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ATP Synthase Complex

ATP Synthase Complex Vol. 270, No. 5, Issue of February 3, pp. 2053-2060, 1995 THE JoURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES* (Received for publication, October 11, 1994, and in revised form, November 21, 1994) Grigory I. Belogrudov:j:, John M. Tomich§, and Youssef Hatefi11 From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the §Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 (Galante et al., 1979; Hekman et al., 1991), exhibits completely The catalytic sector, F and the membrane sector, F , 10 0 of the mitochondrial ATP synthase complex are joined oligomycin-sensitive ATPase and ATP- P; exchange activities together by a 45-A-long stalk. Knowledge of the compo- (Stiggall et al., 1978; Galante et al., 1979), and can drive via sition and structure of the stalk is crucial to investigat- ATP hydrolysis a second mitochondrial proton pump when ing the mechanism of conformational energy transfer coincorporated in liposomes (Eytan et al., 1990). The polypep- between F and F • This paper reports on the near neigh- tide components of the ATP synthase are the a, /3, y, 5, and e 0 1 bor relationships of the stalk subunits with one another subunits of the catalytic sector F v plus OSCP, a (subunit 6), b, and with the subunits ofF and F , as revealed by cross- 1 0 c, d, F , and A6L, which together make up the stalk and the linking experiments. The preparations subjected to membrane sector F . Preparations of the ATP synthase com- cross-linking were bovine heart submitochondrial par- plex also contain substoichiometric amounts of the ATPase ticles (SMP) and F -deficient SMP. The cross-linkers inhibitor protein, IF . The stoichiometry of F subunits is 1 1 were three reagents of different chemical specificities a f3 y5e, and the stoichiometry ofOSCP, d, A6L, b, and F , as 3 3 6 and different lengths of cross-linking from zero to 10 A. determined by radioimmunochemical techniques in well cou- Cross-linked products were identified after gel electro- pled SMP (Hekman et al., 1991), is 1:1:1:2:2. Bovine ATP phoresis of the particles and immunoblotting with sub- synthase also contains multiple copies of subunit c (Graf and unit-specific antibodies to the individual subunits a, p, Sebald, 1978; Sebald et al., 1979; Kiehl and Hatefi, 1980), but y, o, OSCP, F , A6L, a (subunit 6), b, c, and d. The results the precise number remains to be determined. In intact mito- suggested that the two b subunits form the principal chondria, there is 1 mol ofiF /mol ofF (Hekman et al., 1991). 1 1 stem of the stalk to which OSCP, d, and F are bound By comparison, the Escherichia coli ATP synthase is composed independent of one another. Subunits b, OSCP, d, and F of only eight subunits, with the F -F stoichiometry a f3 y5e- cross-linked to a and/or p, but not to y or o. The COOH- 1 0 3 3 terminal half of A6L, which is extramembranous, cross- ab2c10_12 (Fillingame, 1990). Mitochondrial subunits a, /3, and linked to d but not to any other stalk or F subunit. No y have considerable sequence similarity to their E. coli coun- cross-links of subunits a and c with any stalk or F terparts. Mitochondrial OSCP and 5 are analogous to E. coli 5 subunits were detected. In F -deficient SMP, cross- and e, respectively, and, largely on the basis of hydropathy linked b + b and d + F dimers appeared, and the extent profiles, mitochondrial subunits a, b, and c are considered of cross-linking between b and OSCP diminished analogous to the E. coli a, b, and c, respectively (Fillingame, greatly. The addition of F to F cdeficient particles ap- 1990). peared to reverse these changes. Treatment of F -defi- Bovine and the rat F have been crystallized, and quaternary cient particles with trypsin rapidly hydrolyzed away structures, respectively, at 6.5 and 3.6 A resolution have been OSCP and F , fragmented b to membrane-bound 18-, 12-, reported (Abrahams et al., 1993; Bianchet et al., 1991). More and 8-9-kDa antigenic fragments, which cross-linked to recently, the structure of bovine F containing four molecules of d and/or with one another. Trypsin also removed the AppNP, and one molecule of ADP has been published, showing COOH-terminal part of A6L, but the remainder still at 2.8 A resolution the three-dimensional structures of the a cross-linked to subunit d. Models showing the near and f3 subunits plus about 50% of the y (Abrahams et al., 1994). neighbor relationships of the stalk subunits with one However, structural information regarding the remainder of another and with the a and p subunits at a level near the the ATP synthase complex is limited. Subunit cis considered to proximal end (bottom) of F and at the membrane- be shaped like a hairpin, with the hydrophobic arms forming matrix interface are presented. a-helices that traverse the membrane and the hydrophilic hair- pin bend protruding from the membrane on the F side. Recent two-dimensional NMR data for the isolated E. coli subunit c in The bovine heart mitochondrial ATP synthase complex iso- a chloroform-methanol-water solvent are in agreement with lated in our laboratory contains 13 well characterized subunits the hairpin model (Girvin and Fillingame, 1993, 1994). Small- angle neutron scattering studies of bovine OSCP (molecular *This work was supported by United States Public Health Service weight 20,967) have suggested an elongated shape of approxi- Grant DK08126. This is publication 8860-MEM from The Scripps Re- search Institute, La Jolla, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must The abbreviations used are: SMP, bovine heart submitochondrial therefore be hereby marked "advertisement" in accordance with 18 particles; AppNP, 5'-adenylyl imidodiphosphate; ASU particle, F -defi- U.S.C. Section 1734 solely to indicate this fact. cient SMP; EDC, 1-ethyl-3-(dimethylaminopropyl)carbodiimide; DCCD, * On leave from M. M. Shemyakin andY. A. Ovchinnikov Institute of N ,N' -dicyclohexylcarbodiimide; DST, disuccinimidyl tartarate; NHS, Bioorganic Chemistry, Moscow, Russian Federation. N-hydroxysulfosuccinimide; EGS, ethylene glycol bis(succinimidyl '11 To whom correspondence should be addressed: Division of Biochem- succinate); EEDQ, N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline; istry, Dept. of Molecular and Experimental Medicine, The Scripps Re- MBP, 4-(N-maleimido)benzophenone; PAGE, polyacrylamide gel elec- search Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. trophoresis; PMSF, phenylmethylsulfonyl fluoride; TPCK, N-tosyl-L- Tel.: 619-455-9100; Fax: 619-554-6838. phenylalanine chloromethyl ketone. This is an Open Access article under the CC BY license. 2054 ting. There was no evidence of additional cross-linking at the higher mately 90 X 30 X 30 A, with 43% a-helical structure as calcu- particle concentration, but at 0.1 mg of particle proteinlml there was a lated from circular dichroism measurements (Dupuis et al., higher yield of the cross-linked products, consistent with the less shield- 1983). Other than hydropathy profiles, information regarding ing effect of the particles against UV irradiation at the lower protein the structures of other ATP synthase subunits is not available. concentration. Because OSCP can be readily and reversibly removed from Trypsin Treatment of F -depleted SMP-F -depleted ASU particles 1 1 SMP together with F (Steinmeier and Wang, 1979; Matsuno- were suspended at 3-4 mg/ml in 20 mM Tris-HCl, 0.25 M sucrose, pH Yagi and Hatefi, 1984), it is considered to be a stalk component 7.5. TPCK-treated trypsin was added up to 0.8 mg/ml, and the mixture was incubated at 37 oc. At various times, aliquots were withdrawn, required for proper binding ofF to F . In addition, our previ- 1 0 proteolysis was arrested by the addition of 1 mM PMSF plus a 5-fold ous data, based on accessibility of ATP synthase subunits in excess of soybean trypsin inhibitor, and after 10 min on ice the soluble SMP and mitoplasts (mitochondria denuded of outer mem- and particulate fractions were separated by centrifugation. The super- brane) to proteases and subunit-specific antibodies, indicated natant proteins were precipitated by the addition of 10% trichloroacetic that OSCP, b, d, F , and the COOH-terminal half of A6L were acid and were subjected to SDS-PAGE and immunoblotting with spe- cific antibodies to F -stalk subunits. The particulate fraction was accessible from the matrix but not from the cytosolic side of the washed by suspending in buffer and recentrifuging, then a portion was inner membrane, whereas subunits a and c were not accessible suspended in triethanolamine/sucrose buffer plus 1 roM PMSF for cross- to the above reagents from either side of the mitochondrial linking with DST, and another portion in HEPES/sucrose buffer plus 1 inner membrane (Hekman et al., 1991). This paper presents mM PMSF for cross-linking with EDC + NHS. Conditions for cross- the results of cross-linking experiments involving the ATP linking and sample preparation for SDS-PAGE were the same as de- synthase subunits in SMP. The data are consistent with the scribed above. Protein concentrations were determined according to Lowry et al. ( 1951). results of our earlier topography studies described above and Electrophoresis--SDS-PAGE was performed according to Laemmli provide information regarding the near neighbor relationships (1970), routinely using a separating gel containing 15% acrylamide. For ofOSCP, b, d, F , and A6L, which appear to contribute to the optimal separation of high molecular weight cross-linked products, 10% formation of the stalk of the bovine ATP synthase complex. PAGE was employed. Antibody Production and Purification-The polyclonal antisera to EXPERIMENTAL PROCEDURES ATP synthase subunits a, b, d, F , and A6L used here were those Materials-Disuccinimidyl tartarate (DST), 1-ethyl-3-(3-dimethyl- described previously by Hekman et al. (1991). The OSCP antiserum aminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccin- used was from Belogrudov et al. (1988). Antisera to the F subunits a, {3, y, and ll were obtained previously in this laboratory, using homoge- imide (NHS), ethylene glycol bis(succinimidyl succinate) (EGS), and neous preparations of each F subunit as antigen. Antipeptide anti- N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) were ob- bodies were raised in the rabbit against a synthetic peptide correspond- tained from Pierce; nitrocellulose membrane (0.2-J.Lm pore size) from ing to residues 1-10 of subunit c (DCCD-binding protein), using the Schleicher and Schnell; SDS, acrylamide, and prestained SDS-PAGE procedure described elsewhere (Hekman et al., 1991). standards (broad range) from Bio-Rad; TPCK-treated trypsin, soybean trypsin inhibitor, 4-(N-maleimido)benzophenone (MBP) and Tween 20 The IgG fractions used for immunoblotting were affinity purified from Sigma; goat horseradish-conjugated anti-rabbit lgG from Calbio- according to Olmsted ( 1981), using as a source of antigen homogeneous chem; and an Enhanced Chemiluminescence kit from Amersham. F - subunits or subunits transferred to nitrocellulose membranes after ATPase and F -depleted ASU particles, prepared as described previ- SDS-PAGE ofF or purified ATP synthase complex. The affinity-puri- fied antibodies were stored in small aliquots at -20 oc. ously (Matsuno-Yagi and Hatefi, 1984), were gifts of Dr. Akemi lmmunoblotting Conditions-Conditions for immunoblotting were Matsuno-Y agi. the same as before (Belogrudov and Hatefi, 1994), except that electro- Cross-linking Conditions-SMP were diluted and washed with buffer blotting was performed at 100 V for 1 h. Where necessary, nitrocellulose and then adjusted to a protein concentration of 1.0 mg/ml in a medium sheets containing the same cross-linked samples were cut into separate containing 50 mM triethanolamine and 0.25 M sucrose, pH 8.0, and incubated with 1.0 mM DST for 30 min at 20 oc. The reaction was strips for blotting with different antibodies. The immunoblots were terminated by the addition of ammonium acetate to 50 mM. Mter a developed using the Amersham Enhanced Chemiluminescence kit ac- 10-min incubation an equal volume of Laemmli SDS-PAGE sample cording to the manufacturer's instructions. buffer (Laemmli, 1970) containing 6 M urea was added. In other exper- RESULTS iments, 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 Cross-linking of SMP with DST or EDC-Even though our reactions were allowed to proceed for 30 min at 20 oc and were then preparations of the ATP synthase complex display the same quenched by the addition ofTris-HCl, pH 6.8, to 100 mM. After a 10-min activities and inhibitor-response properties as the ATP syn- incubation, samples were treated with SDS-PAGE sample buffer as thase complex in SMP, we chose to carry out our studies of the above. F -depleted ASU particles were cross-linked at 0.5-1.0 mg/ml in near neighbor relationships of the ATP synthase subunits pri- the same manner as SMP. When F was used for cross-linking, an marily on well coupled SMP lest the purification process, which aliquot of the ammonium sulfate suspension of F was desalted on a involves the use detergents, might have introduced some struc- PD-10 column (Pharmacia Biotech Inc.) and adjusted to 0.2 mg/ml in the triethanolamine/sucrose buffer described above, and ATP was tural changes in the isolated enzyme. For example, the highly added to 1 mM. The preparation was then treated with 1 mM DST for 30 purified ATP synthase complex has a low phospholipid content min at 20 oc before the addition of 50 mM ammonium acetate to termi- and an absolute requirement for added phospholipids to dis- nate the cross-linking. Treatment of cross-linked F for SDS-PAGE was play ATPase and ATP- P; exchange activities (Galante et al., the same as described above. 1979). In some cases, however, we used F -depleted ASU par- For cross-linkings involving sulfhydryl groups, the photoactivable 1 ticles to expose to cross-linking stalk subunits that F might heterobifunctional MBP was used. MBP dissolved in dimethylform- amide was added at 1 mM to SMP at I mg/ml in 20 roM HEPES, 0.25 M mask. Among the cross-linkers employed were DST and EGS, sucrose, pH 7.2, and the mixture incubated for 25 min at 20 oc in dim which react with amino groups separated, respectively, by 6.4 light. Unreacted MBP was either removed by centrifugation or first and 16.1 A, and EEDQ and EDC, which activate carboxyl quenched by the addition of 10 mM dithiothreitol and then removed by groups for interaction with a nucleophile (e.g. amino group) to centrifugation. Mter resuspension in HEPES/sucrose buffer, the parti- result in zero length cross-linking. EDC was used in the pres- cles were irradiated with an 8-watt longwave UV lamp at a distance of ence of NHS, which is thought to react with the unstable 5 em for 10 min on ice, with occasional mixing. The particles were then prepared for gel electrophoresis by the addition of an equal volume of 0-acylisourea adduct of EDC to produce the more stable reac- SDS-PAGE buffer as indicated above. For control, particles were pre- tive NHS ester and improve the yield of the EDC-mediated incubated with 5 mM N-ethylmaleimide for 20 min before the addition cross-linked product (Staros et al., 1986). In all cases, cross- of MBP and UV irradiation. Under these conditions, no cross-linking linking conditions were optimized with respect to the concen- was observed. Furthermore, SMP or ASU particles were treated with trations of the cross-linkers and SMP, the duration of cross- 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 immunoblot- linking, and the temperature of the reaction mixture. The ATP Synthase Complex 2055 cross-linked products were identified after SDS-gel electro- anti-lgG phoresis of the samples and electrotransfer to nitrocellulose b OSCP d F AGL sheets by immunoblotting to affinity-purified polyclonal anti- bodies raised to each purified ATP synthase subunit, a , {3, y, S, OSCP, F , a , b, c, and d . The anti-A6L antibodies used were antipeptide antibodies raised to synthetic peptides correspond- ing to residue s 30-43 and 54-66 of this subunit. Attempts to raise antibodies or antipeptide antibodies to the E subunit of bovine F I 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 ·- - -d+AGL three other polypeptides, designated e , f, and g, in their prep- arations of ATP synthase complex, which appear to have no 3 2 ATP- P; exchange activity and only partially oligomycin-sen- sitive 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 -Fs to have complicated the interpretation of the cross-linking data -AGL shown below . 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 8 anti-lgG immunoblots of a 15% SDS gel, which favors transfer to nitro- a b OSCP d cellulose molecular mass species < 80 kDa , and Fig. lB shows immunoblots of a 10% SDS gel , which contains cross-linked products of higher molecular mass . The top of each lane states <X+~\ the antibody with which that nitrocellulose strip was blotted . In these and subsequent figures , the fastest moving (lowest) a+b\ ~+b- protein band is the uncross-linked subunit, except that in Fig. <X+Fsf 1B all of the uncross-linked F and parts of uncross-linked OSCP and d ran out of the gel. It might also be noted that there b+d- are no immunoreactive protein bands below uncross-linked b, OSCP , and d in Fig. 1A and below a and {3 in Fig. lB . 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. In Fig. lA , the cross-linked products of b + OSCP, b + F , FIG. 1. Cross-linking of SMP with DST. SMP at 1 mg/ml were and d + A6L are marked . Also marked are two species of b + incubated with 1 mM DST for 30 min at 20 °C. After termination of t he . h b d h reaction , samples were subjected to a 15% (panel A ) and a 10% (panel B ) d h , w IC are etter separate in Fig. lB. T ese could be the SDS PAGE d te" t t '" d t ·t 11 1 . . - , an pro ms were e 1 ec ro rans.erre o m roce u ose res~lt of two . t~pes_ of cross~lmkiD:g between ~ and the same .ffi!'.vM!4\.\\f,li-.$~y\t.r+)l'e.l~\~ . .f*\N&i,!>'.i,>;W-i.Mw.V:..:~.-,r.fk~,lW,l,r,!>,Q mol:ecut€illl''b 6t4=1ltffetenfc'.1;5'pes9:lf'£rossqirr~tffitWeen, "f1:· · . pro"dm:ts . .wete~lllotwdc'Wltn tfie·a1finit}'¥purrfierrO..antr110'iites":tnait\ateal~o~~-==== and the two molecules of b . The unlabeled top bands in Fig. 1A the top of each lane . The immunoreactive bands were visualized with are cross-linked products of each subunit with a/{3 which are the Enhanced Chem1lummescence detectiOn system. For deta il s, se e . . . ' "Expenmental Procedures ." also clearer m Fig. lB . These species appear to be a+ {3, {3 + b, {3 + OSCP, a+ OSCP, a + b, a + d, and a+ F . The unmarked cross-linked products involving the stalk subunits and the cross-linked bands seen in the upper parts of the lanes in Fig. small subunits ofF I· For example, one would expect the cross- lB are presumably cross-linked trimers and more complex linked products of they subunit ofFI with b, OSCP, and d to products. Essentially a similar cross-linking pattern was ob- produce a protein band immunoreactive to anti-b, anti-OSCP , tained when SMP were treated with EDC in the presence of or an ti-d in the Mr region of 50-60 kDa. However, as is clear in NHS . The product of d + A6L was particularly favored under Fig. lB, this region of these lanes is devoid of any such protein the latter conditions, and the major cross-linked products be- band. To investigate this issue further, SMP and purified F - tween the stalk and F I subunits were a + b, a + OSCP, a + F , ATPase were separately treated with DST and subjected to and {3 + d (data not shown) . Although not shown in Fig. 1, SMP SDS-gel electrophoresis and immunoblotting with affinity-pu- cross-linked with either DST or EDC + NHS and immunoblot- rified antibodies to the individual F I subunits a, {3, y, and S. ted to anti-a or anti-c antibodies showed no cross-linked prod- The results are shown in Fig. 2. The paired lanes compare the ucts involving these subunits. cross-linked products of the F I subunits as marked in the figure A close scrutiny of Fig. 1, A and B, suggests the absence of in SMP and isolated FI-ATPase. In the fir st and third lanes from the left , the bands not seen in the second and fourth lanes are the cross-linked products, respectively, of a and {3 with the Coupling factor B (Sanadi , 1982) with M, of 11-12 X 10 (You and Hatefi, 1976), which is present in SMP and restores ATP- P; exchange stalk subunits (see Fig. lB). Other bands common in the first and ATP-driven transhydrogenation and reverse electron transfer to four lanes are the cross-linked products of a + {3 and a + {3 plus ammonia-EDTA treated SMP (Stiggall et al. , 1979; Sanadi , 1982 ), has one or another small subunit ofF I· The important message of been considered to be a component of the bovine ATP synthase (Sanadi , Fig. 2 is contained, however, in the last four lanes . It is seen 1982). However, we and Walker's laboratory (Walker et al., 1991) have not detected factor B in our respective ATP synthase preparations. that in SMP or isolated F I the cross-linked products of y and S 2056 ATP Synthase Complex anti-a anti-~ anti-y ant Hi anti-b anti-OSCP anti-F SMP F SMP F SMP F SMP F 1 1 2 3 4 5 6 7 8 9 1 1 1 208- --! ... -115 115- 79-- -79 49- -49 34- b+OSCP- 28- b+F - -34 20- -28 b- oscp- -20 7- FIG. 2. Comparison of the cross-linking patterns ofF subunits in purified F and SMP treated with DST. F (0.2 mg/ml) and SMP (1 mg/rnl) were cross-linked with DST as in Fig. 1. Samples of cross- linked SMP and F were subjected to 15% SDS-PAGE in parallel, and -7 proteins were electrotransferred to nitrocellulose membrane and blot- ted 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 FIG. 3. Cross-linking of ATP synthase subunits in SMP with the figure . the heterobifunctional photoactivable sulfhydryl reagent, MBP. SMP at 1 mg/ml were incubated with 1 rnM MBP for 25 min at 20 •c. Particles were sedimented by centrifugation, resuspended in buffered are essentially the same in each case, even though the yields of sucrose solution, and UV irradiated for 10 min or kept on ice in the the cross-linked products of y appear to be less in SMP (per- dark. Samples were subjected to 15% SDS-PAGE, and proteins were haps due to shielding of y) than in isolated F . These results electrotransferred to nitrocellulose and probed with the affinity -puri- suggest, therefore, that in SMP y and a do not cross-link to any fied antibodies indicated on the top of the lanes. Immunore active bands were visualized as in Fig. 1. Lanes 1, 4, and 7, sample of SMP UV stalk subunit in the presence ofDST as the cross-linking agent. irradiated for 10 min; lanes 2, 5, and 8, MBP-treated, nonirradi ated Similar results were obtained when EDC + NHS were u sed. SMP; lanes 3, 6, and 9 , MBP-treated and UV-irradiated SMP. The However, since y and a produced a number of cross-linked positions of prestained mol ecular mass markers are shown in kDa on products common to SMP and isolated F , it is clear that they 1 the right of the figure. are accessible to cross-linkers in SMP. The question, therefore, is how y and a are located in SMP in relation to b, OSCP, d, F , an d OSCP involves this same Cys of b. In isolated OSCP, and A6L. Cysn , which is near the center of the chain, is modifiable by Cross-linking of SMP with MBP-MBP is a photoactivable substitu ted maleimides (Dupuis et al., 1985 ). However, sulfhydryl reagent that can alkylate a thiol via its maleimido whether this cysteine is accessible for modification in SMP is moiety. Then , up on irradiation (at about 350 nm) the aromatic not known. ketone moiety goes into a diradicaloid triplet state, resulting in It was of interest to see wheth er the removal ofF from SMP an electrophilic electron-deficient oxygen that is highly reac- might alter t h e cross-linking of stalk subunits. F -depleted tive, especially toward the C-H bonds of protein backbones ASU particles were prepared essentially according to Racker (Dorman and Prestwich, 1994). The result is the formation of a an d Horstman (1967 ). These preparations are depleted with C-C bond between the ketone carbon and the protein and respect to F and suffer partial loss of OSCP. The addition ofF 1 1 cross-linking at a distance in the case of MBP of about 10 A. + OSCP reconstitutes particles with high ATP synthase activ- Among the ATP synthase subunits under consideration here, ity (Matsuno-Yagi an d Hatefi, 1984). The remainder of OSCP those that contain cysteine residues are the a, y, and E subunits can be removed by further alkaline extraction of ASU particles, ofF plus b, OSCP, d, and c, of which a has 2 cysteine residues/ but this was not done here because ou r aim was to investigate mol and the others 1 cysteine residue each . The experiments the effect ofF removal. When treated with MBP, ASU parti- with MBP were carried out by incubating SMP ( - 1 mg/ml) cles produced two major cross-linked products among the cys- with 1 mM MBP for 25 min. Unreacted MBP was removed by teine-containing subunits of the F -depleted ATP synthase. centrifugation. SMP were resuspended in buffered sucrose so- One was b + F as in Fig. 3, the other was a band at about 44 lution, subjected to UV irradiation as described under "Exper- kDa, wh ose mobility and singular reactivity with only anti-b imental Procedures ," and analyzed by SDS-PAGE and immu- lgG suggested that it is a cross-linked b + b dimer (Fig. 4, lane noblotting with antibodies against 6 of the 7 cysteine- 2 ). There were a lso detectable amounts of b + OSCP (Fig. 4, containing subunits mentioned. As seen in Fig. 3, only two lane 6). The addition to ASU particles ofF (2 and 6 mol/mol F , 1 0 cross-linked products were detected, one between b and OSCP respectively, in the thi rd and fourth lanes of each set in Fig. 4) at about 42 kDa , and another between b and F at about 34 increased the yield of b + OSCP dimer and diminished the kDa. The formation of these produ cts required both the MBP yield of the putative b + b dimer. There are two possible pretreatment of SMP and the subsequent UV irradiation. explanations for t h ese results: (i) removal ofF caused a partial When the SMP were first treate d with 5 mM N-ethylmaleimide separation of b and OSCP so that b-MBP was too distant from before the addition ofMBP and UV irradiation, the cross-linked OSCP to cross-link upon photoactivation or (ii ) removal ofF products shown in Fig. 3 were not forme d (data not sh own) . As resulted in masking of the thiol group of OSCP, making it mentioned above, subunit b contains a single cysteine residue unable to form OSCP-MBP to cross-link with b upon photoir- at position 197, which is close to its COOH terminus, and F radiation. We prefer the first possibility because structural has no cysteine residues . Therefore, the MBP-mediated cross- perturbation of the stalk may also bring the two MBP-modified linking between F and b indicates that the COOH terminus of b subunits closer to one another for cross-linking after photo- b is located near F . It is also possible that cross-linking of b activation of their benzophenones. As seen in Fig. 4, the cys- 6 ATP Synthase Complex 2057 anti-b an ti-d 1991 ), s ubunits a and c were unaffected by trypsin and did not 2 3 4 9 10 11 12 produce a ny cross-linked products when the trypsin-treated -11 5 ASU particles were subjected to cross- linking with DST or EDC -79 + NHS (data not shown) . The COOH-terminal end of A6L was remov ed by tryp sin (Hekman et al. , 1991 ), but the remainder of b+b?\ -4 9 b+OSCP- t h e molecule, including the antigenic re sidues 30-43, survived , -- and this truncated A6L cross-linked to subunit d (see Fig. 5A, b+F6- -- -34 --- b"\. ____ _ lane 9, first arrowhead from bottom ) when the trypsin -treated -28 _ _. _____ _ ASU particles were treated with DST . OSCP - The effect of trypsin (1:25 weight ratio of trypsin to particl e df -20 protein ) on subunits b and d agreed with the results of others (Collinson et al., 1994a; Walker and Collin son , 1994 ), which 166 167 120 121 ha wed that b was hydrolyzed at Arg -Gly , Lys -Arg , 121 122 4 5 and Arg -His , and d at Lys -Leu (see Fig. 5A , lanes 2-4 -7 and 8-10). Hydropathy analysis of sub unit b shows two hydro- phobic clusters of about 20 residues each near the NH termi- FIG. 4. Cross -linking ofF ,-deficient ASU particles with MBP in nus followed by a 130-residue-long hydrophilic region to the the absence and presence of added F • ASU particles (0.5-1.0 1 COOH-termina l end. This suggests that the molecule is an - mg/ml ) were incubated with 1 mM MBP for 25 min at 20 °C , and excess chored to the membrane via its two hydrophobic clusters and reage nt was r emove d by centrifugation. MBP-modified pa rticl es were that its short h ydroph ilic NH terminus - 30 residues ) and its incubate d without a dd ed F, (la nes 1, 2 , 5, 6, 9, and 10 ) or with 325 J.Lg 130-residue-long hydrophilic tail are extramembranous on the ofF ,lm g of particles (lane 3, 7, 11) or 1.0 mg ofF ,fm g of part icles (lanes 4 , 8, a nd 12 ), each for 30 min at 37 °C. Particles were r e isolated by F side . According to this picture , the tryptic cleavages at centrifugation, washed, res uspended in 20 mM Tris-HCI, pH 7.5, con- 166 167 120 121 121 122 Arg -Gly and at Lys -Arg (o r Arg -His ) would taining 0.25 M sucrose a nd UV irradi ated for 10 min on ice. Sample produce two membrane-bound fragments , respectively , about were s ubj ected to 15% SDS-PAGE , and prote in s were e lectrotran ferred 18-19 and 12-14 kDa. These b fragments , which still reacted to nitrocellulose and blotted with the affinity-purified a ntibodi es indi- cated on t h e top of the lanes. Immunoreactive band s were vis ualiz e d as with our polyclonal antibody preparation, are seen in lanes 2-4 in Fig. 1. Lanes 1, 5, a nd 9, MBP-treated, nonirra diated ASU particles; of Fig. 5A . They cross-linked in the presence of DST or EDC + other lanes, MBP-treated ASU particles UV irrad iated for 10 min on ice . NHS with subunit d to produce the two bands marked with The positions of prestained molecular m ass marke1·s are shown in kD a arrowheads in lanes 3, 4, 9, and 10 of Fig. 5A . It may a lso be on the right of the figur e . noted that the yield of the slower moving cro ss -linked product was greater when DST was the cross-linking reagent, and the teine-containing subunit d in ASU particles (or in SMP) did not yie ld of the fa ster moving cross-linked product was greater form any MBP-mediated cross-linked products. As was shown when EDC + NHS were u sed for cross-linking. Other cross- previously, subuni t din SMP and ASU particles is resistant to linked products recognized and marked by arrowheads in Fig. trypsinolysis . It is possible, therefore, that much of subunit d, 5A are the b + b dimer in lane 6 and d + F in lane 11. We including its cysteine residue, is shielded by oth er ATP syn- suspect that the band marked d + A6L, d + xis composed of thase subun it s in SMP and ASU particles. more t h an one cross-linked product of subuni t d. However, Cross-linking ofF rdepleted Particles Treated with Tryp sin- additiona l informatio n is not available at this time. These experiments were carried out with ASU particles t h at The data in Fig. 5B are from an a liquot of ASU particles that had been extracted twice with urea to divest them ofF . They was treated with the higher concentration of trypsin (1: 5 were th e n treated with trypsin under relatively mi ld (1 :25 we ight ratio of trypsin to particle protein ). It is seen in lanes 1 we igh t ratio of trypsin to particle protein for 1 h at 37 °C) or and 2 that subunit b was further degraded to produce an severe ( 1:5 weight ratio of trypsin to particle protein for 1 h at 8-9-kDa antigenic fragment , which was still membrane-bound . 37 °C) cond itions . Proteolys is was arrested as described under Considering that there are about 70 amino acids from the NH "Experimental Procedures, " and the particles were separated terminus to the end of the second hydrophobic cluster of sub- from the soluble protein fragments by centrifugation and wash- unit b, t h e tryptic cleavage site to produce an 8-9-kDa mem- ing. Aliquots of the particles were then subjected to cross- brane-bound fragment must be very close to wh ere the COOH - linking with DST and EDC + NHS, electrophoresed, and im- termina l hydrophilic tail of b exits the membrane . If we assume munoblotted with antibodies to subunits b, d , OSCP , a nd F . that the hydrophobic clusters of b are nonantigenic, then it is Before trypsin treatment cross-linking of the F -depleted par- lik ely that our anti -b IgG recognizes the 30-residue-long NH t icles produced a b + OSCP dimer in a fairl y good yield when terminus of subunit b, which is probably extramembranous . As the cross- linker was DST and in very poor yie ld when it was seen in lane 3 of Fig. 5B, this 8-9-kDa fragment disappeared in EDC + NHS . This agrees with our conclusions regarding cross- the presence of EDC + NHS and produced the cross- linked linking of ASU particles with MBP and suggests that removal products marked with a rro wheads. The uppermost band ofF resu lts in separation of OSCP from b to the extent that marked with an arrowhead a lso appeared to form in lower yield DST (molecular length , 6.4 A.) can still bridge the distance when DST was the cross-linking reagent (Fig. 5B, lane 2). The between t hem, but EDC + NHS (ze ro le ngth cross -linking) proteolysis pattern of bovine b with trypsin as shown in Fig. 5 cannot . By contrast, cross-linking of the F -depleted particles (see also Collinson et al., 1994a; Walker and Collinson, 1994 ) is with either DST or EDC produced a b + F dimer in good yie ld simil ar to that of E . coli subuni t b (Steffens et al. , 1987 ), which (Fig. 5A, lanes 5 and 6, second h eavy band from bottom ), just as is a further in dication of the ana logy between them. it was shown in Fig. 1 for SMP. After treatment of the particles DISCUSSION with the lower concentration of trypsin, OSCP and F were completely degraded in agreement with previous results (Joshi Considerable information is now available regarding the and Burrows, 1990; Hekman et al., 1991 ), and no cross-linked mechanisms of ATP hydrolysis (Al-Shawi et al., 1990; Penefs ky products containing antigenic fragments of these subunits and Cross , 1991; Boyer , 1993) an d synthesis (Matsuno-Yagi were detected by immunoblotting. Also, in agreement with and Hatefi , 1990; Hatefi , 1993) at the level of the catalytic previous re sults (Joshi and Burrows, 1990; Hekman et al., sector, F , of the ATP syn thase complex. Also, the crystal 1 ATP S y nthase Co mplex 205 8 A 8 anti-b anti-b ant i-d ,-----------, DST DST + - + - - - + - + - - + - EDC EDC - + - + - + - + + tryps i n - + ++ ---+++ - trypsin + + + 1 2 3 4 5 6 7 8 9 1011 12 1 2 3 115 - 1 15 - 79 - 79 - 49 - 49 - 34 - ... - .;.. - b + F 34 - 28 - . - .. _ _ f d+A6L 28 - ... = 20 - ---- - l_d+X b (18)__ __ .. _ .. b( 12) __ __ _ b(18)~ ------- --- b(8)7"- b(12)-- 7 - FI G. 5 . Cross-linking of ATP synth as e subunits in F ,-defici e nt ASU parti c les trea t e d with tryps in. ASU pa r ticl es (3 .5 mg/m !) we re in cu bate d wit h t r ypsin (25: 1 r atio by we igh t, panel A , or 5: 1 ratio by weigh t, panel 8 ) fo r 1 h a t 37 •c. Proteo lys is wa s a JTeste d by th e a dd itio n of excess s oy bea n t ryps in inhibi to r plu s 1 mM PMSF , a nd part icles we re reis olated by ce nt ri fugation , was hed , a nd cross-linked wit h 1 m ~·I DST or 5 m M E DC in th e prese nce of 5 m M N H S. Sam pl es we r e s ubj ected to 15% SDS- PAGE , a nd prote in s were electrotr ansfe r red to n itroc e ll u lose memb r a ne a nd blotted wit h t he a ffini ty-p urifi ed a n t ibo die s i ndi cate d on top of t he lan es . l m mu nob lots we re deve loped as describ ed in Fig. 1. Th e tab les of plus a nd minus signs on top of t he immun oblots s how t h e t reatm en t t he ASU pa r t icl e of ea ch lane ha d rece ive d . Arro w heads a re desc r ibe d u n de r "Res ul t s ." Th e des ignation s b(JB), b(12), an d b(8) a r e for m embr ane -bo und try ptic fr agme nt s of s ubu ni t b wit h a ppr ox imate M ,. va lu e of 18, 12 , a nd 8, res pect ive ly. Th e pos itio ns of prestai ned molecul a r mass ma rk e•·s a re s hown in kDa on th e left of panels A a nd B . t h a t OSCP binds to bovi n e F (Hu n da l et al., 1983 ; Dupui s et struct ure of a form of bovi n e h eart F co ntain ing four m olecu les 1 1 of AppNP a nd one mol ecu le of ADP has bee n solve d rece n t ly at al., 1985 ) a nd t hat t h e F -0SC P co mpl ex ca n bind s u buni ts b , 2.8 A. r esoluti on (Abra h a m s et al., 1994 ). In a ddi tio n , it is F , a nd d (Wa lk er an d Collin on , 1994 ). E vid en ce t h at F can kn ow n th a t tran s membra n e prot on s pass only throu gh t h e be r eve r sibl y extr acte d fro m SMP is a lso ava ila ble (Fesse nd en- m embr a n e sector , F , of th e ATP synthase compl ex, a nd e n er gy Ra den , 1972 ). Th ese data a r e con siste n t wit h our ea r lie r stud- communication betwee n F a nd F t ak es pl ace via coup led ies sh owin g t hat b, OSC P , d , an d F a r e accessible t o subun it- 0 1 6 conform ation ch a nges of subunits (for review, see Hate fi , 1993). spec ifi c a ntib odi es in we ll co upl ed SMP , but n ot in mitopl asts, At th e level of F , protein-bound ATP is sy n t h esi ze d fro m a nd t hat in SMP s u buni ts b, OSCP , F , a nd t h e COOH- termi- 1 6 n a l a n t ige ni c pa r t of A6 L co u ld be degrad ed by t ry ps in protein-bound ADP a nd P i with out en er gy u t ili zation , but th e binding of P i a nd ADP t o F a nd th e r elease of ATP fr om F a r e (H ekm a n et al. , 199 1). Th ese studi es a lso indi cate d cert a in 1 1 th e en er gy-requirin g proces ses, which a r e a pp a r ently accom- su bun it access ib ili ty di ffe r en ces betwee n SMP a nd th e purifi ed pli sh ed by a ppropr ia t e conform a tion cha nges of t h e {3 subuni ts . ATP sy n t hase co mpl ex, which decided our choi ce of m ateria l for Th e crystal structure of bovine F (Abra h a ms et al., 1994 ) a nd dete rm inin g t h e stoichi ometry of subunits in F a nd t h e stalk th e cryoe lectron microscop y studies of Ca pa ldi a nd co-workers (H e km a n et a l. , 199 1) as we ll as for ou r studies r eported h er e Wilkin s a nd Ca pa ldi , 1994, a nd re fere nces th er ein ) on t h e E. on t h e nea r n eighb or r ela ti onship s a mon g th e stalk subuni ts co li F s uggest th at s ubuni ts y a nd E are invol ve d in co n veyin g a nd betwee n t h e latter a nd t h e s ubuni ts ofF a nd F . 1 1 0 T a bl e I summ arizes t h e res u lt s obta ined in SMP a nd F - confo r ma ti on ch a nges to a nd from F . However, in bo t h t h e bo vin e a nd E. co li ATP synthases th er e is a 45 -A.-l on g sta lk de pl eted SMP wit h t hree cross -linkin g r eage nts (D ST , MBP , bet wee n F a nd F (Soper et a.l. , 1979 ; Ca pa ldi et al., 1992; see E DC ) of differ en t ch emi cal reactivit ies a nd differ e nt effective a ls o Dub a chev et al. , 1993 ), wh ose stru cture a nd ro le in en er gy distances for cross- linkin g. Co nside rin g fir st t h e n ea r n eighbor communica tion betwee n F a nd F a r e not known . If one as - re lations hip s of t h e s t a lk subunits, it is clea r t h at OSC P cr oss- s umes th a t the ATP sy n t h ase subuni ts th a t a r e not compo- linked only to b, a nd A6L only to d. Th e NH te rmi n us of A6L n e nts of F but are en t irely or pa rtl y extra membra nou s co ntains a sh or t hy dro ph obi c clu ste r ( - 20 residues ), whi ch (H e km a n et al., 1991 ) contribute to t h e co mpo sition of th e s t a lk , a ppea r s to a nchor A6 L to t h e membr a ne . Th e rem a inder of t h e t h en s ubuni ts b, OSCP , d , F , a nd A6L would qu a li fy as ca n- molecu le is hydroph ilic a nd a ppa r en t ly extr a membrano u s. did ates . As possi bl e compone nts of t h e stalk , t hese subuni ts Try psin cleaves off t h e CO OH-termin a l a nti ge ni c part of A6L wo u ld be directly or indirectl y in vo lve d in conform a ti on a l en - (H ekm an et al. , 199 1), but th e r em a inder of t h e molec ul e is still er gy t r a ns fer betwee n F a nd F . Therefor e, information re - close en ough to sub u ni t d to for m a cross -linked produ ct in t h e 0 1 garding t h eir r ela ti ve positi ons in t h e stalk a nd t h eir s pa ti a l prese n ce of DST (Fi g. 5, lan e 9, bottom band ma rk ed wit h a n inter a ct ion with one a n oth er as we ll as wit h F a nd F subuni ts a rrow h ead ). Thi s also suggests t h at su bunit d is prese nt nea r 0 1 wou ld be cru cia l to inves ti gatin g t h e m ech a ni s m of en er gy t h e membra n e. Indee d , t h e NH te rm inu s of d contains a sh ort tra ns fer within th e ATP sy nthase co mpl ex. h ydroph obic clu ste r of a mino acids , whi ch may be in te r ca late d As mention ed ea rli e r , OSCP is eas il y rem ova bl e fro m SMP in to t h e me mbra n e . (Senior , 1979 ) or prepa r a tion s of th e ATP sy n t h ase comp lex As see n in Ta bl e I , a ll of t h e s t a lk subuni ts , exce pt A6L, (Ga la nte et al., 198 1). Remo va l of OSCP a lso r es ul ts in th e cross-link to b in SMP . As men t ion ed a bo ve , t h e hydro pa thy r emova l ofF , but bo t h ca n be a dd ed back t o F to r econ sti t u te a n a lys is of b sh ows tw o hydroph obi c clusters of - 20 r esidu es 1 0 oli gomycin-se nsit ive ATPase activity (Ga la n te et al. , 198 1) or t o eac h located - 30 resi du es fro m t h e NH te rminus . Th ere for e, de pl et ed SMP t o r eco nstit ute oxid a tive phosphorylation a t hi gh on e mi gh t ex pect t h at t h e two hydroph obi c clu sters in te r calate r a t es (Ma t suno-Yagi an d H a t e fi , 1984 ). In a ddi ti on , it is kn own in to t h e membrane, with t h e sh ort NH -ter min a l segm ent a nd 2 2059 TABLE I Cross-linked products of subunits b, OSCP, d, F & and A6L formed in A SMP and F -depleted SMP treated with DST, EDC, or MBP F ,-depleted SMP SMP DST EDC MBP DST EDC MBP b-ba b-ba b-ba b-db b-db b-db b-OSCP b-OSCP b-OSCP b-OSCP b-OSCP b-F b-F b-F b-F b-F b-F 6 6 6 6 6 6 d-A6L d-A6L d-A6L d-A6L b-a/{3 b-a/{3 OSCP-a/{3 OSCP-a/{3 d-a/{3 d-a/{3 Fs-a/f3 F s·a/{3 --;;-Based on Mr and immunoreactivity with only anti-b IgG. b Two species. the long ( ~ 130 residues) COOH-terminal portion beyond the second hydrophobic cluster being extramembranous on the F side (Walker and Collinson, 1994). Furthermore, our study of the stoichiometry ofF 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 -depleted SMP, treated with either DST or EDC, a band that reacted only FIG. 6. Schematic representation of the near neighbor rela- with anti-b IgG and exhibited on SDS gels a mobility consistent tionships of subunits b, OSCP, d, F , and A6L in bovine submi- with that of a cross-linked b + b dimer. tochondrial particles. Cross-linked <timers of subunits were identi- In addition to cross-linking of b to OSCP and F effected by 6 fied where circles representing the subunits overlap. Because DST or EDC, b + OSCP and b + F dimers were also produced knowledge of the structures of these subunits is limited, the diameters of the circles were deliberately made the same to avoid any inference when the cross-linking reagent was the heterobifunctional pho- regarding the relative sizes and shapes of these subunits. A, en~-on toactivable sulfhydryl reagent, MBP (Fig. 3). Because F is view of a cut parallel to the membrane through the bottom (proximal devoid of cysteine residues, the cross-link between b and F end) ofF,. B, end-on view of a cut parallel to the membrane at the must involve Cys of b near its COOH terminus. The same membrane-matrix interface. might be true in the case of the MBP-induced cross-link be- tween band OSCP, or the cysteine in this case might be Cys the proximal end (bottom) of F (A) and at the membrane- of OSCP. However, the fact that the b-MBP adduct did not matrix interface (B). In A, subunits b, OSCP, d, and F are all cross-link upon photoactivation to a, {3, y, or 6 may mean that present because they all cross-link to a and/or {3 (the parallel- the COOH-terminal portion of b, which contains Cys , is ogram labeled a/{3). They also cross-link to one another wher- folded back toward F and away from F • Subunit b of E. coli 0 1 ever the circles representing each subunit overlap. In B (the ATP synthase is also thought to have a folded conformation bottom of the stalk), A6L protrudes from the membrane near d, (Fillingame, 1990; Penefsky and Cross, 1991). It might be and the two b subunits enter the membrane. However, whether added parenthetically that treatment of SMP or ATP synthase OSCP and F extend the length of the stalk from F to F to 1 0 preparations with various mono- and dithiol modifiers results impinge upon the membrane is unclear; therefore, they have in uncoupling (Yagi and Hatefi, 1984), and the studies of Papa not been included. In F -depleted ASU particles, this organi- and co-workers (Zanotti et al., 1988) implicate Cys of subunit zation is somewhat perturbed, as if the removal ofF results in b in this type of uncoupling. Although not shown in Fig. 3, we the inward collapse of the b subunits toward each other, also checked by immunoblotting for MBP-induced cross-links of a, bringing F close to d. As a consequence, in ASU particles, but -y, d, and c, all of which contain cysteine, but none was found. not in SMP, b + b and d + F cross-linked dimers can form (the The cysteine of subunit c is within the membrane and may not latter is marked by an arrowhead in Fig. 5A, lane 11). When react with MBP (although it appears to react withp-chloromer- MBP was used for cross-linking of ASU particles, b + F was curibenzoate).3 Apparently, the cysteines of a, -y, and dare also formed as in MBP-treated SMP, but the yield of b + OSCP unreactive with or inaccessible to MBP. dimer was greatly diminished (Fig. 4, lane 6). Instead, there As seen in Table I, b, OSCP, d, and F , but not A6L, form was the appearance of a band qualifying as a b + b dimer, cross-linked products in SMP with a and/or {3. Furthermore, in which was not seen in MBP-treated SMP. Interestingly, the SMP, OSCP, d, and F can each cross-link to b but not to one addition of F to ASU particles prior to MBP treatment re- another. Therefore, it seems reasonable to assume that the two stored the formation of the b + OSCP product and diminished molecules of subunit b, which extend from the membrane to F , the yield of the b + b dimer (Fig. 5, lanes 3, 4, 7, and 8). These form the principal stem ofthe stalk to which OSCP, d, and F results suggest that (i) F removal diminishes the interaction of bind independently of one another. These considerations are b and OSCP, which is consistent with the fact that extraction of summarized in Fig. 6, which shows the near neighbor relation- F also results in labilization of OSCP, and (ii) the readdition of ships of the stalk subunits in cuts parallel to the membrane at F to ASU particles reestablishes the close association of b and OSCP, which is also consistent with the fact that function can A. Matsuno-Yagi andY. Hatefi, unpublished results. be restored to ASU particles by the addition of F and OSCP 1 2060 Belogrudov, G.!., Ilyina, E. F., Grinkevich, V. A., and Modyanov, N. N. (1988)Biol. (Matsuno-Yagi and Hatefi, 1984). Recent data of Abrahams et Membr. (U.S. S. R) 5, 677-687 al. (1994) on the crystal structure of bovine F indicate that 'Y Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) protrudes 30 A from the core ofF , which the authors believe J. Bioi. Chern. 266, 21197-21201 Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250 forms part of the stalk. Fig. 6A shows a possible location for the Capaldi, R. A., Aggeler, R., Gogo!, E. P., and Wilkens, S. (1992) J. Bioenerg. tail of 'Y surrounded by b, d, and OSCP. This possible location Biomembr. 24, 435-439 Collinson, I. R., Fearnley, I. M., Skehel, J. M., Runswick, M. J., and Walker, J. E. inside the stalk is consistent with our inability to find cross- (1994a) Biochem. J. (Lond.) 330, 639-645 linked products of 'Y with any of the stalk subunits. It also Collinson, I. R., Runswick, M. J., Buchanan, S. K., Fearnley, I. M., Skehel, J. M., agrees with the presumed inward collapse of the stalk to allow van Raaij, M. J., Griffiths, D. E., and Walker, J. E. (1994b) Biochemistry 33, 7971-7978 the formation of b + b and d + F cross-linked products when Dorman, G., and Prestwich, G. D. (1994) Biochemistry 33, 5661-5673 'Y together with the remainder ofF is removed from SMP. As 1 Dubachev, G. E., Lunev, A. V., Barnakov, A. N., Belogrudov, G. I., Grinkevich, V. A., and Demin, V. V. (1993) FEBS Lett. 336, 181-183 was shown in Fig. 2, both 'Y and a form multiple cross-linked Dunn, S.D., and Heppe!, L.A. (1981)Arch. Biochem. Biophys. 210,421-436 products when SMP or isolated F preparations were treated Dunn, S. D., Heppe!, L. A., and Fullmer, C. S. (1980) J. Bioi. Chern. 255, 6891- with DST (or EDC, not shown). However, like y, a also failed to Dupuis, A., Zaccai, G., and Satre, M. (1983) Biochemistry 22, 5951-5956 cross-link to any stalk subunit. The data of Abrahams et al. Dupuis, A., Issartel, J.-P., Lunardi, J., Satre, M., and Vignais, P. V. (1985) (1994) on the crystal structure of bovine F do not show the Biochemistry 24, 728-733 Eytan, G. D., Carlenor, E., and Rydstriim, J. (1990) J. Bioi. Chem. 265, 12949- location of a. It may be of interest to add in this regard that E. coli a and a cross-link via a disulfide bond in the presence of Fessenden-Raden, J. M. (1972) J. Bioi. Chern. 247, 2351-2357 CuCl (Mendel-Hartvig and Capaldi, 1991), a G29D mutation Fillingame, R. H. (1990) in The Bacteria (Krulwich, T. A., ed) vol. 12, pp. 345-391, Academic Press, New York of the E. coli a renders the F a-deficient (Maggio et al., 1988), Galante, Y. M., Wong, S.-Y., and Hatefi, Y. (1979) J. Bioi. Chem. 254, 12372-12378 and tryptic removal of 15 NH -terminal residues of the a sub- 2 Galante, Y. M., Wong, S.-Y., and Hatefi, Y. (1981) Arch. Biochem. Biophys. 211, 643-651 units of E. coli F destroys the ability of the reconstituted Girvin, M. E., and Fillingame, R. H. (1993) Biochemistry 32, 12167-12177 a (3 yE to bind a (Dunn et al., 1980). As mentioned earlier, E. 3 3 Girvin, M. E., and Fillingame, R. H. (1994) Biochemistry 33, 665-674 coli a is considered to be analogous to the bovine OSCP (Walker Graf, T., and Sebald, W. (1978) FEBS Lett. 94, 218-222 Hatefi, Y. (1993) Eur. J. Biochem. 218, 759-767 et al., 1982; Ovchinnikov et al., 1984; see also Dunn and Hekman, C., Tomich, J. M., and Hatefi, Y. (1991) J. Bioi. Chern. 266, 13564-13571 Heppel, 1981). However, the results just described make one Hundal, T., Norling, B., and Ernster, L. (1983) FEBS Lett. 162, 5-10 Joshi, S., and Burrows, R. (1990) J. Bioi. Chern. 265, 14518-14525 wonder about the location of a in relation to the distal end of E. Kiehl, R., and Hatefi, Y. (1980) Biochemistry 19, 541-548 coli F v where by analogy to the bovine F the NH termini of 1 2 Laemmli, U. K. (1970) Nature 227, 680-685 the a subunits would be located (see Abrahams et al., 1994). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bioi. Chern. 193, 265-275 Finally, it should be mentioned that Joshi and Burrows Lutter, R., Saraste, M., van Walraven, H. S., Runswick, M. J., Fine!, M., (1990; see also Archinard et al., 1986) have previously detected Deatherage, J. F., and Walker, J. E. (1993) Biochem. J. 295, 799-806 Maggio, M. B., Parsonage, D., and Senior, A. E. (1988) J. Bioi. Chern. 263, in a preparation of bovine ATP synthase treated with dithio- 4619-4623 bis(succinimidylpropionate), which cross-links amino groups at Matsuno-Yagi, A., and Hatefi, Y. (1984) Biochemistry 23, 3508-3514 a distance of 12 A, many of the cross-linked products we have Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Bioi. Chern. 265, 82-88 Mendel-Hartvig, J., and Capaldi, R. A. (1991) Biochim. Biophys. Acta 1060, 115- characterized here. They employed antibodies to whole F , OSCP, F , A6L, a, and c, but not to the individual subunits of Olmsted, J. B. (1981) J. Bioi. Chern. 256, 11955-11957 Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., F and subunit d. Therefore, some of the cross-linked products Trubetskaya, 0. E., Nazimov, I. V., Hundal, T., and Ernster, L. (1984) FEBS were identified in part on the basis of their mobility on two- Lett. 166, 19-22 dimensional SDS gels. The cross-linked products they report, Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. 64, 173-214 Racker, E., and Horstman, L. L. (1967) J. Bioi. Chern. 242, 2547-2551 which we have not seen in SMP with our three cross-linking Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56 reagents, are those ofF withy, a, and A6L. They also claim the Sebald, W., Graf, T., and Lukins, H. B. (1979) Eur. J. Biochem. 93, 587-599 Senior, A. E. (1979) Methods Enzymol. 55, 391-397 formation of F + d dimer (they refer to d as the 20-kDa Soper, J. W., Decker, G. L., and Pedersen, P. L. (1979) J. Bioi. Chern. 254, subunit), which we only find in DST-treated ASU particles, but 11170-11176 not in intact SMP. Staros, J. V., Wright, R. W., and Swingle, D. M. (1986) Anal. Biochem. 156, 220-222 Steffens, K., Schneider, E., Deckers-Heberstreit, G., and Altendorf, K. (1987) Acknowledgments-We thank C. Munoz for the preparation of mito- J. Bioi. Chern. 262, 5866-5869 chondria and Drs. Akemi Matsuno-Yagi and Mutsuo Yamaguchi for Steinmeier, R. C., and Wang, J. H. (1979) Biochemistry 18, 11-18 helpful discussions. Stiggall, D. L., Galante, Y. M., and Hatefi, Y. (1978) J. Bioi. Chern. 253, 956-964 Stiggall, D. L., Galante, Y. M., Kiehl, R., and Hatefi, Y. (1979) Arch. Biochem. REFERENCES Biophys. 196, 638-644 Walker, J. E., and Collinson, I. R. (1994) FEBS Lett. 346, 39-43 Abrahams, J. P., Lutter, R., Todd, R. J., van Raaij, M. J., Leslie, A. G. W., and Walker, J. E. (1993) EMBO J. 12, 1775-1780 Walker, J. E., Runswick, M. J., and Saraste, M. (1982) FEBS Lett. 146, 393-396 Walker, J. E., Lutter, R., Dupuis, A., and Runswick, M. J. (1991) Biochemistry 30, Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621-628 5369-5378 Wilkens, S., and Capaldi, R. A. (1994) Bioi. Chern. Hoppe-Seyler 375, 43-51 Al-Shawi, M. K., Parsonage, D., and Senior, A. E. (1990) J. Bioi. Chern. 265, Yagi, T., and Hatefi, Y. (1984) Biochemistry 23, 2449-2455 4402-4410 Archinard, P., Godinot, C., Comte, J., and Gautheron, D. C. (1986) Biochemistry You, K.-S., and Hatefi, Y. (1976) Biochim. Biophys. Acta 423, 398-412 Zanotti, F., Guerrieu, F., Capozza, G., Houstek, J., Ronchi, S., and Papa, S. (1988) 25,3397-3404 Belogrudov, G. I., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576 FEBS Lett. 237, 9-14 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

ATP Synthase Complex

Belogrudov, Grigory I.; Tomich, John M.; Hatefi, Youssef
Journal of Biological Chemistry – Feb 1, 1995
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10.1074/jbc.270.5.2053
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Vol. 270, No. 5, Issue of February 3, pp. 2053-2060, 1995 THE JoURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. PROXIMITIES OF SUBUNITS IN BOVINE SUBMITOCHONDRIAL PARTICLES* (Received for publication, October 11, 1994, and in revised form, November 21, 1994) Grigory I. Belogrudov:j:, John M. Tomich§, and Youssef Hatefi11 From the Division of Biochemistry, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the §Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506 (Galante et al., 1979; Hekman et al., 1991), exhibits completely The catalytic sector, F and the membrane sector, F , 10 0 of the mitochondrial ATP synthase complex are joined oligomycin-sensitive ATPase and ATP- P; exchange activities together by a 45-A-long stalk. Knowledge of the compo- (Stiggall et al., 1978; Galante et al., 1979), and can drive via sition and structure of the stalk is crucial to investigat- ATP hydrolysis a second mitochondrial proton pump when ing the mechanism of conformational energy transfer coincorporated in liposomes (Eytan et al., 1990). The polypep- between F and F • This paper reports on the near neigh- tide components of the ATP synthase are the a, /3, y, 5, and e 0 1 bor relationships of the stalk subunits with one another subunits of the catalytic sector F v plus OSCP, a (subunit 6), b, and with the subunits ofF and F , as revealed by cross- 1 0 c, d, F , and A6L, which together make up the stalk and the linking experiments. The preparations subjected to membrane sector F . Preparations of the ATP synthase com- cross-linking were bovine heart submitochondrial par- plex also contain substoichiometric amounts of the ATPase ticles (SMP) and F -deficient SMP. The cross-linkers inhibitor protein, IF . The stoichiometry of F subunits is 1 1 were three reagents of different chemical specificities a f3 y5e, and the stoichiometry ofOSCP, d, A6L, b, and F , as 3 3 6 and different lengths of cross-linking from zero to 10 A. determined by radioimmunochemical techniques in well cou- Cross-linked products were identified after gel electro- pled SMP (Hekman et al., 1991), is 1:1:1:2:2. Bovine ATP phoresis of the particles and immunoblotting with sub- synthase also contains multiple copies of subunit c (Graf and unit-specific antibodies to the individual subunits a, p, Sebald, 1978; Sebald et al., 1979; Kiehl and Hatefi, 1980), but y, o, OSCP, F , A6L, a (subunit 6), b, c, and d. The results the precise number remains to be determined. In intact mito- suggested that the two b subunits form the principal chondria, there is 1 mol ofiF /mol ofF (Hekman et al., 1991). 1 1 stem of the stalk to which OSCP, d, and F are bound By comparison, the Escherichia coli ATP synthase is composed independent of one another. Subunits b, OSCP, d, and F of only eight subunits, with the F -F stoichiometry a f3 y5e- cross-linked to a and/or p, but not to y or o. The COOH- 1 0 3 3 terminal half of A6L, which is extramembranous, cross- ab2c10_12 (Fillingame, 1990). Mitochondrial subunits a, /3, and linked to d but not to any other stalk or F subunit. No y have considerable sequence similarity to their E. coli coun- cross-links of subunits a and c with any stalk or F terparts. Mitochondrial OSCP and 5 are analogous to E. coli 5 subunits were detected. In F -deficient SMP, cross- and e, respectively, and, largely on the basis of hydropathy linked b + b and d + F dimers appeared, and the extent profiles, mitochondrial subunits a, b, and c are considered of cross-linking between b and OSCP diminished analogous to the E. coli a, b, and c, respectively (Fillingame, greatly. The addition of F to F cdeficient particles ap- 1990). peared to reverse these changes. Treatment of F -defi- Bovine and the rat F have been crystallized, and quaternary cient particles with trypsin rapidly hydrolyzed away structures, respectively, at 6.5 and 3.6 A resolution have been OSCP and F , fragmented b to membrane-bound 18-, 12-, reported (Abrahams et al., 1993; Bianchet et al., 1991). More and 8-9-kDa antigenic fragments, which cross-linked to recently, the structure of bovine F containing four molecules of d and/or with one another. Trypsin also removed the AppNP, and one molecule of ADP has been published, showing COOH-terminal part of A6L, but the remainder still at 2.8 A resolution the three-dimensional structures of the a cross-linked to subunit d. Models showing the near and f3 subunits plus about 50% of the y (Abrahams et al., 1994). neighbor relationships of the stalk subunits with one However, structural information regarding the remainder of another and with the a and p subunits at a level near the the ATP synthase complex is limited. Subunit cis considered to proximal end (bottom) of F and at the membrane- be shaped like a hairpin, with the hydrophobic arms forming matrix interface are presented. a-helices that traverse the membrane and the hydrophilic hair- pin bend protruding from the membrane on the F side. Recent two-dimensional NMR data for the isolated E. coli subunit c in The bovine heart mitochondrial ATP synthase complex iso- a chloroform-methanol-water solvent are in agreement with lated in our laboratory contains 13 well characterized subunits the hairpin model (Girvin and Fillingame, 1993, 1994). Small- angle neutron scattering studies of bovine OSCP (molecular *This work was supported by United States Public Health Service weight 20,967) have suggested an elongated shape of approxi- Grant DK08126. This is publication 8860-MEM from The Scripps Re- search Institute, La Jolla, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must The abbreviations used are: SMP, bovine heart submitochondrial therefore be hereby marked "advertisement" in accordance with 18 particles; AppNP, 5'-adenylyl imidodiphosphate; ASU particle, F -defi- U.S.C. Section 1734 solely to indicate this fact. cient SMP; EDC, 1-ethyl-3-(dimethylaminopropyl)carbodiimide; DCCD, * On leave from M. M. Shemyakin andY. A. Ovchinnikov Institute of N ,N' -dicyclohexylcarbodiimide; DST, disuccinimidyl tartarate; NHS, Bioorganic Chemistry, Moscow, Russian Federation. N-hydroxysulfosuccinimide; EGS, ethylene glycol bis(succinimidyl '11 To whom correspondence should be addressed: Division of Biochem- succinate); EEDQ, N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline; istry, Dept. of Molecular and Experimental Medicine, The Scripps Re- MBP, 4-(N-maleimido)benzophenone; PAGE, polyacrylamide gel elec- search Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. trophoresis; PMSF, phenylmethylsulfonyl fluoride; TPCK, N-tosyl-L- Tel.: 619-455-9100; Fax: 619-554-6838. phenylalanine chloromethyl ketone. This is an Open Access article under the CC BY license. 2054 ting. There was no evidence of additional cross-linking at the higher mately 90 X 30 X 30 A, with 43% a-helical structure as calcu- particle concentration, but at 0.1 mg of particle proteinlml there was a lated from circular dichroism measurements (Dupuis et al., higher yield of the cross-linked products, consistent with the less shield- 1983). Other than hydropathy profiles, information regarding ing effect of the particles against UV irradiation at the lower protein the structures of other ATP synthase subunits is not available. concentration. Because OSCP can be readily and reversibly removed from Trypsin Treatment of F -depleted SMP-F -depleted ASU particles 1 1 SMP together with F (Steinmeier and Wang, 1979; Matsuno- were suspended at 3-4 mg/ml in 20 mM Tris-HCl, 0.25 M sucrose, pH Yagi and Hatefi, 1984), it is considered to be a stalk component 7.5. TPCK-treated trypsin was added up to 0.8 mg/ml, and the mixture was incubated at 37 oc. At various times, aliquots were withdrawn, required for proper binding ofF to F . In addition, our previ- 1 0 proteolysis was arrested by the addition of 1 mM PMSF plus a 5-fold ous data, based on accessibility of ATP synthase subunits in excess of soybean trypsin inhibitor, and after 10 min on ice the soluble SMP and mitoplasts (mitochondria denuded of outer mem- and particulate fractions were separated by centrifugation. The super- brane) to proteases and subunit-specific antibodies, indicated natant proteins were precipitated by the addition of 10% trichloroacetic that OSCP, b, d, F , and the COOH-terminal half of A6L were acid and were subjected to SDS-PAGE and immunoblotting with spe- cific antibodies to F -stalk subunits. The particulate fraction was accessible from the matrix but not from the cytosolic side of the washed by suspending in buffer and recentrifuging, then a portion was inner membrane, whereas subunits a and c were not accessible suspended in triethanolamine/sucrose buffer plus 1 roM PMSF for cross- to the above reagents from either side of the mitochondrial linking with DST, and another portion in HEPES/sucrose buffer plus 1 inner membrane (Hekman et al., 1991). This paper presents mM PMSF for cross-linking with EDC + NHS. Conditions for cross- the results of cross-linking experiments involving the ATP linking and sample preparation for SDS-PAGE were the same as de- synthase subunits in SMP. The data are consistent with the scribed above. Protein concentrations were determined according to Lowry et al. ( 1951). results of our earlier topography studies described above and Electrophoresis--SDS-PAGE was performed according to Laemmli provide information regarding the near neighbor relationships (1970), routinely using a separating gel containing 15% acrylamide. For ofOSCP, b, d, F , and A6L, which appear to contribute to the optimal separation of high molecular weight cross-linked products, 10% formation of the stalk of the bovine ATP synthase complex. PAGE was employed. Antibody Production and Purification-The polyclonal antisera to EXPERIMENTAL PROCEDURES ATP synthase subunits a, b, d, F , and A6L used here were those Materials-Disuccinimidyl tartarate (DST), 1-ethyl-3-(3-dimethyl- described previously by Hekman et al. (1991). The OSCP antiserum aminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysulfosuccin- used was from Belogrudov et al. (1988). Antisera to the F subunits a, {3, y, and ll were obtained previously in this laboratory, using homoge- imide (NHS), ethylene glycol bis(succinimidyl succinate) (EGS), and neous preparations of each F subunit as antigen. Antipeptide anti- N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) were ob- bodies were raised in the rabbit against a synthetic peptide correspond- tained from Pierce; nitrocellulose membrane (0.2-J.Lm pore size) from ing to residues 1-10 of subunit c (DCCD-binding protein), using the Schleicher and Schnell; SDS, acrylamide, and prestained SDS-PAGE procedure described elsewhere (Hekman et al., 1991). standards (broad range) from Bio-Rad; TPCK-treated trypsin, soybean trypsin inhibitor, 4-(N-maleimido)benzophenone (MBP) and Tween 20 The IgG fractions used for immunoblotting were affinity purified from Sigma; goat horseradish-conjugated anti-rabbit lgG from Calbio- according to Olmsted ( 1981), using as a source of antigen homogeneous chem; and an Enhanced Chemiluminescence kit from Amersham. F - subunits or subunits transferred to nitrocellulose membranes after ATPase and F -depleted ASU particles, prepared as described previ- SDS-PAGE ofF or purified ATP synthase complex. The affinity-puri- fied antibodies were stored in small aliquots at -20 oc. ously (Matsuno-Yagi and Hatefi, 1984), were gifts of Dr. Akemi lmmunoblotting Conditions-Conditions for immunoblotting were Matsuno-Y agi. the same as before (Belogrudov and Hatefi, 1994), except that electro- Cross-linking Conditions-SMP were diluted and washed with buffer blotting was performed at 100 V for 1 h. Where necessary, nitrocellulose and then adjusted to a protein concentration of 1.0 mg/ml in a medium sheets containing the same cross-linked samples were cut into separate containing 50 mM triethanolamine and 0.25 M sucrose, pH 8.0, and incubated with 1.0 mM DST for 30 min at 20 oc. The reaction was strips for blotting with different antibodies. The immunoblots were terminated by the addition of ammonium acetate to 50 mM. Mter a developed using the Amersham Enhanced Chemiluminescence kit ac- 10-min incubation an equal volume of Laemmli SDS-PAGE sample cording to the manufacturer's instructions. buffer (Laemmli, 1970) containing 6 M urea was added. In other exper- RESULTS iments, 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 Cross-linking of SMP with DST or EDC-Even though our reactions were allowed to proceed for 30 min at 20 oc and were then preparations of the ATP synthase complex display the same quenched by the addition ofTris-HCl, pH 6.8, to 100 mM. After a 10-min activities and inhibitor-response properties as the ATP syn- incubation, samples were treated with SDS-PAGE sample buffer as thase complex in SMP, we chose to carry out our studies of the above. F -depleted ASU particles were cross-linked at 0.5-1.0 mg/ml in near neighbor relationships of the ATP synthase subunits pri- the same manner as SMP. When F was used for cross-linking, an marily on well coupled SMP lest the purification process, which aliquot of the ammonium sulfate suspension of F was desalted on a involves the use detergents, might have introduced some struc- PD-10 column (Pharmacia Biotech Inc.) and adjusted to 0.2 mg/ml in the triethanolamine/sucrose buffer described above, and ATP was tural changes in the isolated enzyme. For example, the highly added to 1 mM. The preparation was then treated with 1 mM DST for 30 purified ATP synthase complex has a low phospholipid content min at 20 oc before the addition of 50 mM ammonium acetate to termi- and an absolute requirement for added phospholipids to dis- nate the cross-linking. Treatment of cross-linked F for SDS-PAGE was play ATPase and ATP- P; exchange activities (Galante et al., the same as described above. 1979). In some cases, however, we used F -depleted ASU par- For cross-linkings involving sulfhydryl groups, the photoactivable 1 ticles to expose to cross-linking stalk subunits that F might heterobifunctional MBP was used. MBP dissolved in dimethylform- amide was added at 1 mM to SMP at I mg/ml in 20 roM HEPES, 0.25 M mask. Among the cross-linkers employed were DST and EGS, sucrose, pH 7.2, and the mixture incubated for 25 min at 20 oc in dim which react with amino groups separated, respectively, by 6.4 light. Unreacted MBP was either removed by centrifugation or first and 16.1 A, and EEDQ and EDC, which activate carboxyl quenched by the addition of 10 mM dithiothreitol and then removed by groups for interaction with a nucleophile (e.g. amino group) to centrifugation. Mter resuspension in HEPES/sucrose buffer, the parti- result in zero length cross-linking. EDC was used in the pres- cles were irradiated with an 8-watt longwave UV lamp at a distance of ence of NHS, which is thought to react with the unstable 5 em for 10 min on ice, with occasional mixing. The particles were then prepared for gel electrophoresis by the addition of an equal volume of 0-acylisourea adduct of EDC to produce the more stable reac- SDS-PAGE buffer as indicated above. For control, particles were pre- tive NHS ester and improve the yield of the EDC-mediated incubated with 5 mM N-ethylmaleimide for 20 min before the addition cross-linked product (Staros et al., 1986). In all cases, cross- of MBP and UV irradiation. Under these conditions, no cross-linking linking conditions were optimized with respect to the concen- was observed. Furthermore, SMP or ASU particles were treated with trations of the cross-linkers and SMP, the duration of cross- 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 immunoblot- linking, and the temperature of the reaction mixture. The ATP Synthase Complex 2055 cross-linked products were identified after SDS-gel electro- anti-lgG phoresis of the samples and electrotransfer to nitrocellulose b OSCP d F AGL sheets by immunoblotting to affinity-purified polyclonal anti- bodies raised to each purified ATP synthase subunit, a , {3, y, S, OSCP, F , a , b, c, and d . The anti-A6L antibodies used were antipeptide antibodies raised to synthetic peptides correspond- ing to residue s 30-43 and 54-66 of this subunit. Attempts to raise antibodies or antipeptide antibodies to the E subunit of bovine F I 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 ·- - -d+AGL three other polypeptides, designated e , f, and g, in their prep- arations of ATP synthase complex, which appear to have no 3 2 ATP- P; exchange activity and only partially oligomycin-sen- sitive 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 -Fs to have complicated the interpretation of the cross-linking data -AGL shown below . 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 8 anti-lgG immunoblots of a 15% SDS gel, which favors transfer to nitro- a b OSCP d cellulose molecular mass species < 80 kDa , and Fig. lB shows immunoblots of a 10% SDS gel , which contains cross-linked products of higher molecular mass . The top of each lane states <X+~\ the antibody with which that nitrocellulose strip was blotted . In these and subsequent figures , the fastest moving (lowest) a+b\ ~+b- protein band is the uncross-linked subunit, except that in Fig. <X+Fsf 1B all of the uncross-linked F and parts of uncross-linked OSCP and d ran out of the gel. It might also be noted that there b+d- are no immunoreactive protein bands below uncross-linked b, OSCP , and d in Fig. 1A and below a and {3 in Fig. lB . 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. In Fig. lA , the cross-linked products of b + OSCP, b + F , FIG. 1. Cross-linking of SMP with DST. SMP at 1 mg/ml were and d + A6L are marked . Also marked are two species of b + incubated with 1 mM DST for 30 min at 20 °C. After termination of t he . h b d h reaction , samples were subjected to a 15% (panel A ) and a 10% (panel B ) d h , w IC are etter separate in Fig. lB. T ese could be the SDS PAGE d te" t t '" d t ·t 11 1 . . - , an pro ms were e 1 ec ro rans.erre o m roce u ose res~lt of two . t~pes_ of cross~lmkiD:g between ~ and the same .ffi!'.vM!4\.\\f,li-.$~y\t.r+)l'e.l~\~ . .f*\N&i,!>'.i,>;W-i.Mw.V:..:~.-,r.fk~,lW,l,r,!>,Q mol:ecut€illl''b 6t4=1ltffetenfc'.1;5'pes9:lf'£rossqirr~tffitWeen, "f1:· · . pro"dm:ts . .wete~lllotwdc'Wltn tfie·a1finit}'¥purrfierrO..antr110'iites":tnait\ateal~o~~-==== and the two molecules of b . The unlabeled top bands in Fig. 1A the top of each lane . The immunoreactive bands were visualized with are cross-linked products of each subunit with a/{3 which are the Enhanced Chem1lummescence detectiOn system. For deta il s, se e . . . ' "Expenmental Procedures ." also clearer m Fig. lB . These species appear to be a+ {3, {3 + b, {3 + OSCP, a+ OSCP, a + b, a + d, and a+ F . The unmarked cross-linked products involving the stalk subunits and the cross-linked bands seen in the upper parts of the lanes in Fig. small subunits ofF I· For example, one would expect the cross- lB are presumably cross-linked trimers and more complex linked products of they subunit ofFI with b, OSCP, and d to products. Essentially a similar cross-linking pattern was ob- produce a protein band immunoreactive to anti-b, anti-OSCP , tained when SMP were treated with EDC in the presence of or an ti-d in the Mr region of 50-60 kDa. However, as is clear in NHS . The product of d + A6L was particularly favored under Fig. lB, this region of these lanes is devoid of any such protein the latter conditions, and the major cross-linked products be- band. To investigate this issue further, SMP and purified F - tween the stalk and F I subunits were a + b, a + OSCP, a + F , ATPase were separately treated with DST and subjected to and {3 + d (data not shown) . Although not shown in Fig. 1, SMP SDS-gel electrophoresis and immunoblotting with affinity-pu- cross-linked with either DST or EDC + NHS and immunoblot- rified antibodies to the individual F I subunits a, {3, y, and S. ted to anti-a or anti-c antibodies showed no cross-linked prod- The results are shown in Fig. 2. The paired lanes compare the ucts involving these subunits. cross-linked products of the F I subunits as marked in the figure A close scrutiny of Fig. 1, A and B, suggests the absence of in SMP and isolated FI-ATPase. In the fir st and third lanes from the left , the bands not seen in the second and fourth lanes are the cross-linked products, respectively, of a and {3 with the Coupling factor B (Sanadi , 1982) with M, of 11-12 X 10 (You and Hatefi, 1976), which is present in SMP and restores ATP- P; exchange stalk subunits (see Fig. lB). Other bands common in the first and ATP-driven transhydrogenation and reverse electron transfer to four lanes are the cross-linked products of a + {3 and a + {3 plus ammonia-EDTA treated SMP (Stiggall et al. , 1979; Sanadi , 1982 ), has one or another small subunit ofF I· The important message of been considered to be a component of the bovine ATP synthase (Sanadi , Fig. 2 is contained, however, in the last four lanes . It is seen 1982). However, we and Walker's laboratory (Walker et al., 1991) have not detected factor B in our respective ATP synthase preparations. that in SMP or isolated F I the cross-linked products of y and S 2056 ATP Synthase Complex anti-a anti-~ anti-y ant Hi anti-b anti-OSCP anti-F SMP F SMP F SMP F SMP F 1 1 2 3 4 5 6 7 8 9 1 1 1 208- --! ... -115 115- 79-- -79 49- -49 34- b+OSCP- 28- b+F - -34 20- -28 b- oscp- -20 7- FIG. 2. Comparison of the cross-linking patterns ofF subunits in purified F and SMP treated with DST. F (0.2 mg/ml) and SMP (1 mg/rnl) were cross-linked with DST as in Fig. 1. Samples of cross- linked SMP and F were subjected to 15% SDS-PAGE in parallel, and -7 proteins were electrotransferred to nitrocellulose membrane and blot- ted 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 FIG. 3. Cross-linking of ATP synthase subunits in SMP with the figure . the heterobifunctional photoactivable sulfhydryl reagent, MBP. SMP at 1 mg/ml were incubated with 1 rnM MBP for 25 min at 20 •c. Particles were sedimented by centrifugation, resuspended in buffered are essentially the same in each case, even though the yields of sucrose solution, and UV irradiated for 10 min or kept on ice in the the cross-linked products of y appear to be less in SMP (per- dark. Samples were subjected to 15% SDS-PAGE, and proteins were haps due to shielding of y) than in isolated F . These results electrotransferred to nitrocellulose and probed with the affinity -puri- suggest, therefore, that in SMP y and a do not cross-link to any fied antibodies indicated on the top of the lanes. Immunore active bands were visualized as in Fig. 1. Lanes 1, 4, and 7, sample of SMP UV stalk subunit in the presence ofDST as the cross-linking agent. irradiated for 10 min; lanes 2, 5, and 8, MBP-treated, nonirradi ated Similar results were obtained when EDC + NHS were u sed. SMP; lanes 3, 6, and 9 , MBP-treated and UV-irradiated SMP. The However, since y and a produced a number of cross-linked positions of prestained mol ecular mass markers are shown in kDa on products common to SMP and isolated F , it is clear that they 1 the right of the figure. are accessible to cross-linkers in SMP. The question, therefore, is how y and a are located in SMP in relation to b, OSCP, d, F , an d OSCP involves this same Cys of b. In isolated OSCP, and A6L. Cysn , which is near the center of the chain, is modifiable by Cross-linking of SMP with MBP-MBP is a photoactivable substitu ted maleimides (Dupuis et al., 1985 ). However, sulfhydryl reagent that can alkylate a thiol via its maleimido whether this cysteine is accessible for modification in SMP is moiety. Then , up on irradiation (at about 350 nm) the aromatic not known. ketone moiety goes into a diradicaloid triplet state, resulting in It was of interest to see wheth er the removal ofF from SMP an electrophilic electron-deficient oxygen that is highly reac- might alter t h e cross-linking of stalk subunits. F -depleted tive, especially toward the C-H bonds of protein backbones ASU particles were prepared essentially according to Racker (Dorman and Prestwich, 1994). The result is the formation of a an d Horstman (1967 ). These preparations are depleted with C-C bond between the ketone carbon and the protein and respect to F and suffer partial loss of OSCP. The addition ofF 1 1 cross-linking at a distance in the case of MBP of about 10 A. + OSCP reconstitutes particles with high ATP synthase activ- Among the ATP synthase subunits under consideration here, ity (Matsuno-Yagi an d Hatefi, 1984). The remainder of OSCP those that contain cysteine residues are the a, y, and E subunits can be removed by further alkaline extraction of ASU particles, ofF plus b, OSCP, d, and c, of which a has 2 cysteine residues/ but this was not done here because ou r aim was to investigate mol and the others 1 cysteine residue each . The experiments the effect ofF removal. When treated with MBP, ASU parti- with MBP were carried out by incubating SMP ( - 1 mg/ml) cles produced two major cross-linked products among the cys- with 1 mM MBP for 25 min. Unreacted MBP was removed by teine-containing subunits of the F -depleted ATP synthase. centrifugation. SMP were resuspended in buffered sucrose so- One was b + F as in Fig. 3, the other was a band at about 44 lution, subjected to UV irradiation as described under "Exper- kDa, wh ose mobility and singular reactivity with only anti-b imental Procedures ," and analyzed by SDS-PAGE and immu- lgG suggested that it is a cross-linked b + b dimer (Fig. 4, lane noblotting with antibodies against 6 of the 7 cysteine- 2 ). There were a lso detectable amounts of b + OSCP (Fig. 4, containing subunits mentioned. As seen in Fig. 3, only two lane 6). The addition to ASU particles ofF (2 and 6 mol/mol F , 1 0 cross-linked products were detected, one between b and OSCP respectively, in the thi rd and fourth lanes of each set in Fig. 4) at about 42 kDa , and another between b and F at about 34 increased the yield of b + OSCP dimer and diminished the kDa. The formation of these produ cts required both the MBP yield of the putative b + b dimer. There are two possible pretreatment of SMP and the subsequent UV irradiation. explanations for t h ese results: (i) removal ofF caused a partial When the SMP were first treate d with 5 mM N-ethylmaleimide separation of b and OSCP so that b-MBP was too distant from before the addition ofMBP and UV irradiation, the cross-linked OSCP to cross-link upon photoactivation or (ii ) removal ofF products shown in Fig. 3 were not forme d (data not sh own) . As resulted in masking of the thiol group of OSCP, making it mentioned above, subunit b contains a single cysteine residue unable to form OSCP-MBP to cross-link with b upon photoir- at position 197, which is close to its COOH terminus, and F radiation. We prefer the first possibility because structural has no cysteine residues . Therefore, the MBP-mediated cross- perturbation of the stalk may also bring the two MBP-modified linking between F and b indicates that the COOH terminus of b subunits closer to one another for cross-linking after photo- b is located near F . It is also possible that cross-linking of b activation of their benzophenones. As seen in Fig. 4, the cys- 6 ATP Synthase Complex 2057 anti-b an ti-d 1991 ), s ubunits a and c were unaffected by trypsin and did not 2 3 4 9 10 11 12 produce a ny cross-linked products when the trypsin-treated -11 5 ASU particles were subjected to cross- linking with DST or EDC -79 + NHS (data not shown) . The COOH-terminal end of A6L was remov ed by tryp sin (Hekman et al. , 1991 ), but the remainder of b+b?\ -4 9 b+OSCP- t h e molecule, including the antigenic re sidues 30-43, survived , -- and this truncated A6L cross-linked to subunit d (see Fig. 5A, b+F6- -- -34 --- b"\. ____ _ lane 9, first arrowhead from bottom ) when the trypsin -treated -28 _ _. _____ _ ASU particles were treated with DST . OSCP - The effect of trypsin (1:25 weight ratio of trypsin to particl e df -20 protein ) on subunits b and d agreed with the results of others (Collinson et al., 1994a; Walker and Collin son , 1994 ), which 166 167 120 121 ha wed that b was hydrolyzed at Arg -Gly , Lys -Arg , 121 122 4 5 and Arg -His , and d at Lys -Leu (see Fig. 5A , lanes 2-4 -7 and 8-10). Hydropathy analysis of sub unit b shows two hydro- phobic clusters of about 20 residues each near the NH termi- FIG. 4. Cross -linking ofF ,-deficient ASU particles with MBP in nus followed by a 130-residue-long hydrophilic region to the the absence and presence of added F • ASU particles (0.5-1.0 1 COOH-termina l end. This suggests that the molecule is an - mg/ml ) were incubated with 1 mM MBP for 25 min at 20 °C , and excess chored to the membrane via its two hydrophobic clusters and reage nt was r emove d by centrifugation. MBP-modified pa rticl es were that its short h ydroph ilic NH terminus - 30 residues ) and its incubate d without a dd ed F, (la nes 1, 2 , 5, 6, 9, and 10 ) or with 325 J.Lg 130-residue-long hydrophilic tail are extramembranous on the ofF ,lm g of particles (lane 3, 7, 11) or 1.0 mg ofF ,fm g of part icles (lanes 4 , 8, a nd 12 ), each for 30 min at 37 °C. Particles were r e isolated by F side . According to this picture , the tryptic cleavages at centrifugation, washed, res uspended in 20 mM Tris-HCI, pH 7.5, con- 166 167 120 121 121 122 Arg -Gly and at Lys -Arg (o r Arg -His ) would taining 0.25 M sucrose a nd UV irradi ated for 10 min on ice. Sample produce two membrane-bound fragments , respectively , about were s ubj ected to 15% SDS-PAGE , and prote in s were e lectrotran ferred 18-19 and 12-14 kDa. These b fragments , which still reacted to nitrocellulose and blotted with the affinity-purified a ntibodi es indi- cated on t h e top of the lanes. Immunoreactive band s were vis ualiz e d as with our polyclonal antibody preparation, are seen in lanes 2-4 in Fig. 1. Lanes 1, 5, a nd 9, MBP-treated, nonirra diated ASU particles; of Fig. 5A . They cross-linked in the presence of DST or EDC + other lanes, MBP-treated ASU particles UV irrad iated for 10 min on ice . NHS with subunit d to produce the two bands marked with The positions of prestained molecular m ass marke1·s are shown in kD a arrowheads in lanes 3, 4, 9, and 10 of Fig. 5A . It may a lso be on the right of the figur e . noted that the yield of the slower moving cro ss -linked product was greater when DST was the cross-linking reagent, and the teine-containing subunit d in ASU particles (or in SMP) did not yie ld of the fa ster moving cross-linked product was greater form any MBP-mediated cross-linked products. As was shown when EDC + NHS were u sed for cross-linking. Other cross- previously, subuni t din SMP and ASU particles is resistant to linked products recognized and marked by arrowheads in Fig. trypsinolysis . It is possible, therefore, that much of subunit d, 5A are the b + b dimer in lane 6 and d + F in lane 11. We including its cysteine residue, is shielded by oth er ATP syn- suspect that the band marked d + A6L, d + xis composed of thase subun it s in SMP and ASU particles. more t h an one cross-linked product of subuni t d. However, Cross-linking ofF rdepleted Particles Treated with Tryp sin- additiona l informatio n is not available at this time. These experiments were carried out with ASU particles t h at The data in Fig. 5B are from an a liquot of ASU particles that had been extracted twice with urea to divest them ofF . They was treated with the higher concentration of trypsin (1: 5 were th e n treated with trypsin under relatively mi ld (1 :25 we ight ratio of trypsin to particle protein ). It is seen in lanes 1 we igh t ratio of trypsin to particle protein for 1 h at 37 °C) or and 2 that subunit b was further degraded to produce an severe ( 1:5 weight ratio of trypsin to particle protein for 1 h at 8-9-kDa antigenic fragment , which was still membrane-bound . 37 °C) cond itions . Proteolys is was arrested as described under Considering that there are about 70 amino acids from the NH "Experimental Procedures, " and the particles were separated terminus to the end of the second hydrophobic cluster of sub- from the soluble protein fragments by centrifugation and wash- unit b, t h e tryptic cleavage site to produce an 8-9-kDa mem- ing. Aliquots of the particles were then subjected to cross- brane-bound fragment must be very close to wh ere the COOH - linking with DST and EDC + NHS, electrophoresed, and im- termina l hydrophilic tail of b exits the membrane . If we assume munoblotted with antibodies to subunits b, d , OSCP , a nd F . that the hydrophobic clusters of b are nonantigenic, then it is Before trypsin treatment cross-linking of the F -depleted par- lik ely that our anti -b IgG recognizes the 30-residue-long NH t icles produced a b + OSCP dimer in a fairl y good yield when terminus of subunit b, which is probably extramembranous . As the cross- linker was DST and in very poor yie ld when it was seen in lane 3 of Fig. 5B, this 8-9-kDa fragment disappeared in EDC + NHS . This agrees with our conclusions regarding cross- the presence of EDC + NHS and produced the cross- linked linking of ASU particles with MBP and suggests that removal products marked with a rro wheads. The uppermost band ofF resu lts in separation of OSCP from b to the extent that marked with an arrowhead a lso appeared to form in lower yield DST (molecular length , 6.4 A.) can still bridge the distance when DST was the cross-linking reagent (Fig. 5B, lane 2). The between t hem, but EDC + NHS (ze ro le ngth cross -linking) proteolysis pattern of bovine b with trypsin as shown in Fig. 5 cannot . By contrast, cross-linking of the F -depleted particles (see also Collinson et al., 1994a; Walker and Collinson, 1994 ) is with either DST or EDC produced a b + F dimer in good yie ld simil ar to that of E . coli subuni t b (Steffens et al. , 1987 ), which (Fig. 5A, lanes 5 and 6, second h eavy band from bottom ), just as is a further in dication of the ana logy between them. it was shown in Fig. 1 for SMP. After treatment of the particles DISCUSSION with the lower concentration of trypsin, OSCP and F were completely degraded in agreement with previous results (Joshi Considerable information is now available regarding the and Burrows, 1990; Hekman et al., 1991 ), and no cross-linked mechanisms of ATP hydrolysis (Al-Shawi et al., 1990; Penefs ky products containing antigenic fragments of these subunits and Cross , 1991; Boyer , 1993) an d synthesis (Matsuno-Yagi were detected by immunoblotting. Also, in agreement with and Hatefi , 1990; Hatefi , 1993) at the level of the catalytic previous re sults (Joshi and Burrows, 1990; Hekman et al., sector, F , of the ATP syn thase complex. Also, the crystal 1 ATP S y nthase Co mplex 205 8 A 8 anti-b anti-b ant i-d ,-----------, DST DST + - + - - - + - + - - + - EDC EDC - + - + - + - + + tryps i n - + ++ ---+++ - trypsin + + + 1 2 3 4 5 6 7 8 9 1011 12 1 2 3 115 - 1 15 - 79 - 79 - 49 - 49 - 34 - ... - .;.. - b + F 34 - 28 - . - .. _ _ f d+A6L 28 - ... = 20 - ---- - l_d+X b (18)__ __ .. _ .. b( 12) __ __ _ b(18)~ ------- --- b(8)7"- b(12)-- 7 - FI G. 5 . Cross-linking of ATP synth as e subunits in F ,-defici e nt ASU parti c les trea t e d with tryps in. ASU pa r ticl es (3 .5 mg/m !) we re in cu bate d wit h t r ypsin (25: 1 r atio by we igh t, panel A , or 5: 1 ratio by weigh t, panel 8 ) fo r 1 h a t 37 •c. Proteo lys is wa s a JTeste d by th e a dd itio n of excess s oy bea n t ryps in inhibi to r plu s 1 mM PMSF , a nd part icles we re reis olated by ce nt ri fugation , was hed , a nd cross-linked wit h 1 m ~·I DST or 5 m M E DC in th e prese nce of 5 m M N H S. Sam pl es we r e s ubj ected to 15% SDS- PAGE , a nd prote in s were electrotr ansfe r red to n itroc e ll u lose memb r a ne a nd blotted wit h t he a ffini ty-p urifi ed a n t ibo die s i ndi cate d on top of t he lan es . l m mu nob lots we re deve loped as describ ed in Fig. 1. Th e tab les of plus a nd minus signs on top of t he immun oblots s how t h e t reatm en t t he ASU pa r t icl e of ea ch lane ha d rece ive d . Arro w heads a re desc r ibe d u n de r "Res ul t s ." Th e des ignation s b(JB), b(12), an d b(8) a r e for m embr ane -bo und try ptic fr agme nt s of s ubu ni t b wit h a ppr ox imate M ,. va lu e of 18, 12 , a nd 8, res pect ive ly. Th e pos itio ns of prestai ned molecul a r mass ma rk e•·s a re s hown in kDa on th e left of panels A a nd B . t h a t OSCP binds to bovi n e F (Hu n da l et al., 1983 ; Dupui s et struct ure of a form of bovi n e h eart F co ntain ing four m olecu les 1 1 of AppNP a nd one mol ecu le of ADP has bee n solve d rece n t ly at al., 1985 ) a nd t hat t h e F -0SC P co mpl ex ca n bind s u buni ts b , 2.8 A. r esoluti on (Abra h a m s et al., 1994 ). In a ddi tio n , it is F , a nd d (Wa lk er an d Collin on , 1994 ). E vid en ce t h at F can kn ow n th a t tran s membra n e prot on s pass only throu gh t h e be r eve r sibl y extr acte d fro m SMP is a lso ava ila ble (Fesse nd en- m embr a n e sector , F , of th e ATP synthase compl ex, a nd e n er gy Ra den , 1972 ). Th ese data a r e con siste n t wit h our ea r lie r stud- communication betwee n F a nd F t ak es pl ace via coup led ies sh owin g t hat b, OSC P , d , an d F a r e accessible t o subun it- 0 1 6 conform ation ch a nges of subunits (for review, see Hate fi , 1993). spec ifi c a ntib odi es in we ll co upl ed SMP , but n ot in mitopl asts, At th e level of F , protein-bound ATP is sy n t h esi ze d fro m a nd t hat in SMP s u buni ts b, OSCP , F , a nd t h e COOH- termi- 1 6 n a l a n t ige ni c pa r t of A6 L co u ld be degrad ed by t ry ps in protein-bound ADP a nd P i with out en er gy u t ili zation , but th e binding of P i a nd ADP t o F a nd th e r elease of ATP fr om F a r e (H ekm a n et al. , 199 1). Th ese studi es a lso indi cate d cert a in 1 1 th e en er gy-requirin g proces ses, which a r e a pp a r ently accom- su bun it access ib ili ty di ffe r en ces betwee n SMP a nd th e purifi ed pli sh ed by a ppropr ia t e conform a tion cha nges of t h e {3 subuni ts . ATP sy n t hase co mpl ex, which decided our choi ce of m ateria l for Th e crystal structure of bovine F (Abra h a ms et al., 1994 ) a nd dete rm inin g t h e stoichi ometry of subunits in F a nd t h e stalk th e cryoe lectron microscop y studies of Ca pa ldi a nd co-workers (H e km a n et a l. , 199 1) as we ll as for ou r studies r eported h er e Wilkin s a nd Ca pa ldi , 1994, a nd re fere nces th er ein ) on t h e E. on t h e nea r n eighb or r ela ti onship s a mon g th e stalk subuni ts co li F s uggest th at s ubuni ts y a nd E are invol ve d in co n veyin g a nd betwee n t h e latter a nd t h e s ubuni ts ofF a nd F . 1 1 0 T a bl e I summ arizes t h e res u lt s obta ined in SMP a nd F - confo r ma ti on ch a nges to a nd from F . However, in bo t h t h e bo vin e a nd E. co li ATP synthases th er e is a 45 -A.-l on g sta lk de pl eted SMP wit h t hree cross -linkin g r eage nts (D ST , MBP , bet wee n F a nd F (Soper et a.l. , 1979 ; Ca pa ldi et al., 1992; see E DC ) of differ en t ch emi cal reactivit ies a nd differ e nt effective a ls o Dub a chev et al. , 1993 ), wh ose stru cture a nd ro le in en er gy distances for cross- linkin g. Co nside rin g fir st t h e n ea r n eighbor communica tion betwee n F a nd F a r e not known . If one as - re lations hip s of t h e s t a lk subunits, it is clea r t h at OSC P cr oss- s umes th a t the ATP sy n t h ase subuni ts th a t a r e not compo- linked only to b, a nd A6L only to d. Th e NH te rmi n us of A6L n e nts of F but are en t irely or pa rtl y extra membra nou s co ntains a sh or t hy dro ph obi c clu ste r ( - 20 residues ), whi ch (H e km a n et al., 1991 ) contribute to t h e co mpo sition of th e s t a lk , a ppea r s to a nchor A6 L to t h e membr a ne . Th e rem a inder of t h e t h en s ubuni ts b, OSCP , d , F , a nd A6L would qu a li fy as ca n- molecu le is hydroph ilic a nd a ppa r en t ly extr a membrano u s. did ates . As possi bl e compone nts of t h e stalk , t hese subuni ts Try psin cleaves off t h e CO OH-termin a l a nti ge ni c part of A6L wo u ld be directly or indirectl y in vo lve d in conform a ti on a l en - (H ekm an et al. , 199 1), but th e r em a inder of t h e molec ul e is still er gy t r a ns fer betwee n F a nd F . Therefor e, information re - close en ough to sub u ni t d to for m a cross -linked produ ct in t h e 0 1 garding t h eir r ela ti ve positi ons in t h e stalk a nd t h eir s pa ti a l prese n ce of DST (Fi g. 5, lan e 9, bottom band ma rk ed wit h a n inter a ct ion with one a n oth er as we ll as wit h F a nd F subuni ts a rrow h ead ). Thi s also suggests t h at su bunit d is prese nt nea r 0 1 wou ld be cru cia l to inves ti gatin g t h e m ech a ni s m of en er gy t h e membra n e. Indee d , t h e NH te rm inu s of d contains a sh ort tra ns fer within th e ATP sy nthase co mpl ex. h ydroph obic clu ste r of a mino acids , whi ch may be in te r ca late d As mention ed ea rli e r , OSCP is eas il y rem ova bl e fro m SMP in to t h e me mbra n e . (Senior , 1979 ) or prepa r a tion s of th e ATP sy n t h ase comp lex As see n in Ta bl e I , a ll of t h e s t a lk subuni ts , exce pt A6L, (Ga la nte et al., 198 1). Remo va l of OSCP a lso r es ul ts in th e cross-link to b in SMP . As men t ion ed a bo ve , t h e hydro pa thy r emova l ofF , but bo t h ca n be a dd ed back t o F to r econ sti t u te a n a lys is of b sh ows tw o hydroph obi c clusters of - 20 r esidu es 1 0 oli gomycin-se nsit ive ATPase activity (Ga la n te et al. , 198 1) or t o eac h located - 30 resi du es fro m t h e NH te rminus . Th ere for e, de pl et ed SMP t o r eco nstit ute oxid a tive phosphorylation a t hi gh on e mi gh t ex pect t h at t h e two hydroph obi c clu sters in te r calate r a t es (Ma t suno-Yagi an d H a t e fi , 1984 ). In a ddi ti on , it is kn own in to t h e membrane, with t h e sh ort NH -ter min a l segm ent a nd 2 2059 TABLE I Cross-linked products of subunits b, OSCP, d, F & and A6L formed in A SMP and F -depleted SMP treated with DST, EDC, or MBP F ,-depleted SMP SMP DST EDC MBP DST EDC MBP b-ba b-ba b-ba b-db b-db b-db b-OSCP b-OSCP b-OSCP b-OSCP b-OSCP b-F b-F b-F b-F b-F b-F 6 6 6 6 6 6 d-A6L d-A6L d-A6L d-A6L b-a/{3 b-a/{3 OSCP-a/{3 OSCP-a/{3 d-a/{3 d-a/{3 Fs-a/f3 F s·a/{3 --;;-Based on Mr and immunoreactivity with only anti-b IgG. b Two species. the long ( ~ 130 residues) COOH-terminal portion beyond the second hydrophobic cluster being extramembranous on the F side (Walker and Collinson, 1994). Furthermore, our study of the stoichiometry ofF 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 -depleted SMP, treated with either DST or EDC, a band that reacted only FIG. 6. Schematic representation of the near neighbor rela- with anti-b IgG and exhibited on SDS gels a mobility consistent tionships of subunits b, OSCP, d, F , and A6L in bovine submi- with that of a cross-linked b + b dimer. tochondrial particles. Cross-linked <timers of subunits were identi- In addition to cross-linking of b to OSCP and F effected by 6 fied where circles representing the subunits overlap. Because DST or EDC, b + OSCP and b + F dimers were also produced knowledge of the structures of these subunits is limited, the diameters of the circles were deliberately made the same to avoid any inference when the cross-linking reagent was the heterobifunctional pho- regarding the relative sizes and shapes of these subunits. A, en~-on toactivable sulfhydryl reagent, MBP (Fig. 3). Because F is view of a cut parallel to the membrane through the bottom (proximal devoid of cysteine residues, the cross-link between b and F end) ofF,. B, end-on view of a cut parallel to the membrane at the must involve Cys of b near its COOH terminus. The same membrane-matrix interface. might be true in the case of the MBP-induced cross-link be- tween band OSCP, or the cysteine in this case might be Cys the proximal end (bottom) of F (A) and at the membrane- of OSCP. However, the fact that the b-MBP adduct did not matrix interface (B). In A, subunits b, OSCP, d, and F are all cross-link upon photoactivation to a, {3, y, or 6 may mean that present because they all cross-link to a and/or {3 (the parallel- the COOH-terminal portion of b, which contains Cys , is ogram labeled a/{3). They also cross-link to one another wher- folded back toward F and away from F • Subunit b of E. coli 0 1 ever the circles representing each subunit overlap. In B (the ATP synthase is also thought to have a folded conformation bottom of the stalk), A6L protrudes from the membrane near d, (Fillingame, 1990; Penefsky and Cross, 1991). It might be and the two b subunits enter the membrane. However, whether added parenthetically that treatment of SMP or ATP synthase OSCP and F extend the length of the stalk from F to F to 1 0 preparations with various mono- and dithiol modifiers results impinge upon the membrane is unclear; therefore, they have in uncoupling (Yagi and Hatefi, 1984), and the studies of Papa not been included. In F -depleted ASU particles, this organi- and co-workers (Zanotti et al., 1988) implicate Cys of subunit zation is somewhat perturbed, as if the removal ofF results in b in this type of uncoupling. Although not shown in Fig. 3, we the inward collapse of the b subunits toward each other, also checked by immunoblotting for MBP-induced cross-links of a, bringing F close to d. As a consequence, in ASU particles, but -y, d, and c, all of which contain cysteine, but none was found. not in SMP, b + b and d + F cross-linked dimers can form (the The cysteine of subunit c is within the membrane and may not latter is marked by an arrowhead in Fig. 5A, lane 11). When react with MBP (although it appears to react withp-chloromer- MBP was used for cross-linking of ASU particles, b + F was curibenzoate).3 Apparently, the cysteines of a, -y, and dare also formed as in MBP-treated SMP, but the yield of b + OSCP unreactive with or inaccessible to MBP. dimer was greatly diminished (Fig. 4, lane 6). Instead, there As seen in Table I, b, OSCP, d, and F , but not A6L, form was the appearance of a band qualifying as a b + b dimer, cross-linked products in SMP with a and/or {3. Furthermore, in which was not seen in MBP-treated SMP. Interestingly, the SMP, OSCP, d, and F can each cross-link to b but not to one addition of F to ASU particles prior to MBP treatment re- another. Therefore, it seems reasonable to assume that the two stored the formation of the b + OSCP product and diminished molecules of subunit b, which extend from the membrane to F , the yield of the b + b dimer (Fig. 5, lanes 3, 4, 7, and 8). These form the principal stem ofthe stalk to which OSCP, d, and F results suggest that (i) F removal diminishes the interaction of bind independently of one another. These considerations are b and OSCP, which is consistent with the fact that extraction of summarized in Fig. 6, which shows the near neighbor relation- F also results in labilization of OSCP, and (ii) the readdition of ships of the stalk subunits in cuts parallel to the membrane at F to ASU particles reestablishes the close association of b and OSCP, which is also consistent with the fact that function can A. Matsuno-Yagi andY. Hatefi, unpublished results. be restored to ASU particles by the addition of F and OSCP 1 2060 Belogrudov, G.!., Ilyina, E. F., Grinkevich, V. A., and Modyanov, N. N. (1988)Biol. (Matsuno-Yagi and Hatefi, 1984). Recent data of Abrahams et Membr. (U.S. S. R) 5, 677-687 al. (1994) on the crystal structure of bovine F indicate that 'Y Bianchet, M., Ysern, X., Hullihen, J., Pedersen, P. L., and Amzel, L. M. (1991) protrudes 30 A from the core ofF , which the authors believe J. Bioi. Chern. 266, 21197-21201 Boyer, P. D. (1993) Biochim. Biophys. Acta 1140, 215-250 forms part of the stalk. Fig. 6A shows a possible location for the Capaldi, R. A., Aggeler, R., Gogo!, E. P., and Wilkens, S. (1992) J. Bioenerg. tail of 'Y surrounded by b, d, and OSCP. This possible location Biomembr. 24, 435-439 Collinson, I. R., Fearnley, I. M., Skehel, J. M., Runswick, M. J., and Walker, J. E. inside the stalk is consistent with our inability to find cross- (1994a) Biochem. J. (Lond.) 330, 639-645 linked products of 'Y with any of the stalk subunits. It also Collinson, I. R., Runswick, M. J., Buchanan, S. K., Fearnley, I. M., Skehel, J. M., agrees with the presumed inward collapse of the stalk to allow van Raaij, M. J., Griffiths, D. E., and Walker, J. E. (1994b) Biochemistry 33, 7971-7978 the formation of b + b and d + F cross-linked products when Dorman, G., and Prestwich, G. D. (1994) Biochemistry 33, 5661-5673 'Y together with the remainder ofF is removed from SMP. As 1 Dubachev, G. E., Lunev, A. V., Barnakov, A. N., Belogrudov, G. I., Grinkevich, V. A., and Demin, V. V. (1993) FEBS Lett. 336, 181-183 was shown in Fig. 2, both 'Y and a form multiple cross-linked Dunn, S.D., and Heppe!, L.A. (1981)Arch. Biochem. Biophys. 210,421-436 products when SMP or isolated F preparations were treated Dunn, S. D., Heppe!, L. A., and Fullmer, C. S. (1980) J. Bioi. Chern. 255, 6891- with DST (or EDC, not shown). However, like y, a also failed to Dupuis, A., Zaccai, G., and Satre, M. (1983) Biochemistry 22, 5951-5956 cross-link to any stalk subunit. The data of Abrahams et al. Dupuis, A., Issartel, J.-P., Lunardi, J., Satre, M., and Vignais, P. V. (1985) (1994) on the crystal structure of bovine F do not show the Biochemistry 24, 728-733 Eytan, G. D., Carlenor, E., and Rydstriim, J. (1990) J. Bioi. Chem. 265, 12949- location of a. It may be of interest to add in this regard that E. coli a and a cross-link via a disulfide bond in the presence of Fessenden-Raden, J. M. (1972) J. Bioi. Chern. 247, 2351-2357 CuCl (Mendel-Hartvig and Capaldi, 1991), a G29D mutation Fillingame, R. H. (1990) in The Bacteria (Krulwich, T. A., ed) vol. 12, pp. 345-391, Academic Press, New York of the E. coli a renders the F a-deficient (Maggio et al., 1988), Galante, Y. M., Wong, S.-Y., and Hatefi, Y. (1979) J. Bioi. Chem. 254, 12372-12378 and tryptic removal of 15 NH -terminal residues of the a sub- 2 Galante, Y. M., Wong, S.-Y., and Hatefi, Y. (1981) Arch. Biochem. Biophys. 211, 643-651 units of E. coli F destroys the ability of the reconstituted Girvin, M. E., and Fillingame, R. H. (1993) Biochemistry 32, 12167-12177 a (3 yE to bind a (Dunn et al., 1980). As mentioned earlier, E. 3 3 Girvin, M. E., and Fillingame, R. H. (1994) Biochemistry 33, 665-674 coli a is considered to be analogous to the bovine OSCP (Walker Graf, T., and Sebald, W. (1978) FEBS Lett. 94, 218-222 Hatefi, Y. (1993) Eur. J. Biochem. 218, 759-767 et al., 1982; Ovchinnikov et al., 1984; see also Dunn and Hekman, C., Tomich, J. M., and Hatefi, Y. (1991) J. Bioi. Chern. 266, 13564-13571 Heppel, 1981). However, the results just described make one Hundal, T., Norling, B., and Ernster, L. (1983) FEBS Lett. 162, 5-10 Joshi, S., and Burrows, R. (1990) J. Bioi. Chern. 265, 14518-14525 wonder about the location of a in relation to the distal end of E. Kiehl, R., and Hatefi, Y. (1980) Biochemistry 19, 541-548 coli F v where by analogy to the bovine F the NH termini of 1 2 Laemmli, U. K. (1970) Nature 227, 680-685 the a subunits would be located (see Abrahams et al., 1994). Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Bioi. Chern. 193, 265-275 Finally, it should be mentioned that Joshi and Burrows Lutter, R., Saraste, M., van Walraven, H. S., Runswick, M. J., Fine!, M., (1990; see also Archinard et al., 1986) have previously detected Deatherage, J. F., and Walker, J. E. (1993) Biochem. J. 295, 799-806 Maggio, M. B., Parsonage, D., and Senior, A. E. (1988) J. Bioi. Chern. 263, in a preparation of bovine ATP synthase treated with dithio- 4619-4623 bis(succinimidylpropionate), which cross-links amino groups at Matsuno-Yagi, A., and Hatefi, Y. (1984) Biochemistry 23, 3508-3514 a distance of 12 A, many of the cross-linked products we have Matsuno-Yagi, A., and Hatefi, Y. (1990) J. Bioi. Chern. 265, 82-88 Mendel-Hartvig, J., and Capaldi, R. A. (1991) Biochim. Biophys. Acta 1060, 115- characterized here. They employed antibodies to whole F , OSCP, F , A6L, a, and c, but not to the individual subunits of Olmsted, J. B. (1981) J. Bioi. Chern. 256, 11955-11957 Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., F and subunit d. Therefore, some of the cross-linked products Trubetskaya, 0. E., Nazimov, I. V., Hundal, T., and Ernster, L. (1984) FEBS were identified in part on the basis of their mobility on two- Lett. 166, 19-22 dimensional SDS gels. The cross-linked products they report, Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. 64, 173-214 Racker, E., and Horstman, L. L. (1967) J. Bioi. Chern. 242, 2547-2551 which we have not seen in SMP with our three cross-linking Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56 reagents, are those ofF withy, a, and A6L. They also claim the Sebald, W., Graf, T., and Lukins, H. B. (1979) Eur. J. Biochem. 93, 587-599 Senior, A. E. (1979) Methods Enzymol. 55, 391-397 formation of F + d dimer (they refer to d as the 20-kDa Soper, J. W., Decker, G. L., and Pedersen, P. L. (1979) J. Bioi. Chern. 254, subunit), which we only find in DST-treated ASU particles, but 11170-11176 not in intact SMP. Staros, J. V., Wright, R. W., and Swingle, D. M. (1986) Anal. Biochem. 156, 220-222 Steffens, K., Schneider, E., Deckers-Heberstreit, G., and Altendorf, K. (1987) Acknowledgments-We thank C. Munoz for the preparation of mito- J. Bioi. Chern. 262, 5866-5869 chondria and Drs. Akemi Matsuno-Yagi and Mutsuo Yamaguchi for Steinmeier, R. C., and Wang, J. H. (1979) Biochemistry 18, 11-18 helpful discussions. Stiggall, D. L., Galante, Y. M., and Hatefi, Y. (1978) J. Bioi. Chern. 253, 956-964 Stiggall, D. L., Galante, Y. M., Kiehl, R., and Hatefi, Y. (1979) Arch. Biochem. REFERENCES Biophys. 196, 638-644 Walker, J. E., and Collinson, I. R. (1994) FEBS Lett. 346, 39-43 Abrahams, J. P., Lutter, R., Todd, R. J., van Raaij, M. J., Leslie, A. G. W., and Walker, J. E. (1993) EMBO J. 12, 1775-1780 Walker, J. E., Runswick, M. J., and Saraste, M. (1982) FEBS Lett. 146, 393-396 Walker, J. E., Lutter, R., Dupuis, A., and Runswick, M. J. (1991) Biochemistry 30, Abrahams, J.P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994)Nature 370, 621-628 5369-5378 Wilkens, S., and Capaldi, R. A. (1994) Bioi. Chern. Hoppe-Seyler 375, 43-51 Al-Shawi, M. K., Parsonage, D., and Senior, A. E. (1990) J. Bioi. Chern. 265, Yagi, T., and Hatefi, Y. (1984) Biochemistry 23, 2449-2455 4402-4410 Archinard, P., Godinot, C., Comte, J., and Gautheron, D. C. (1986) Biochemistry You, K.-S., and Hatefi, Y. (1976) Biochim. Biophys. Acta 423, 398-412 Zanotti, F., Guerrieu, F., Capozza, G., Houstek, J., Ronchi, S., and Papa, S. (1988) 25,3397-3404 Belogrudov, G. I., and Hatefi, Y. (1994) Biochemistry 33, 4571-4576 FEBS Lett. 237, 9-14

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