAGE (Figs. 4A and 5A). These cross-links were recognized by anti-b antibodies (Figs. 4B and 5B), suggesting that in each case an internal cross-link had been formed in b24-156. This internal cross-link was not formed to the same extent in the absence of F1.

Some cross-linked products of higher apparent molecular weight were observed with both the E155C and the 158C mutations (Figs. 4A and 5A). In each case, one of these cross-links was recognized by the anti-delta antibodies, whereas the other was not (Figs. 4B and 5B). In the absence of other data we have tentatively identified one of these bands as a (b24-156)2-delta cross-link. The other high molecular weight cross-linked product was recognized by an anti-alpha antibody (data not shown) and therefore could correspond to a b24-156-alpha cross-link.

Peptide Analysis of b24-156-delta Cross-links-- To identify the region of delta  involved in the cross-links to b24-156, a slice containing the cross-linked product from each cysteine mutation was cut from an SDS-polyacrylamide gel and treated with cyanogen bromide as outlined under "Experimental Procedures." CNBr cleavage of b24-156 should give rise to fragments of 2, 12, and 126 residues, with the large C-terminal fragment (residues Ala-31 to Leu-156) containing the site of the cross-link. The delta  subunit should give rise to fragments between 10 and 49 residues in length. The difference between b24-156 and its largest CNBr fragment is readily apparent on SDS-PAGE (Fig. 6). CNBr cleavage of each cross-linked product gave rise to a predominant fragment that is markedly larger than the 126-residue fragment derived from b24-156 alone (marked by an arrow in Fig. 6).


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Fig. 6.   Cyanogen bromide digestion of b24-156-delta cross-linked products. The bands corresponding to the b24-156-delta cross-links obtained with the E155C and the 158C proteins were excised from an SDS-polyacrylamide gel, treated with cyanogen bromide as described under "Experimental Procedures," and analyzed by SDS-PAGE. The wt lane shows the position of unmodified b24-156. The arrow indicates the position of the bands that were cut from a subsequent blot and analyzed by protein sequencing.

After blotting a gel containing the cross-linked b24-156-delta fragments to a PVDF membrane, the major CNBr product from each cross-link was cut from the membrane and analyzed by Edman degradation. In most cycles, both cross-links gave rise to two residues, corresponding to sequences from b and delta  (Table II). As expected, one set of amino acids was the sequence of b beginning after Met-30. The other set represented the sequence of delta  starting after Met-148. Interestingly, the delta  fragment sequence in both the E155C and the 158C cross-links continued past residue Met-158, indicating that delta  had not been cleaved at this position by the CNBr. Met-158 was not observed in the appropriate cycle of the Edman degradation of either of the cross-links.

                              
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Table II
Peptide sequencing of CNBr-treated cross-linking products
Cross-linked proteins were treated with CNBr, blotted onto PVDF, and sequenced by Edman degradation as described under "Experimental Procedures" and the text.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The results provided demonstrate that the residues at the C terminus of b are essential for proper interaction with F1, that the interaction of b with delta  is weakened when these residues are lacking, and that the C-terminal region of b is proximal to that of delta . Together these subunits are believed to form a "second stalk" reaching from the membrane to near the top of the F1 sector, which may function to hold the alpha 3beta 3 subunits stationary during rotation of gamma  and epsilon  driven by proton flow through F0 (48, 49). Earlier truncation and mutagenesis studies showed that the C terminus of b was essential for proper assembly of ATP synthase (10) and implied that the C terminus of delta  played a role in interaction of F1 with the F0 sector (21-24).

Our present studies confirm and extend this earlier work by providing additional information about the bdelta interaction. The major site of cross-linking of the E155C and 158C proteins to delta  was shown to be C-terminal to Met-148. It is interesting that the cross-linked delta  subunit was not cleaved at Met-158 by cyanogen bromide treatment and that no signal corresponding to this residue was observed during the appropriate cycle of peptide sequencing. The most straightforward interpretation of these results is that the major site of benzophenone linkage was through the side chain of Met-158. The benzophenone moiety may have formed a cross-link at Cgamma of this methionine to create a tertiary carbon center that would be relatively unreactive to cleavage by CNBr. It is notable that methylene groups adjacent to nitrogen or sulfur are particularly reactive to benzophenone (50).

Modeling of the C-terminal region of b as an alpha -helix reveals an amphipathic structure (Fig.7). The cross-linking results imply that the hydrophobic face of this helix on one of the b subunits interacts with delta . We suspect that the hydrophobic face of the second b subunit is in contact with another region of b. Removal of one or more hydrophobic residues might disrupt this interaction, causing a significant conformational change in the soluble b protein. Such a conformational change to a more asymmetric shape would explain why the sedimentation coefficients of b24-155 and b24-152 were markedly lower than that observed for b24-156 (Table I). In the absence of delta , the hydrophobic face of the first b subunit would not have its normal partner and might interact nonspecifically with the similarly exposed hydrophobic face of another b24-156 dimer. This would explain why the slight aggregation of b24-156 observed during sedimentation equilibrium centrifugation at high concentrations (34) is not seen with b24-152.


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Fig. 7.   Depiction of residues 146 to 156 of b in a helical wheel.

Hydrophobic residues are conserved at positions corresponding to E. coli residues 153 and 156 in the b subunit of many organisms (51). It is tempting to suggest that the importance of these hydrophobic residues in the b-delta interaction may provide the explanation for the observation that low ionic strength disrupts the binding of F1 to F0. However, other regions of b must also be involved in b-F1 interactions, since b24-152 was able to compete to a minor extent with the affinity resin for binding to F1 (Fig. 2). In this regard it is also noteworthy that some bacteria have b subunits that lack the hydrophobic residues at the C terminus. For example, the b subunit of the thermophile PS3 ends at the residue corresponding to position 148 in E. coli b (52).

The difference in sedimentation coefficients observed between the (b24-156)2delta and the (b34-156)2delta complexes (Table I) was surprising. One possible explanation is that flexibility in the N-terminal region of the soluble b construct allows the hydrophobic residues Y24VWPPLMAAI33, present in b24-156 but absent in b34-156, to loop back and interact with delta . Such an arrangement would be more compact, and the complex would therefore sediment faster than if the N-terminal helices were in a more extended conformation. Because the N termini of the b subunits are anchored in the membrane in ATP synthase, it seems unlikely that residues 24-33 of b normally interact with delta  in the intact complex.

Takeyama and co-workers (10) showed that one aspect of the defective ATP synthase assembly caused by deletion of residues from the C terminus of b was the failure of F0 to form a functional proton pore in vivo. In subsequent work, Brusilow and co-workers (53, 54) demonstrated that the delta  subunit was required for the formation of the proton pore from the cloned F0 subunits. Here we have shown that the same region of b implicated in the F0 assembly process is essential for binding to delta , strengthening the argument that the b-delta interaction is critical for the assembly of functional F0. At present, however, we have no evidence of how a signal arising from the interaction may be transmitted from the C-terminal end of b to the membrane, where interaction with the other F0 subunits would occur.

The solution structure of residues 1-105 of delta ', a proteolytic fragment consisting of residues 1-134 of delta , has been solved by NMR spectroscopy, but the structure of the entire 177-residue subunit could not be determined (49). Although the C-terminal regions of both proteins are predicted to be largely alpha -helical, in the absence of concrete structural information from either region it is difficult to propose specific interactions between amino acid residues in b and delta . The proposed site of cross-linking, Met-158, lies before a predicted C-terminal helix encompassing residues delta 167-175 (55).

Our current results demonstrate that b and delta  interact via their C-terminal regions, and a recent study from our laboratory has shown that the b2delta complex is extended enough to span the distance from the membrane to the N-terminal domain of alpha  (31). Thus our work is consistent with the hypothesis that the b and delta  subunits form a stator to prevent alpha 3beta 3 from moving relative to gamma . Since the stator must resist an appreciable torque (15), and since the Kd of the b-delta interaction is relatively high (5-10 µM; Ref. 31), it is likely that the interaction of these subunits is stabilized by the presence of the other parts of the ATP synthase complex. A further possibility is that b makes direct contact with other subunits, such as alpha  or beta , to stabilize the b-F1 interaction. The newly developed competition assay for b-F1 binding provides a simpler, more quantitative, and more sensitive way of detecting minor changes in b-F1 affinity compared with earlier methods. We are currently using this assay to define other residues of b that affect b-F1 interactions.

    ACKNOWLEDGEMENTS

We thank Drs. Gabriele Deckers-Hebestreit and Karlheinz Altendorf for providing the 10-1A4 and 10-6D1 monoclonal antibodies; Drs. Kimberly McCormick and Brian Cain for providing E. coli strain KM2; Matthew Revington for helpful discussions and construction of plasmid pMR2; Hanna Abou Alfa for helpful discussions; and Faye Males for technical assistance.

    FOOTNOTES

* This work was supported by Grant MT-10237 from the Medical Research Council of Canada. The Beckman XL-A analytical ultracentrifuge was acquired through support from the Academic Development Fund of the University of Western Ontario.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by an Ontario Graduate Scholarship.

§ To whom correspondence should be addressed. Tel.: 519-661-3055; Fax: 519-661-3175; E-mail: sdunn{at}julian.uwo.ca.

1 The abbreviations used are: OSCP, oligomycin sensitivity conferral protein; bMERC, form of the b subunit containing residues Tyr-24 to Leu-156 with the N-terminal leader sequence MERCSNSH; b24-156, b24-155, and b24-152, forms of the b subunit containing the residues indicated with the N-terminal leader sequence MTMITNSH; b24-158 or 158C, form of the b subunit identical to b24-156 but with Gly and Cys added to the C terminus; DTT, dithiothreitol; TEA, triethanolamine; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; BPM, benzophenone-4-maleimide; PCR, polymerase chain reaction; DMF, dimethylformamide; PVDF, polyvinylidene difluoride.

    REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
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