©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunits Coupling H Transport and ATP Synthesis in the Escherichia coli ATP Synthase
Cys-Cys CROSS-LINKING OF F(1) SUBUNIT TO THE POLAR LOOP OF F(0) SUBUNIT c(*)

(Received for publication, June 5, 1995)

Ying Zhang (§) Robert H. Fillingame (¶)

From the Department of Biomolecular Chemistry, University of Wisconsin Medical School, Madison, Wisconsin 53706

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Second site suppressor mutations at position 31 of F(1) subunit recouple ATP-driven H translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F(1)F(0) ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H.(1994) J. Biol. Chem. 269, 10221-10224). This finding suggests a functional interaction between subunit c and subunit during the coupling of H transport through F(0) to ATP synthesis of F(1). However, the physical proximity of the two subunits remained to be defined. In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit to see whether the subunits could be cross-linked. Disulfide bridge formation between subunit c and subunit was observed in membranes of three double mutants, i.e. cA40C/E31C, cQ42C/E31C, and cP43C/E31C, but not in wild type membranes or in membranes of the cA39C/E31C double mutant. These results indicate that the polar loop of subunit c and the region around residue 31 of subunit are physically close to each other in the F(1)F(0) complex and support the hypothesis that these two subunits interact directly in the coupling of H transport to ATP synthesis. Disulfide cross-linking of the Q42C subunit c and E31C subunit leads to inhibition of ATPase coupled H transport, as might be expected in a model where the catalytic sites of the F(1)ATPase alternate during H transport-coupled ATP hydrolysis/synthesis. However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the -c cross-linked F(1)F(0) complex retains residual H transporting activity.


INTRODUCTION

The H-transporting, F(1)F(0) ATP synthase of Escherichia coli utilizes an H electrochemical gradient to drive ATP synthesis during oxidative phosphorylation (Senior, 1988). Similar enzymes are found in mitochondria, chloroplasts, and other bacteria. The enzymes are composed of two sectors, termed F(1) and F(0). The F(1) sector contains the catalytic sites for ATP synthesis, and when released from membrane, it shows ATPase activity. The F(0) sector traverses the membrane and functions as the H transporter. When F(1) is bound to F(0), the complex acts as a reversible, H-transporting ATP synthase or ATPase. In E. coli, F(1) is composed of five types of subunits in an alpha(3)beta(3) stoichiometry and F(0) is composed of three types of subunits in an a(1)bc stoichiometry (Foster and Fillingame, 1982). Each subunit is encoded by a single gene of the unc operon (Walker et al., 1984).

Subunit c is a protein of 79 amino acid residues which folds in the membrane with a hairpin-like structure. The two, hydrophobic transmembrane alpha-helices are joined by a more polar loop region that is exposed to the F(1) binding side of the membrane (Fillingame, 1990). Asp, lying in the center of transmembrane helix-2, is believed to be essential for H transport. The coupling of H transport in F(0) to ATP synthesis/hydrolysis in F(1) is thought to occur via conformational changes transmitted through the complex, since the catalytic sites in F(1) may lie more than 50 Å away from the surface of the membrane (Abraham et al., 1993, 1994; Lucken et al., 1990). The Q42E mutation in the universally conserved Arg-Gln(or Asn)-Pro sequence of the polar loop of subunit c causes an uncoupling of H transport and ATP hydrolysis/synthesis (Mosher et al., 1985). This ``uncoupled'' phenotype of the cQ42E mutant can be suppressed by second site substitutions in Glu of F(1) subunit (Zhang et al., 1994). A functional interaction between the polar loop of subunit c and subunit could play a key role in the coupling process There is however no physical evidence that subunit c and lie close to each other in the F(1)F(0) complex. In this study, we report on experiments where the polar loop region of subunit c and residue 31 of subunit were cross-linked by disulfide bridge formation following introduction of Cys residues into these regions. Wild type subunits c and lack Cys, so the site of cross-link formation must be between the sites of Cys substitution. The experiments provide the first evidence of a contacting interface between subunit c and subunit and support the hypothesis that the subunits may interact directly during the coupling of H transport to ATP synthesis.


EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis and Plasmid Construction

All the plasmids used in this study are derivatives of plasmid pBR322. Plasmid pYZ201 (Fig. 1), which carries the eight structural genes of unc operon (bases 870-10172), (^1)is an equivalent of plasmid pMO142 (Zhang et al., 1994) except that it lacks an NcoI site at base 1305 due to a silent mutation introduced in the subunit a His codon. The subunit c polar loop Cys substitutions and subunit E31C mutation were introduced using the polymerase chain reaction, as described by Herlitze and Koenen(1990), with several 21-27-base oligonucleotides (Table 1). The mutated polymerase chain reaction fragments for the uncE gene were digested with PstI(1561) and HpaI(2162) and ligated into the equivalent sites of pDF163 (Fig. 1). The mutated polymerase chain reaction fragment for the uncC gene, containing the E31C mutation, was digested with NcoI (at bases 6376 and 7386) and ligated into the equivalent sites of pYZ201 to generate plasmid pYZ202 (Fig. 1). The DNA of the ligated regions was sequenced to confirm introduction of the intended substitution and absence of additional mutations. Whole operon plasmids containing single Cys substitutions in the polor loop of subunit c, or double Cys substitutions in both the polar loop of subunit c and at position 31 of subunit , were constructed by ligation of the SphI fragment of DNA (bases 3216-10172 coding the uncAGDC genes) from plasmid pMO142 or pYZ202 (E31C) into the unique SphI site of the plasmid pDF163 mutant derivatives with Cys substitutions at subunit c position 39, 40 42, 43, and 44. Plasmid pMO142 is the wild type control plasmid used in this study.


Figure 1: Plasmids used in polymerase chain reaction mutagenesis and in construction of mutagenized unc operon plasmids. Key restriction digest sites are shown. The unc fragments in the plasmids are shaded.





Complementation of Chromosomal unc Deletion Mutant

Strain MO204 (AN346, Deltaunc (B-C), Ilv) (Fraga et al., 1994) was transformed with pMO142 and its mutant derivatives. Transformant colonies were transferred to minimal medium 63 agar plates (Miller, 1972), containing 22 mM succinate as carbon source and incubated at 37 °C with scoring for growth after 1-5 days.

Membrane Preparations, Assays, and Buffers

Cells were grown on M63 minimal medium containing 1% glucose, 2 mg/liter thiamine, 0.2 mML-arginine, 0.2 mM uracil, 0.02 mM dihydroxybenzoic acid, and supplemented with 10% LB (Miller, 1972). Ampicillin was added to a final concentration of 100 mg/liter. Membranes were prepared in TMG buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl(2), and 10% glycerol) by rupture of cells in a French press (Mosher et al., 1985). Protein and ATPase activity were determined as described (Fillingame, 1975). HMK buffer (10 mM HEPES-NaOH, pH 7.5, 5 mM MgCl(2), 300 mM KCl) was used in the ACMA (^2)quenching experiments. Fluorescence measurements were made as described in Zhang and Fillingame(1994).

Gel Electrophoresis and Immunoblotting Analysis

Membrane vesicles were incubated in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 0.01% bromphenol blue) in the absence of reducing agent at 5 mg of protein/ml at room temperature for 1 h. The solubilized membrane proteins were separated by SDS-polyacrylamide gel electrophoresis using a 12% acrylamide gel and the Tris-Tricine buffer prepared according to Schagger and von Jagow(1987). Proteins in the gel were transferred electrophoretically onto nitrocellulose paper (Towbin et al., 1979). Immunostaining was carried out using the ``Enhanced Chemiluminescence System'' (Amersham Corp.). The rabbit antiserum to subunit c used was that described by Girvin et al.(1989). Antibodies that nonspecifically cross-reacted with E. coli membrane proteins were removed by preabsorption with membranes prepared from a mutant strain with a deleted unc operon (Girvin et al. 1989). The mouse monoclonal antibody to subunit (13-A7, II; Aggeler et al., 1990) was a gift from Dr. R. Capaldi (University of Oregon, Eugene, OR). Subunit c was purified as described by Hermolin and Fillingame (1989). Purified subunit was a gift from Dr. S. Dunn (University of Western Ontario, Canada).

Cross-linking and Purification by Immunoprecipitation

Membrane vesicles at 10 mg/ml in TMG buffer were treated with 1.5 mM Cu(II)-(1,10-phenanthroline)(3) to catalyze disulfide bond formation. After a 1-h incubation at room temperature, EDTA was added to a final concentration of 15 mM, and N-ethylmaleimide (from a 0.5 M stock in dimethyl sulfoxide) was added to a final concentration of 20 mM, and the sample was incubated for a further 20 min. F(0) was isolated from 5 mg of these oxidized membrane vesicles by the method of Schneider and Altendorf(1984), following stripping of F(1) from the membranes, except that dithiothreitol was omitted in all buffers. The isolated F(0) was resolubilized in 50 µl of radioimmune precipitation buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholate). Immunoprecipitation was performed according to the method described by Sambrook et al.(1989) with some modifications. The solubilized F(0) mixture was first diluted to 0.5 ml with IPP150 buffer (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, 0.1% Nonidet P-40, 0.02% NaN(3)) and incubated for 4 h at 4 °C with mouse monoclonal antibody to subunit which had been preabsorbed to protein A-Sepharose beads. The immunoprecipitates were centrifuged and washed twice with 1 ml of radioimmune precipitation buffer, three times with 1 ml of IPP150 buffer, and finally once with 1 ml of 10 mM Tris-HCl, pH 7.5. The beads were suspended in 40 µl of SDS sample buffer in the absence of reducing agent and incubated 30 min at 50 °C. Following centrifugation for 20 s in a microcentrifuge, the supernatant solution was collected and analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting; half of the sample was made 10% in beta-mercaptoethanol to reduce disulfide cross-links prior to electrophoresis.


RESULTS

Substitutions and Their Effect on Function

Cys residues were introduced at positions 39, 40, 42, 43, and 44 in the polar loop of subunit c or/and position 31 of subunit . Plasmids coding the whole unc operon were transformed into strain MO204, which carries a deletion of the chromosomal unc operon. The growth of transformants was tested using a succinate carbon source, where growth depends upon a functional oxidative phosphorylation system. As shown in Table 2, each of the single mutant plasmids and most of the double mutant plasmids promoted growth as well as the wild type plasmid. The cP43C/E31C plasmid transformant grew less well.



The ATPase and ATP-driven ACMA quenching activities of membrane vesicles from these transformant strains are shown in Table 3. Membrane vesicles were prepare under nonreducing conditions in buffer lacking dithiothreitol. The E31C substitution by itself led to a 2-fold elevation in ATPase activity, whereas the polar loop substitutions by themselves had little effect on ATPase activity. The combined effect of mutations on ATPase activity was variable. The cP43C mutation by itself caused a significant reduction in the ATP-driven ACMA quenching response. Three of the four polar loop mutations, when combined with the E31C mutation, also led to significant reductions in the ACMA quenching response. The significance of these changes in activity is considered in greater detail below.



Spontaneous Sulfhydryl Cross-link Formation in the Membrane Vesicles

Membrane vesicles prepared under nonreducing conditions (TMG buffer lacking dithiothreitol) were solubilized with SDS and analyzed by electrophoresis and immunoblotting. A single band corresponding to subunit was detected with monoclonal antibody to subunit in each of the single Cys mutant membranes and in wild type membranes (Fig. 2). A slower moving protein band with a molecular mass around 25 kDa was detected in three of the double Cys mutants, cA40C/E31C, cQ42C/E31C, and cP43C/E31C, but not in the cA39C/E31C double Cys mutant. (^3)The apparent molecular mass of this band (25 kDa) corresponds to the size expected for a heterodimer of subunit and c (i.e. 24 kDa). When the gel was analyzed by immunoblotting using antibodies to subunit c, a 25-kDa protein band of apparently corresponding mobility was observed in the same three double Cys mutants, although the patterns were more complicated due to subunit c oligomer formation (Fig. 2). A heavy protein band with a mobility around 15 kDa was also detected with subunit c antiserum in all the subunit c Cys mutant membranes, but not in wild type or the E31C single mutant membranes. This protein band likely results from cross-linking of the polar loop Cys of two neighboring subunit c in the membrane. These results indicate formation of a disulfide bridge between Cys in positions 40, 42, and 43 of subunit c and the Cys of subunit . The components of the 25-kDa cross-linked product are verified below.


Figure 2: Immunoblots of SDS-polyacrylamide gel of membrane vesicles. Membrane vesicles (60 µg of protein) were prepared and electrophoresed under nonreducing conditions before being transferred to nitrocellulose paper. The blot was first probed with monoclonal antibody against subunit . The blot was then stripped of bound antibodies by submerging it in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 2% SDS, and 100 mM beta-mercaptoethanol for 30 min at 50 °C, and the same blot was reprobed with antiserum against subunit c. Mutations are indicated by referring to the Cys residues at the positions of subunit c or/and . Dashes indicate wild type subunit c or . The positions of the subunit monomers and the putative homo- or heterodimers are shown by arrows. The positions of prestained molecular mass markers, with the molecular mass given in kDa, is shown at the side of the blots.



Confirmation of Covalent Cross-linking of Subunit c and

In the experiments that follow, we reasoned that it should be possible to purify a cross-linked c- product from F(1)-depleted F(0) by immunoprecipitation using a monoclonal antibody to subunit . Subunit covalently bound to subunit c should not be removed from membrane vesicles during stripping with the rest of the F(1) subunits. To increase the yield of cross-linked product, membrane vesicles were first treated with the oxidant, Cu(II)-(1,10-phenanthroline)(3), before removing F(1) and purification of F(0). The partially purified F(0) preparations were analyzed by SDS gel electrophoresis and immunoblotting (Fig. 3). In the F(0) preparations of three double Cys mutants (cA40C/E31C, cQ42C/E31C, and cP43C/E31C), a single 25-kDa protein band was detected by both antibody to subunit and antiserum to subunit c. The 25-kDa band was not detected in any of the other F(0) preparations. A 15-kDa protein band, corresponding in size to a subunit c homodimer, was detected by subunit c antiserum in the F(0) preparations of all the subunit c Cys mutants.


Figure 3: Immunoblots of SDS-polyacrylamide gel of partially purified F(0) from oxidized membrane vesicles. The F(0) were prepared and electrophoresed under nonreducing conditions. Separate gels were run for each blot. The blots are presented and marked in the same way as described in the legend for Fig. 2. The purified subunit was a gift from Dr. S. Dunn (University of Western Ontario, Canada).



Following dissociation of F(0) subunits with detergent, the putative c- cross-linked product was precipitated by antibody to subunit . The immunoprecipitates were analyzed by immunoblotting (Fig. 4). In the three double Cys mutants shown, the 25-kDa protein band was detected both with antiserum against subunit c and antibody to subunit . A corresponding band was not detected in equivalent preparations from wild type or E31C single Cys mutant membranes (Fig. 4A). The 25-kDa protein band was not observed if the sample was treated with beta-mercaptoethanol before electrophoresis. Instead, two protein bands were observed, and they migrated to the positions corresponding to subunit c and subunit monomers (Fig. 4B). From these results we conclude that a disulfide bridge is indeed formed between polar loop residues of subunit c and position 31 of subunit , after introduction of Cys groups at these positions, and that these two regions lie physically close to each other at the surface of the membrane.


Figure 4: Confirmation of components of 25-kDa cross-linked product as subunits c and . Partially purified F(0) prepared from oxidized membrane vesicles were immunoprecipitated with subunit monoclonal antibody preabsorbed to protein A-Sepharose. Immunoprecipitates were solubilized in SDS sample buffer in the absence (A) or presence (B) of 10% beta-mercaptoethanol and analyzed by electrophoresis and immunoblotting. Separate gels were run for each blot. The blots are presented and marked the same way as in the Fig. 2. The ``control w/o Ab'' was prepared by mock-precipitation of cA40C/E31C F(0) with protein A beads lacking antibody. Purified subunit c was prepared as described (Hermolin and Fillingame, 1989).



Effect of Cross-linking on Function of the Enzyme

To exam the effect of disulfide cross-link formation on the function of the enzyme, membrane vesicles from one of the double mutants, cQ42C/E31C, were prepared under either nonreducing or reducing conditions, i.e. in the presence or absence of 5 mM dithiothreitol. The proportions of subunit in the 25-kDa product versus monomeric subunit were compared after solubilization of membranes in SDS sample buffer lacking beta-mercaptoethanol. In the membrane prepared under reducing conditions, antibody staining of the 25-kDa band was nearly undetectable; >95% of the stain was estimated to track with monomeric subunit (Fig. 5B). In membranes prepared under nonreducing conditions the yield of cross-linked product was estimated by comparing of the staining density of the two bands and found to vary in the range of 63-78% with different gels and different sample loadings.


Figure 5: Effect of cross-linking on ATPase-coupled H transport function of the enzyme. A, ATP-driven quenching of ACMA fluorescence by cQ42C/E31C membranes prepared in the absence (trace 1) or presence (trace 2) of 5 mM dithiothreitol. Membranes were diluted to 0.25 mg/ml in HMK buffer, pH 7.5, and incubated with 0.3 µg/ml ACMA. ATP was added to 0.94 mM and the uncoupler SF6847 added to 0.3 µM. Membranes prepared in the absence of dithiothreitol were incubated with 10 mM dithiothreitol in HMK buffer for 30 min prior to the addition of ATP (trace 3). The quenching response of cQ42C membranes prepared under nonreducing conditions without dithiothreitol is also indicated (trace 4). B, immunoblots of SDS-polyacrylamide gel of the cQ42C/E31C membrane vesicles prepared in the absence or presence of 5 mM dithiothreitol. Membrane vesicles (50 µg of protein) were electrophoresed under nonreducing conditions before being transferred to nitrocellulose paper for probing with monoclonal antiboby against subunit . Arrows indicate bands corresponding to subunit and the c- dimer. The positions of prestained molecular mass markers are shown.



The ATP-driven ACMA quenching response of the cQ42C/E31C membrane vesicles, prepared under reducing and nonreducing conditions, was compared to assess the effect of cross-linking on ATPase-coupled H transport function.^4 As shown in Fig. 5A, the ATP-driven ACMA quenching response of cQ42C/E31C membranes prepared under nonreducing conditions was approximately 70% of that given by membranes prepared under reducing conditions (curve 1 versus curve 2). A nearly complete quenching response was restored when the oxidized preparation was assayed in HMK assay buffer with 10 mM dithiothreitol (Fig. 5A, curve 3). The final experiment shown in Fig. 5A (curve 4) is the quenching response of cQ42C membrane vesicles prepared under nonreducing conditions. These vesicles show a normal quenching response. This control suggests that the oxidation induced inhibition of quenching with cQ42C/E31C vesicles can be attributed to formation of the c- cross-link rather than c-c cross-links.

Unfortunately, the ACMA quenching response does not decrease linearly with decreases in ATPase activity (Miller et al., 1990). Using the calibration curves previously described by Miller et al.(1990), we estimate that a 30% reduction in quenching response would occur under conditions where activity was reduced by 50-90%. The reduction in quenching response is thus approximately that expected from the extent of cross-link formation, if the c- cross-linked F(1)F(0)is inactive in proton pumping. The results therefore do indicate that c- cross-link formation markedly reduces enzyme function, but do not rule out the possibility that the c- cross-linked enzyme retains residual proton pumping activity.

ATPase-coupled H transport by other membrane vesicles prepared under nonreducing conditions was also measured. As shown in Table 3, the quenching response by the cA39C and cA40C single mutant membranes approached that of wild type membranes, even though subunit c homodimer formation was observed in both preparations (Fig. 2). The quenching responses by the double mutant membranes, i.e. cA40C/E31C and cP43C/E31C, were decreased compared with that of their subunit c single mutant membrane counterparts, whereas the quenching response by the cA39C/E31C mutant membranes was not significantly different from that of cA39C single mutant membranes. The cA39C/E31C mutant was the only double mutant not showing formation of a cross-link between subunit c and subunit . These results provide further support to the above conclusion that cross-linking between subunit c and inhibits the function of the enzyme.


DISCUSSION

In a previous study (Zhang et al., 1994), we demonstrated that the uncoupled phenotype of the cQ42E mutation could be suppressed by second site mutations in Glu of F(1) subunit . This discovery suggested a possible functional interaction between the polar loop of subunit c and subunit in the coupling process. To test whether the loop region of subunit c and residue 31 of subunit are actually physically close to each other in the F(1)F(0) complex, Cys residues were introduced into both regions and cross-linking attempted by oxidation. Heterodimeric cross-linked products were formed in the membrane vesicles of three of the double Cys mutants, i.e.cA40C/E31C, cQ42C/E31C, and cP43C/E31C, but not in the membrane vesicles of the cA39C/E31C mutant. These experiments provide the first evidence of a contacting interface between the polar loop of subunit c and subunit and support the hypothesis that these two subunits may interact directly. Our conclusions are consistent with the previous cross-linking experiments of Suss(1986), who concluded that subunit c and of chloroplast F(1)F(0) could be cross-linked with bifunctional imidoesters.

The cA39C/E31C mutant was the only double mutant not forming a cross-link between subunit c and subunit . The Cys of the A39C subunit c does appear to be reactive in forming subunit c dimers (see Fig. 2and Fig. 3). The residue also appears to be accessible to Cu(II)-(1,10-phenanthroline)(3) oxidation since c-c dimerization was increased by this reagent. On the other hand, the A39C sulfhydryl reacted less readily with N-[^3H]ethylmaleimide than the other polar loop Cys residues (data not shown). The Cys sulhydryl may be buried in the F(0) complex in a less reactive, more hydrophobic environment, relative to the other polar loop substitutions.

A direct interaction between the polar loop of subunit c and subunit has important implications in the mechanism of coupling H transport to ATP synthesis. The conformation of the polar loop of subunit c is hypothesized to change with the ionization state of Asp in the middle of the membrane (Fillingame, 1990; Fillingame et al., 1992). The conformation of subunit and its position within F(1) also changes with the occupancy of catalytic sites (Capaldi et al., 1992). Binding of Mg-ADP-P(i) at catalytic sites results in a simultaneous association of and with beta (Gogol et al., 1990), an association that is manifested by increased N-ethyl-N`-dimethylaminopropylcarbodiimide catalyzed cross-link formation between Ser of subunit and Glu in the conserved DELSEED sequence of subunit beta (Dallman et al., 1992; Mendel-Hartvig and Capaldi, 1991). According to the recently published, atomic resolution crystal structure of the alpha(3)beta(3) domain of beef heart mitochondrial F(1) ATPase (Abraham et al., 1994), the DELSEED sequence lies below the catalytic site at the bottom of subunit beta. When ATP is bound in the catalytic site of subunit beta, the DELSEED region of the same subunit directly contacts several residues of subunit to form a ``catch.'' We envision that a conformational change in the polar loop of subunit c, caused by protonation or deprotonation of Asp, is transmitted to subunit via a direct interaction with the position 31 region. Further conformational changes may then be transmitted, perhaps through subunit , to subunits beta and/or . Movement of helical bundles initiated by changes in the DELSEED region could then be envisioned as altering the conformation of the catalytic site to promote release of ATP product. The role of the Ser region of subunit in the coupling process is still unclear. Kuki et al.(1988) have shown that mutant F(1)F(0) with truncated versions of subunit , terminating after residue 78, are still active in oxidative phosphorylation and ATP-driven proton transport.

The ATPase-coupled H transport function of three double Cys mutants membranes, i.e.cA40C/E31C, cQ42C/E31C and cP43C/E31C, was decreased under conditions where cross-link formation between subunits c and was observed. The inhibition of H transport appears to relate to c- heterodimer formation rather than c-c dimer formation. In the cA39C, cA40C, and cQ42C single mutants, c-c dimers were formed but normal activity observed. Normal activity was also observed in the cA39C/E31C mutant where c-c dimers, but not c- dimers, were formed. Formation of c- dimers might be expected to inhibit activity by fixing the conformation of the two regions and preventing further coupling at alternating catalytic sites (Boyer, 1993).

We were not able to proportionally relate the extent of inhibition of ATPase-coupled H transport function to the extent of c- cross-link formation by the methods used here. The major problem stems from the nonlinear decrease in ACMA quenching response with ATPase function (Miller et al., 1990). The cross-linked F(1)F(0) studied most thoroughly in cQ42C/E31C membranes was generated by spontaneous oxidation during membrane preparation, and the extent of incorporation into the -c dimer was approximately 70%. It might be possible to relate -c cross-link formation to inhibition more easily under conditions where the cross-linking of subunits to c approached 100%. Although further cross-linking could be achieved by mild treatment with Cu(II)-(1,10-phenanthroline)(3) and activity further reduced, the inhibition following this treatment was not effectively reversed by dithiothreitol treatment. In conclusion, cross-link formation between subunit and a single subunit c might be expected to totally inhibit ATPase-coupled proton transport by preventing alternation between catalytic sites in F(1). The inhibition observed here is suggestive of such a phenomenon, but we cannot rule out residual activity in an c- cross-linked complex. The relatively high ATPase activity of the cross-linked F(1)F(0) complexes may reflect mutationally induced uncoupling of F(1) from F(0).


FOOTNOTES

*
This study was supported by United States Public Health Service Grant GM23105 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by a gift from the Lucille P. Markey Charitable Trust to the University of Wisconsin Medical School.

To whom correspondence should be addressed: Dept. of Biomolecular Chemistry, 587 Medical Sciences Bldg., University of Wisconsin-Madison, Madison, WI 53706. Tel.: 608-262-1439; Fax: 608-262-5253.

(^1)
The unc DNA numbering system corresponds to that used by Walker et al.(1984).

(^2)
The abbreviations used are: ACMA, 9-amino-6-chloro-2-methoxyacridine; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

(^3)
The properties of cD44C mutant membranes are described in a separate communication (S. D. Watts, Y. Zhang, R. H. Fillingame, and R. A. Capaldi, submitted for publication).

(^4)
The ATPase activity of cQ42C/E31C membranes prepared without dithiothreitol was also significantly lower than those prepared under reducing conditions, i.e. 0.23 ± 0.01 versus 0.32 ± 0.03 µmolbulletmgbulletmin (where n = 4 sets of assays on the two membrane preparations).


ACKNOWLEDGEMENTS

We thank Dr. Rod Capaldi (University of Oregon) and Dr. Stanley Dunn (University of Western Ontario) for gifts of anti- monoclonal antibody and purified subunit , respectively.


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