©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ATP Synthase Complex
PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES (*)

(Received for publication, October 11, 1994; and in revised form, November 21, 1994)

Grigory I. Belogrudov (§) John M. Tomich (1) Youssef Hatefi (¶)

From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The catalytic sector, F(1), and the membrane sector, F(0), of the mitochondrial ATP synthase complex are joined together by a 45-Å-long stalk. Knowledge of the composition and structure of the stalk is crucial to investigating the mechanism of conformational energy transfer between F(0) and F(1). This paper reports on the near neighbor relationships of the stalk subunits with one another and with the subunits of F(1) and F(0), as revealed by cross-linking experiments. The preparations subjected to cross-linking were bovine heart submitochondrial particles (SMP) and F(1)-deficient SMP. The cross-linkers were three reagents of different chemical specificities and different lengths of cross-linking from zero to 10 Å. Cross-linked products were identified after gel electrophoresis of the particles and immunoblotting with subunit-specific antibodies to the individual subunits alpha, beta, , , OSCP, F(6), A6L, a (subunit 6), b, c, and d. The results suggested that the two b subunits form the principal stem of the stalk to which OSCP, d, and F(6) are bound independent of one another. Subunits b, OSCP, d, and F(6) cross-linked to alpha and/or beta, but not to or . The COOH-terminal half of A6L, which is extramembranous, cross-linked to d but not to any other stalk or F(1) subunit. No cross-links of subunits a and c with any stalk or F(1) subunits were detected. In F(1)-deficient SMP, cross-linked b + b and d + F(6) dimers appeared, and the extent of cross-linking between b and OSCP diminished greatly. The addition of F(1) to F(1)-deficient particles appeared to reverse these changes. Treatment of F(1)-deficient particles with trypsin rapidly hydrolyzed away OSCP and F(6), fragmented b to membrane-bound 18-, 12-, and 8-9-kDa antigenic fragments, which cross-linked to d and/or with one another. Trypsin also removed the COOH-terminal part of A6L, but the remainder still cross-linked to subunit d. Models showing the near neighbor relationships of the stalk subunits with one another and with the alpha and beta subunits at a level near the proximal end (bottom) of F(1) and at the membranematrix interface are presented.


INTRODUCTION

The bovine heart mitochondrial ATP synthase complex isolated in our laboratory contains 13 well characterized subunits (Galante et al., 1979; Hekman et al., 1991), exhibits completely oligomycin-sensitive ATPase and ATP-P(i) exchange activities (Stiggall et al., 1978; Galante et al., 1979), and can drive via ATP hydrolysis a second mitochondrial proton pump when coincorporated in liposomes (Eytan et al., 1990). The polypeptide components of the ATP synthase are the alpha, beta, , , and subunits of the catalytic sector F(1), plus OSCP, a (subunit 6), b, c, d, F(6), and A6L, which together make up the stalk and the membrane sector F(0). Preparations of the ATP synthase complex also contain substoichiometric amounts of the ATPase inhibitor protein, IF(1). The stoichiometry of F(1) subunits is alpha(3)beta(3), and the stoichiometry of OSCP, d, A6L, b, and F(6), as determined by radioimmunochemical techniques in well coupled SMP (^1)(Hekman et al., 1991), is 1:1:1:2:2. Bovine ATP synthase also contains multiple copies of subunit c (Graf and Sebald, 1978; Sebald et al., 1979; Kiehl and Hatefi, 1980), but the precise number remains to be determined. In intact mitochondria, there is 1 mol of IF(1)/mol of F(1) (Hekman et al., 1991). By comparison, the Escherichia coli ATP synthase is composed of only eight subunits, with the F(1)-F(0) stoichiometry alpha(3)beta(3)-ab(2)c (Fillingame, 1990). Mitochondrial subunits alpha, beta, and have considerable sequence similarity to their E. coli counterparts. Mitochondrial OSCP and are analogous to E. coli and , respectively, and, largely on the basis of hydropathy profiles, mitochondrial subunits a, b, and c are considered analogous to the E. coli a, b, and c, respectively (Fillingame, 1990).

Bovine and the rat F(1) have been crystallized, and quaternary structures, respectively, at 6.5 and 3.6 Å resolution have been reported (Abrahams et al., 1993; Bianchet et al., 1991). More recently, the structure of bovine F(1) containing four molecules of AppNP, and one molecule of ADP has been published, showing at 2.8 Å resolution the three-dimensional structures of the alpha and beta subunits plus about 50% of the (Abrahams et al., 1994). However, structural information regarding the remainder of the ATP synthase complex is limited. Subunit c is considered to be shaped like a hairpin, with the hydrophobic arms forming alpha-helices that traverse the membrane and the hydrophilic hairpin bend protruding from the membrane on the F(1) side. Recent two-dimensional NMR data for the isolated E. coli subunit c in a chloroform-methanol-water solvent are in agreement with the hairpin model (Girvin and Fillingame, 1993, 1994). Small-angle neutron scattering studies of bovine OSCP (molecular weight 20,967) have suggested an elongated shape of approximately 90 times 30 times 30 Å, with 43% alpha-helical structure as calculated from circular dichroism measurements (Dupuis et al., 1983). Other than hydropathy profiles, information regarding the structures of other ATP synthase subunits is not available.

Because OSCP can be readily and reversibly removed from SMP together with F(1) (Steinmeier and Wang, 1979; Matsuno-Yagi and Hatefi, 1984), it is considered to be a stalk component required for proper binding of F(1) to F(0). In addition, our previous data, based on accessibility of ATP synthase subunits in SMP and mitoplasts (mitochondria denuded of outer membrane) to proteases and subunit-specific antibodies, indicated that OSCP, b, d, F(6), and the COOH-terminal half of A6L were accessible from the matrix but not from the cytosolic side of the inner membrane, whereas subunits a and c were not accessible to the above reagents from either side of the mitochondrial inner membrane (Hekman et al., 1991). This paper presents the results of cross-linking experiments involving the ATP synthase subunits in SMP. The data are consistent with the results of our earlier topography studies described above and provide information regarding the near neighbor relationships of OSCP, b, d, F(6), and A6L, which appear to contribute to the formation of the stalk of the bovine ATP synthase complex.


EXPERIMENTAL PROCEDURES

Materials

Disuccinimidyl tartarate (DST), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), ethylene glycol bis(succinimidyl succinate) (EGS), and N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) were obtained from Pierce; nitrocellulose membrane (0.2-µm pore size) from Schleicher and Schuell; SDS, acrylamide, and prestained SDS-PAGE standards (broad range) from Bio-Rad; TPCK-treated trypsin, soybean trypsin inhibitor, 4-(N-maleimido)benzophenone (MBP) and Tween 20 from Sigma; goat horseradish-conjugated anti-rabbit IgG from Calbiochem; and an Enhanced Chemiluminescence kit from Amersham. F(1)-ATPase and F(1)-depleted ASU particles, prepared as described previously (Matsuno-Yagi and Hatefi, 1984), were gifts of Dr. Akemi Matsuno-Yagi.

Cross-linking Conditions

SMP were diluted and washed with buffer and then adjusted to a protein concentration of 1.0 mg/ml in a medium containing 50 mM triethanolamine and 0.25 M sucrose, pH 8.0, and incubated with 1.0 mM DST for 30 min at 20 °C. The reaction was terminated by the addition of ammonium acetate to 50 mM. After a 10-min incubation an equal volume of Laemmli SDS-PAGE sample buffer (Laemmli, 1970) containing 6 M urea was added. In other experiments, samples of SMP at 1.0 mg/ml in 50 mM HEPES, 0.25 M sucrose, pH 7.2, were treated with 5 mM EDC in the presence of 5 mM NHS. The reactions were allowed to proceed for 30 min at 20 °C and were then quenched by the addition of Tris-HCl, pH 6.8, to 100 mM. After a 10-min incubation, samples were treated with SDS-PAGE sample buffer as above. F(1)-depleted ASU particles were cross-linked at 0.5-1.0 mg/ml in the same manner as SMP. When F(1) was used for cross-linking, an aliquot of the ammonium sulfate suspension of F(1) was desalted on a PD-10 column (Pharmacia Biotech Inc.) and adjusted to 0.2 mg/ml in the triethanolamine/sucrose buffer described above, and ATP was added to 1 mM. The preparation was then treated with 1 mM DST for 30 min at 20 °C before the addition of 50 mM ammonium acetate to terminate the cross-linking. Treatment of cross-linked F(1) for SDS-PAGE was the same as described above.

For cross-linkings involving sulfhydryl groups, the photoactivable heterobifunctional MBP was used. MBP dissolved in dimethylformamide was added at 1 mM to SMP at l mg/ml in 20 mM HEPES, 0.25 M sucrose, pH 7.2, and the mixture incubated for 25 min at 20 °C in dim light. Unreacted MBP was either removed by centrifugation or first quenched by the addition of 10 mM dithiothreitol and then removed by centrifugation. After resuspension in HEPES/sucrose buffer, the particles were irradiated with an 8-watt longwave UV lamp at a distance of 5 cm for 10 min on ice, with occasional mixing. The particles were then prepared for gel electrophoresis by the addition of an equal volume of SDS-PAGE buffer as indicated above. For control, particles were preincubated with 5 mMN-ethylmaleimide for 20 min before the addition of MBP and UV irradiation. Under these conditions, no cross-linking was observed. Furthermore, SMP or ASU particles were treated with MBP and then subjected to UV irradiation at 1.0 as well as 0.1 mg/ml and analyzed for cross-linked products by SDS-PAGE and immunoblotting. There was no evidence of additional cross-linking at the higher particle concentration, but at 0.1 mg of particle protein/ml there was a higher yield of the cross-linked products, consistent with the less shielding effect of the particles against UV irradiation at the lower protein concentration.

Trypsin Treatment of F(1)-depleted SMP

F(1)-depleted ASU particles were suspended at 3-4 mg/ml in 20 mM Tris-HCl, 0.25 M sucrose, pH 7.5. TPCK-treated trypsin was added up to 0.8 mg/ml, and the mixture was incubated at 37 °C. At various times, aliquots were withdrawn, proteolysis was arrested by the addition of 1 mM PMSF plus a 5-fold excess of soybean trypsin inhibitor, and after 10 min on ice the soluble and particulate fractions were separated by centrifugation. The supernatant proteins were precipitated by the addition of 10% trichloroacetic acid and were subjected to SDS-PAGE and immunoblotting with specific antibodies to F(0)-stalk subunits. The particulate fraction was washed by suspending in buffer and recentrifuging, then a portion was suspended in triethanolamine/sucrose buffer plus 1 mM PMSF for cross-linking with DST, and another portion in HEPES/sucrose buffer plus 1 mM PMSF for cross-linking with EDC + NHS. Conditions for cross-linking and sample preparation for SDS-PAGE were the same as described above. Protein concentrations were determined according to Lowry et al. (1951).

Electrophoresis

SDS-PAGE was performed according to Laemmli (1970), routinely using a separating gel containing 15% acrylamide. For optimal separation of high molecular weight cross-linked products, 10% PAGE was employed.

Antibody Production and Purification

The polyclonal antisera to ATP synthase subunits a, b, d, F(6), and A6L used here were those described previously by Hekman et al.(1991). The OSCP antiserum used was from Belogrudov et al.(1988). Antisera to the F(1) subunits alpha, beta, , and were obtained previously in this laboratory, using homogeneous preparations of each F(1) subunit as antigen. Antipeptide antibodies were raised in the rabbit against a synthetic peptide corresponding to residues 1-10 of subunit c (DCCD-binding protein), using the procedure described elsewhere (Hekman et al., 1991).

The IgG fractions used for immunoblotting were affinity purified according to Olmsted(1981), using as a source of antigen homogeneous subunits or subunits transferred to nitrocellulose membranes after SDS-PAGE of F(1) or purified ATP synthase complex. The affinity-purified antibodies were stored in small aliquots at -20 °C.

Immunoblotting Conditions

Conditions for immunoblotting were the same as before (Belogrudov and Hatefi, 1994), except that electroblotting was performed at 100 V for 1 h. Where necessary, nitrocellulose sheets containing the same cross-linked samples were cut into separate strips for blotting with different antibodies. The immunoblots were developed using the Amersham Enhanced Chemiluminescence kit according to the manufacturer's instructions.


RESULTS

Cross-linking of SMP with DST or EDC

Even though our preparations of the ATP synthase complex display the same activities and inhibitor-response properties as the ATP synthase complex in SMP, we chose to carry out our studies of the near neighbor relationships of the ATP synthase subunits primarily on well coupled SMP lest the purification process, which involves the use detergents, might have introduced some structural changes in the isolated enzyme. For example, the highly purified ATP synthase complex has a low phospholipid content and an absolute requirement for added phospholipids to display ATPase and ATP-P(i) exchange activities (Galante et al., 1979). In some cases, however, we used F(1)-depleted ASU particles to expose to cross-linking stalk subunits that F(1) might mask. Among the cross-linkers employed were DST and EGS, which react with amino groups separated, respectively, by 6.4 and 16.1 Å, and EEDQ and EDC, which activate carboxyl groups for interaction with a nucleophile (e.g. amino group) to result in zero length cross-linking. EDC was used in the presence of NHS, which is thought to react with the unstable O-acylisourea adduct of EDC to produce the more stable reactive NHS ester and improve the yield of the EDC-mediated cross-linked product (Staros et al., 1986). In all cases, cross-linking conditions were optimized with respect to the concentrations of the cross-linkers and SMP, the duration of cross-linking, and the temperature of the reaction mixture. The cross-linked products were identified after SDS-gel electrophoresis of the samples and electrotransfer to nitrocellulose sheets by immunoblotting to affinity-purified polyclonal antibodies raised to each purified ATP synthase subunit, alpha, beta, , , OSCP, F(6), a, b, c, and d. The anti-A6L antibodies used were antipeptide antibodies raised to synthetic peptides corresponding to residues 30-43 and 54-66 of this subunit. Attempts to raise antibodies or antipeptide antibodies to the subunit of bovine F(1) have not been successful to date. It should also be mentioned that Walker and co-workers (Walker et al., 1991; Collinson et al., 1994b) have recently reported the presence of three other polypeptides, designated e, f, and g, in their preparations of ATP synthase complex, which appear to have no ATP-P(i) exchange activity and only partially oligomycin-sensitive ATPase activity (Lutter et al., 1993; Collinson et al., 1994b). Whether e, f, and g are present in stoichiometric amounts in our highly purified ATP synthase complex is not known at this time, but their presence in SMP does not appear to have complicated the interpretation of the cross-linking data shown below. (^2)

Representative of the cross-linking pattern observed with the reagents mentioned above are the results shown in Fig. 1, where DST was the cross-linking reagent. Fig. 1A exhibits immunoblots of a 15% SDS gel, which favors transfer to nitrocellulose molecular mass species <80 kDa, and Fig. 1B shows immunoblots of a 10% SDS gel, which contains cross-linked products of higher molecular mass. The top of each lane states the antibody with which that nitrocellulose strip was blotted. In these and subsequent figures, the fastest moving (lowest) protein band is the uncross-linked subunit, except that in Fig. 1B all of the uncross-linked F(6) and parts of uncross-linked OSCP and d ran out of the gel. It might also be noted that there are no immunoreactive protein bands below uncross-linked b, OSCP, and d in Fig. 1A and below alpha and beta in Fig. 1B. This indicates the absence of immunoreactive fragments (e.g. due to proteolysis) of these subunits in the SMP preparations used, which is an important consideration in the identification of cross-linked products.



Figure 1: Cross-linking of SMP with DST. SMP at 1 mg/ml were incubated with 1 mM DST for 30 min at 20 °C. After termination of the reaction, samples were subjected to a 15% (panel A) and a 10% (panel B) SDS-PAGE, and proteins were electrotransferred to nitrocellulose membranes. Strips of nitrocellulose containing identical cross-linked products were blotted with the affinity-purified antibodies indicated on the top of each lane. The immunoreactive bands were visualized with the Enhanced Chemiluminescence detection system. For details, see ``Experimental Procedures.''



In Fig. 1A, the cross-linked products of b + OSCP, b + F(6), and d + A6L are marked. Also marked are two species of b + d, which are better separated in Fig. 1B. These could be the result of two types of cross-linking between d and the same molecule of b or of different types of cross-linking between d and the two molecules of b. The unlabeled top bands in Fig. 1A are cross-linked products of each subunit with alpha/beta, which are also clearer in Fig. 1B. These species appear to be alpha + beta, beta + b, beta + OSCP, alpha + OSCP, alpha + b, alpha + d, and alpha + F(6). The unmarked cross-linked bands seen in the upper parts of the lanes in Fig. 1B are presumably cross-linked trimers and more complex products. Essentially a similar cross-linking pattern was obtained when SMP were treated with EDC in the presence of NHS. The product of d + A6L was particularly favored under the latter conditions, and the major cross-linked products between the stalk and F(1) subunits were alpha + b, alpha + OSCP, alpha + F(6), and beta + d (data not shown). Although not shown in Fig. 1, SMP cross-linked with either DST or EDC + NHS and immunoblotted to anti-a or anti-c antibodies showed no cross-linked products involving these subunits.

A close scrutiny of Fig. 1, A and B, suggests the absence of cross-linked products involving the stalk subunits and the small subunits of F(1). For example, one would expect the cross-linked products of the subunit of F(1) with b, OSCP, and d to produce a protein band immunoreactive to anti-b, anti-OSCP, or anti-d in the M(r) region of 50-60 kDa. However, as is clear in Fig. 1B, this region of these lanes is devoid of any such protein band. To investigate this issue further, SMP and purified F(1)-ATPase were separately treated with DST and subjected to SDS-gel electrophoresis and immunoblotting with affinity-purified antibodies to the individual F(1) subunits alpha, beta, , and . The results are shown in Fig. 2. The paired lanes compare the cross-linked products of the F(1) subunits as marked in the figure in SMP and isolated F(1)-ATPase. In the first and thirdlanes from the left, the bands not seen in the second and fourth lanes are the cross-linked products, respectively, of alpha and beta with the stalk subunits (see Fig. 1B). Other bands common in the first four lanes are the cross-linked products of alpha + beta and alpha + beta plus one or another small subunit of F(1). The important message of Fig. 2is contained, however, in the last four lanes. It is seen that in SMP or isolated F(1) the cross-linked products of and are essentially the same in each case, even though the yields of the cross-linked products of appear to be less in SMP (perhaps due to shielding of ) than in isolated F(1). These results suggest, therefore, that in SMP and do not cross-link to any stalk subunit in the presence of DST as the cross-linking agent. Similar results were obtained when EDC + NHS were used. However, since and produced a number of cross-linked products common to SMP and isolated F(1), it is clear that they are accessible to cross-linkers in SMP. The question, therefore, is how and are located in SMP in relation to b, OSCP, d, F(6), and A6L.


Figure 2: Comparison of the cross-linking patterns of F(1) subunits in purified F(1) and SMP treated with DST. F(1) (0.2 mg/ml) and SMP (1 mg/ml) were cross-linked with DST as in Fig. 1. Samples of cross-linked SMP and F(1) were subjected to 15% SDS-PAGE in parallel, and proteins were electrotransferred to nitrocellulose membrane and blotted with the affinity-purified antibodies indicated on top of the paired lanes. Immunoblots were developed as indicated in Fig. 1. The positions of prestained molecular mass markers are shown in kDa on the left of the figure.



Cross-linking of SMP with MBP

MBP is a photoactivable sulfhydryl reagent that can alkylate a thiol via its maleimido moiety. Then, upon irradiation (at about 350 nm) the aromatic ketone moiety goes into a diradicaloid triplet state, resulting in an electrophilic electron-deficient oxygen that is highly reactive, especially toward the C-H bonds of protein backbones (Dormán and Prestwich, 1994). The result is the formation of a C-C bond between the ketone carbon and the protein and cross-linking at a distance in the case of MBP of about 10 Å. Among the ATP synthase subunits under consideration here, those that contain cysteine residues are the alpha, , and subunits of F(1) plus b, OSCP, d, and c, of which alpha has 2 cysteine residues/mol and the others 1 cysteine residue each. The experiments with MBP were carried out by incubating SMP (1 mg/ml) with 1 mM MBP for 25 min. Unreacted MBP was removed by centrifugation. SMP were resuspended in buffered sucrose solution, subjected to UV irradiation as described under ``Experimental Procedures,'' and analyzed by SDS-PAGE and immunoblotting with antibodies against 6 of the 7 cysteine-containing subunits mentioned. As seen in Fig. 3, only two cross-linked products were detected, one between b and OSCP at about 42 kDa, and another between b and F(6) at about 34 kDa. The formation of these products required both the MBP pretreatment of SMP and the subsequent UV irradiation. When the SMP were first treated with 5 mMN-ethylmaleimide before the addition of MBP and UV irradiation, the cross-linked products shown in Fig. 3were not formed (data not shown). As mentioned above, subunit b contains a single cysteine residue at position 197, which is close to its COOH terminus, and F(6) has no cysteine residues. Therefore, the MBP-mediated cross-linking between F(6) and b indicates that the COOH terminus of b is located near F(6). It is also possible that cross-linking of b and OSCP involves this same Cys of b. In isolated OSCP, Cys, which is near the center of the chain, is modifiable by substituted maleimides (Dupuis et al., 1985). However, whether this cysteine is accessible for modification in SMP is not known.


Figure 3: Cross-linking of ATP synthase subunits in SMP with the heterobifunctional photoactivable sulfhydryl reagent, MBP. SMP at 1 mg/ml were incubated with 1 mM MBP for 25 min at 20 °C. Particles were sedimented by centrifugation, resuspended in buffered sucrose solution, and UV irradiated for 10 min or kept on ice in the dark. Samples were subjected to 15% SDS-PAGE, and proteins were electrotransferred to nitrocellulose and probed with the affinity-purified antibodies indicated on the top of the lanes. Immunoreactive bands were visualized as in Fig. 1. Lanes 1, 4, and 7, sample of SMP UV irradiated for 10 min; lanes 2, 5, and 8, MBP-treated, nonirradiated SMP; lanes 3, 6, and 9, MBP-treated and UV-irradiated SMP. The positions of prestained molecular mass markers are shown in kDa on the right of the figure.



It was of interest to see whether the removal of F(1) from SMP might alter the cross-linking of stalk subunits. F(1)-depleted ASU particles were prepared essentially according to Racker and Horstman(1967). These preparations are depleted with respect to F(1) and suffer partial loss of OSCP. The addition of F(1) + OSCP reconstitutes particles with high ATP synthase activity (Matsuno-Yagi and Hatefi, 1984). The remainder of OSCP can be removed by further alkaline extraction of ASU particles, but this was not done here because our aim was to investigate the effect of F(1) removal. When treated with MBP, ASU particles produced two major cross-linked products among the cysteine-containing subunits of the F(1)-depleted ATP synthase. One was b + F(6) as in Fig. 3, the other was a band at about 44 kDa, whose mobility and singular reactivity with only anti-b IgG suggested that it is a cross-linked b + b dimer (Fig. 4, lane 2). There were also detectable amounts of b + OSCP (Fig. 4, lane 6). The addition to ASU particles of F(1) (2 and 6 mol/mol F(0), respectively, in the third and fourth lanes of each set in Fig. 4) increased the yield of b + OSCP dimer and diminished the yield of the putative b + b dimer. There are two possible explanations for these results: (i) removal of F(1) caused a partial separation of b and OSCP so that b-MBP was too distant from OSCP to cross-link upon photoactivation or (ii) removal of F(1) resulted in masking of the thiol group of OSCP, making it unable to form OSCP-MBP to cross-link with b upon photoirradiation. We prefer the first possibility because structural perturbation of the stalk may also bring the two MBP-modified b subunits closer to one another for cross-linking after photoactivation of their benzophenones. As seen in Fig. 4, the cysteine-containing subunit d in ASU particles (or in SMP) did not form any MBP-mediated cross-linked products. As was shown previously, subunit d in SMP and ASU particles is resistant to trypsinolysis. It is possible, therefore, that much of subunit d, including its cysteine residue, is shielded by other ATP synthase subunits in SMP and ASU particles.


Figure 4: Cross-linking of F(1)-deficient ASU particles with MBP in the absence and presence of added F(1). ASU particles (0.5-1.0 mg/ml) were incubated with 1 mM MBP for 25 min at 20 °C, and excess reagent was removed by centrifugation. MBP-modified particles were incubated without added F(1) (lanes 1, 2, 5, 6, 9, and 10) or with 325 µg of F(1)/mg of particles (lanes 3, 7, 11) or 1.0 mg of F(1)/mg of particles (lanes 4, 8, and 12), each for 30 min at 37 °C. Particles were reisolated by centrifugation, washed, resuspended in 20 mM Tris-HCl, pH 7.5, containing 0.25 M sucrose and UV irradiated for 10 min on ice. Samples were subjected to 15% SDS-PAGE, and proteins were electrotransferred to nitrocellulose and blotted with the affinity-purified antibodies indicated on the top of the lanes. Immunoreactive bands were visualized as in Fig. 1. Lanes 1, 5, and 9, MBP-treated, nonirradiated ASU particles; other lanes, MBP-treated ASU particles UV irradiated for 10 min on ice. The positions of prestained molecular mass markers are shown in kDa on the right of the figure.



Cross-linking of F(1)-depleted Particles Treated with Trypsin

These experiments were carried out with ASU particles that had been extracted twice with urea to divest them of F(1). They were then treated with trypsin under relatively mild (1:25 weight ratio of trypsin to particle protein for 1 h at 37 °C) or severe (1:5 weight ratio of trypsin to particle protein for 1 h at 37 °C) conditions. Proteolysis was arrested as described under ``Experimental Procedures,'' and the particles were separated from the soluble protein fragments by centrifugation and washing. Aliquots of the particles were then subjected to cross-linking with DST and EDC + NHS, electrophoresed, and immunoblotted with antibodies to subunits b, d, OSCP, and F(6). Before trypsin treatment cross-linking of the F(1)-depleted particles produced a b + OSCP dimer in a fairly good yield when the cross-linker was DST and in very poor yield when it was EDC + NHS. This agrees with our conclusions regarding cross-linking of ASU particles with MBP and suggests that removal of F(1) results in separation of OSCP from b to the extent that DST (molecular length, 6.4 Å) can still bridge the distance between them, but EDC + NHS (zero length cross-linking) cannot. By contrast, cross-linking of the F(1)-depleted particles with either DST or EDC produced a b + F(6) dimer in good yield (Fig. 5A, lanes 5 and 6, second heavy band from bottom), just as it was shown in Fig. 1for SMP. After treatment of the particles with the lower concentration of trypsin, OSCP and F(6) were completely degraded in agreement with previous results (Joshi and Burrows, 1990; Hekman et al., 1991), and no cross-linked products containing antigenic fragments of these subunits were detected by immunoblotting. Also, in agreement with previous results (Joshi and Burrows, 1990; Hekman et al., 1991), subunits a and c were unaffected by trypsin and did not produce any cross-linked products when the trypsin-treated ASU particles were subjected to cross-linking with DST or EDC + NHS (data not shown). The COOH-terminal end of A6L was removed by trypsin (Hekman et al., 1991), but the remainder of the molecule, including the antigenic residues 30-43, survived, and this truncated A6L cross-linked to subunit d (see Fig. 5A, lane 9, first arrowhead from bottom) when the trypsin-treated ASU particles were treated with DST.


Figure 5: Cross-linking of ATP synthase subunits in F(1)-deficient ASU particles treated with trypsin. ASU particles (3.5 mg/ml) were incubated with trypsin (25:1 ratio by weight, panel A, or 5:1 ratio by weight, panel B) for 1 h at 37 °C. Proteolysis was arrested by the addition of excess soybean trypsin inhibitor plus 1 mM PMSF, and particles were reisolated by centrifugation, washed, and cross-linked with 1 mM DST or 5 mM EDC in the presence of 5 mM NHS. Samples were subjected to 15% SDS-PAGE, and proteins were electrotransferred to nitrocellulose membrane and blotted with the affinity-purified antibodies indicated on top of the lanes. Immunoblots were developed as described in Fig. 1. The tables of plus and minus signs on top of the immunoblots show the treatments the ASU particle of each lane had received. Arrowheads are described under ``Results.'' The designations b(18), b(12), and b(8) are for membrane-bound tryptic fragments of subunit b with approximate M(r) values of 18, 12, and 8, respectively. The positions of prestained molecular mass markers are shown in kDa on the left of panels A and B.



The effect of trypsin (1:25 weight ratio of trypsin to particle protein) on subunits b and d agreed with the results of others (Collinson et al., 1994a; Walker and Collinson, 1994), which showed that b was hydrolyzed at Arg-Gly, Lys-Arg, and Arg-His, and d at Lys^4-Leu^5 (see Fig. 5A, lanes 2-4 and 8-10). Hydropathy analysis of subunit b shows two hydrophobic clusters of about 20 residues each near the NH(2) terminus followed by a 130-residue-long hydrophilic region to the COOH-terminal end. This suggests that the molecule is anchored to the membrane via its two hydrophobic clusters and that its short hydrophilic NH(2) terminus 30 residues) and its 130-residue-long hydrophilic tail are extramembranous on the F(1) side. According to this picture, the tryptic cleavages at Arg-Gly and at Lys-Arg (or Arg-His) would produce two membrane-bound fragments, respectively, about 18-19 and 12-14 kDa. These b fragments, which still reacted with our polyclonal antibody preparation, are seen in lanes 2-4 of Fig. 5A. They cross-linked in the presence of DST or EDC + NHS with subunit d to produce the two bands marked with arrowheads in lanes3, 4, 9, and 10 of Fig. 5A. It may also be noted that the yield of the slower moving cross-linked product was greater when DST was the cross-linking reagent, and the yield of the faster moving cross-linked product was greater when EDC + NHS were used for cross-linking. Other cross-linked products recognized and marked by arrowheads in Fig. 5A are the b + b dimer in lane 6 and d + F(6) in lane 11. We suspect that the band marked d + A6L, d + x is composed of more than one cross-linked product of subunit d. However, additional information is not available at this time.

The data in Fig. 5B are from an aliquot of ASU particles that was treated with the higher concentration of trypsin (1:5 weight ratio of trypsin to particle protein). It is seen in lanes 1 and 2 that subunit b was further degraded to produce an 8-9-kDa antigenic fragment, which was still membrane-bound. Considering that there are about 70 amino acids from the NH(2) terminus to the end of the second hydrophobic cluster of subunit b, the tryptic cleavage site to produce an 8-9-kDa membrane-bound fragment must be very close to where the COOH-terminal hydrophilic tail of b exits the membrane. If we assume that the hydrophobic clusters of b are nonantigenic, then it is likely that our anti-b IgG recognizes the 30-residue-long NH(2) terminus of subunit b, which is probably extramembranous. As seen in lane 3 of Fig. 5B, this 8-9-kDa fragment disappeared in the presence of EDC + NHS and produced the cross-linked products marked with arrowheads. The uppermost band marked with an arrowhead also appeared to form in lower yield when DST was the cross-linking reagent (Fig. 5B, lane 2). The proteolysis pattern of bovine b with trypsin as shown in Fig. 5(see also Collinson et al., 1994a; Walker and Collinson, 1994) is similar to that of E. coli subunit b (Steffens et al., 1987), which is a further indication of the analogy between them.


DISCUSSION

Considerable information is now available regarding the mechanisms of ATP hydrolysis (Al-Shawi et al., 1990; Penefsky and Cross, 1991; Boyer, 1993) and synthesis (Matsuno-Yagi and Hatefi, 1990; Hatefi, 1993) at the level of the catalytic sector, F(1), of the ATP synthase complex. Also, the crystal structure of a form of bovine heart F(1) containing four molecules of AppNP and one molecule of ADP has been solved recently at 2.8 Å resolution (Abrahams et al., 1994). In addition, it is known that transmembrane protons pass only through the membrane sector, F(0), of the ATP synthase complex, and energy communication between F(0) and F(1) takes place via coupled conformation changes of subunits (for review, see Hatefi, 1993). At the level of F(1), protein-bound ATP is synthesized from protein-bound ADP and P(i) without energy utilization, but the binding of P(i) and ADP to F(1) and the release of ATP from F(1) are the energy-requiring processes, which are apparently accomplished by appropriate conformation changes of the beta subunits. The crystal structure of bovine F(1) (Abrahams et al., 1994) and the cryoelectron microscopy studies of Capaldi and co-workers Wilkins and Capaldi, 1994, and references therein) on the E. coli F(1) suggest that subunits and are involved in conveying conformation changes to and from F(1). However, in both the bovine and E. coli ATP synthases there is a 45-Å-long stalk between F(1) and F(0) (Soper et al., 1979; Capaldi et al., 1992; see also Dubachev et al., 1993), whose structure and role in energy communication between F(0) and F(1) are not known. If one assumes that the ATP synthase subunits that are not components of F(1) but are entirely or partly extramembranous (Hekman et al., 1991) contribute to the composition of the stalk, then subunits b, OSCP, d, F(6), and A6L would qualify as candidates. As possible components of the stalk, these subunits would be directly or indirectly involved in conformational energy transfer between F(0) and F(1). Therefore, information regarding their relative positions in the stalk and their spatial interaction with one another as well as with F(0) and F(1) subunits would be crucial to investigating the mechanism of energy transfer within the ATP synthase complex.

As mentioned earlier, OSCP is easily removable from SMP (Senior, 1979) or preparations of the ATP synthase complex (Galante et al., 1981). Removal of OSCP also results in the removal of F(1), but both can be added back to F(0) to reconstitute oligomycin-sensitive ATPase activity (Galante et al., 1981) or to depleted SMP to reconstitute oxidative phosphorylation at high rates (Matsuno-Yagi and Hatefi, 1984). In addition, it is known that OSCP binds to bovine F(1) (Hundal et al., 1983; Dupuis et al., 1985) and that the F(1)-OSCP complex can bind subunits b, F(6), and d (Walker and Collinson, 1994). Evidence that F(6) can be reversibly extracted from SMP is also available (Fessenden-Raden, 1972). These data are consistent with our earlier studies showing that b, OSCP, d, and F(6) are accessible to subunit-specific antibodies in well coupled SMP, but not in mitoplasts, and that in SMP subunits b, OSCP, F(6), and the COOH-terminal antigenic part of A6L could be degraded by trypsin (Hekman et al., 1991). These studies also indicated certain subunit accessibility differences between SMP and the purified ATP synthase complex, which decided our choice of material for determining the stoichiometry of subunits in F(0) and the stalk (Hekman et al., 1991) as well as for our studies reported here on the near neighbor relationships among the stalk subunits and between the latter and the subunits of F(1) and F(0).

Table 1summarizes the results obtained in SMP and F(1)-depleted SMP with three cross-linking reagents (DST, MBP, EDC) of different chemical reactivities and different effective distances for cross-linking. Considering first the near neighbor relationships of the stalk subunits, it is clear that OSCP cross-linked only to b, and A6L only to d. The NH(2) terminus of A6L contains a short hydrophobic cluster (20 residues), which appears to anchor A6L to the membrane. The remainder of the molecule is hydrophilic and apparently extramembranous. Trypsin cleaves off the COOH-terminal antigenic part of A6L (Hekman et al., 1991), but the remainder of the molecule is still close enough to subunit d to form a cross-linked product in the presence of DST (Fig. 5, lane 9, bottom band marked with an arrowhead). This also suggests that subunit d is present near the membrane. Indeed, the NH(2) terminus of d contains a short hydrophobic cluster of amino acids, which may be intercalated into the membrane.



As seen in Table 1, all of the stalk subunits, except A6L, cross-link to b in SMP. As mentioned above, the hydropathy analysis of b shows two hydrophobic clusters of 20 residues each located 30 residues from the NH(2) terminus. Therefore, one might expect that the two hydrophobic clusters intercalate into the membrane, with the short NH(2)-terminal segment and the long (130 residues) COOH-terminal portion beyond the second hydrophobic cluster being extramembranous on the F(1) side (Walker and Collinson, 1994). Furthermore, our study of the stoichiometry of F(0) stalk subunits indicated that there are two molecules of b/ATP synthase (Hekman et al., 1991), which agreed with the stoichiometry of the structurally analogous subunit b of the E. coli ATP synthase (Fillingame, 1990). In further agreement with this stoichiometry, we found in the present study two cross-linked species of b + d in the presence of either DST or EDC, even when b was partially degraded by trypsin to form membrane-bound fragments of about 18 and 12 kDa (Fig. 5, lanes 3, 4, 9, and 10). We also found in F(1)-depleted SMP, treated with either DST or EDC, a band that reacted only with anti-b IgG and exhibited on SDS gels a mobility consistent with that of a cross-linked b + b dimer.

In addition to cross-linking of b to OSCP and F(6) effected by DST or EDC, b + OSCP and b + F(6) dimers were also produced when the cross-linking reagent was the heterobifunctional photoactivable sulfhydryl reagent, MBP (Fig. 3). Because F(6) is devoid of cysteine residues, the cross-link between b and F(6) must involve Cys of b near its COOH terminus. The same might be true in the case of the MBP-induced cross-link between b and OSCP, or the cysteine in this case might be Cys of OSCP. However, the fact that the b-MBP adduct did not cross-link upon photoactivation to alpha, beta, , or may mean that the COOH-terminal portion of b, which contains Cys, is folded back toward F(0) and away from F(1). Subunit b of E. coli ATP synthase is also thought to have a folded conformation (Fillingame, 1990; Penefsky and Cross, 1991). It might be added parenthetically that treatment of SMP or ATP synthase preparations with various mono- and dithiol modifiers results in uncoupling (Yagi and Hatefi, 1984), and the studies of Papa and co-workers (Zanotti et al., 1988) implicate Cys of subunit b in this type of uncoupling. Although not shown in Fig. 3, we checked by immunoblotting for MBP-induced cross-links of alpha, , d, and c, all of which contain cysteine, but none was found. The cysteine of subunit c is within the membrane and may not react with MBP (although it appears to react with p-chloromercuribenzoate). (^3)Apparently, the cysteines of alpha, , and d are also unreactive with or inaccessible to MBP.

As seen in Table 1, b, OSCP, d, and F(6), but not A6L, form cross-linked products in SMP with alpha and/or beta. Furthermore, in SMP, OSCP, d, and F(6) can each cross-link to b but not to one another. Therefore, it seems reasonable to assume that the two molecules of subunit b, which extend from the membrane to F(1), form the principal stem of the stalk to which OSCP, d, and F(6) bind independently of one another. These considerations are summarized in Fig. 6, which shows the near neighbor relationships of the stalk subunits in cuts parallel to the membrane at the proximal end (bottom) of F(1) (A) and at the membrane-matrix interface (B). In A, subunits b, OSCP, d, and F(6) are all present because they all cross-link to alpha and/or beta (the parallelogram labeled alpha/beta). They also cross-link to one another wherever the circles representing each subunit overlap. In B (the bottom of the stalk), A6L protrudes from the membrane near d, and the two b subunits enter the membrane. However, whether OSCP and F(6) extend the length of the stalk from F(1) to F(0) to impinge upon the membrane is unclear; therefore, they have not been included. In F(1)-depleted ASU particles, this organization is somewhat perturbed, as if the removal of F(1) results in the inward collapse of the b subunits toward each other, also bringing F(6) close to d. As a consequence, in ASU particles, but not in SMP, b + b and d + F(6) cross-linked dimers can form (the latter is marked by an arrowhead in Fig. 5A, lane 11). When MBP was used for cross-linking of ASU particles, b + F(6) was formed as in MBP-treated SMP, but the yield of b + OSCP dimer was greatly diminished (Fig. 4, lane 6). Instead, there was the appearance of a band qualifying as a b + b dimer, which was not seen in MBP-treated SMP. Interestingly, the addition of F(1) to ASU particles prior to MBP treatment restored the formation of the b + OSCP product and diminished the yield of the b + b dimer (Fig. 5, lanes 3, 4, 7, and 8). These results suggest that (i) F(1) removal diminishes the interaction of b and OSCP, which is consistent with the fact that extraction of F(1) also results in labilization of OSCP, and (ii) the readdition of F(1) to ASU particles reestablishes the close association of b and OSCP, which is also consistent with the fact that function can be restored to ASU particles by the addition of F(1) and OSCP (Matsuno-Yagi and Hatefi, 1984). Recent data of Abrahams et al.(1994) on the crystal structure of bovine F(1) indicate that protrudes 30 Å from the core of F(1), which the authors believe forms part of the stalk. Fig. 6A shows a possible location for the tail of surrounded by b, d, and OSCP. This possible location inside the stalk is consistent with our inability to find cross-linked products of with any of the stalk subunits. It also agrees with the presumed inward collapse of the stalk to allow the formation of b + b and d + F(6) cross-linked products when together with the remainder of F(1) is removed from SMP. As was shown in Fig. 2, both and form multiple cross-linked products when SMP or isolated F(1) preparations were treated with DST (or EDC, not shown). However, like , also failed to cross-link to any stalk subunit. The data of Abrahams et al.(1994) on the crystal structure of bovine F(1) do not show the location of . It may be of interest to add in this regard that E. coli alpha and cross-link via a disulfide bond in the presence of CuCl(2) (Mendel-Hartvig and Capaldi, 1991), a G29D mutation of the E. coli alpha renders the F(1) -deficient (Maggio et al., 1988), and tryptic removal of 15 NH(2)-terminal residues of the alpha subunits of E. coli F(1) destroys the ability of the reconstituted alpha(3)beta(3) to bind (Dunn et al., 1980). As mentioned earlier, E. coli is considered to be analogous to the bovine OSCP (Walker et al., 1982; Ovchinnikov et al., 1984; see also Dunn and Heppel, 1981). However, the results just described make one wonder about the location of in relation to the distal end of E. coli F(1), where by analogy to the bovine F(1) the NH(2) termini of the alpha subunits would be located (see Abrahams et al., 1994).


Figure 6: Schematic representation of the near neighbor relationships of subunits b, OSCP, d, F(6), and A6L in bovine submitochondrial particles. Cross-linked dimers of subunits were identified where circles representing the subunits overlap. Because knowledge of the structures of these subunits is limited, the diameters of the circles were deliberately made the same to avoid any inference regarding the relative sizes and shapes of these subunits. A, end-on view of a cut parallel to the membrane through the bottom (proximal end) of F(1). B, end-on view of a cut parallel to the membrane at the membrane-matrix interface.



Finally, it should be mentioned that Joshi and Burrows (1990; see also Archinard et al., 1986) have previously detected in a preparation of bovine ATP synthase treated with dithiobis(succinimidylpropionate), which cross-links amino groups at a distance of 12 Å, many of the cross-linked products we have characterized here. They employed antibodies to whole F(1), OSCP, F(6), A6L, a, and c, but not to the individual subunits of F(1) and subunit d. Therefore, some of the cross-linked products were identified in part on the basis of their mobility on two-dimensional SDS gels. The cross-linked products they report, which we have not seen in SMP with our three cross-linking reagents, are those of F(6) with , , and A6L. They also claim the formation of F(6) + d dimer (they refer to d as the 20-kDa subunit), which we only find in DST-treated ASU particles, but not in intact SMP.


FOOTNOTES

*
This work was supported by United States Public Health Service Grant DK08126. This is publication 8860-MEM from The Scripps Research Institute, La Jolla, CA. 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.

§
On leave from M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russian Federation.

To whom correspondence should be addressed: Division of Biochemistry, Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-455-9100; Fax: 619-554-6838.

(^1)
The abbreviations used are: SMP, bovine heart submitochondrial particles; AppNP, 5`-adenylyl imidodiphosphate; ASU particle, F(1)-deficient SMP; EDC, 1-ethyl-3-(dimethylaminopropyl)carbodiimide; DCCD, N,N`-dicyclohexylcarbodiimide; DST, disuccinimidyl tartarate; NHS, N-hydroxysulfosuccinimide; EGS, ethylene glycol bis(succinimidyl succinate); EEDQ, N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline; MBP, 4-(N-maleimido)benzophenone; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone.

(^2)
Coupling factor B (Sanadi, 1982) with M(r) of 11-12 times 10^3 (You and Hatefi, 1976), which is present in SMP and restores ATP-P(i) exchange and ATP-driven transhydrogenation and reverse electron transfer to ammonia-EDTA treated SMP (Stiggall et al., 1979; Sanadi, 1982), has been considered to be a component of the bovine ATP synthase (Sanadi, 1982). However, we and Walker's laboratory (Walker et al., 1991) have not detected factor B in our respective ATP synthase preparations.

(^3)
A. Matsuno-Yagi and Y. Hatefi, unpublished results.


ACKNOWLEDGEMENTS

We thank C. Munoz for the preparation of mitochondria and Drs. Akemi Matsuno-Yagi and Mutsuo Yamaguchi for helpful discussions.


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