F0 of ATP Synthase Is a Rotary Proton Channel
2002; Elsevier BV; Volume: 277; Issue: 15 Linguagem: Inglês
10.1074/jbc.m111210200
ISSN1083-351X
AutoresToshiharu Suzuki, Hiroshi Ueno, Noriyo Mitome, Junko Suzuki, Masasuke Yoshida,
Tópico(s)Electron Spin Resonance Studies
ResumoCoupling of proton flow and rotation in the F0 motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl2 to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F0, either in the F1-stripped inverted vesicles or in the reconstituted F0-proteoliposomes, hardly mediated passive proton translocation through F0. Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F0. Coupling of proton flow and rotation in the F0 motor of ATP synthase was investigated using the thermophilic Bacillus PS3 enzyme expressed functionally in Escherichia coli cells. Cysteine residues introduced into the N-terminal regions of subunits b and c of ATP synthase (bL2C/cS2C) were readily oxidized by treating the expressing cells with CuCl2 to form predominantly a b-c cross-link with b-b and c-c cross-links being minor products. The oxidized ATP synthases, either in the inverted membrane vesicles or in the reconstituted proteoliposomes, showed drastically decreased proton pumping and ATPase activities compared with the reduced ones. Also, the oxidized F0, either in the F1-stripped inverted vesicles or in the reconstituted F0-proteoliposomes, hardly mediated passive proton translocation through F0. Careful analysis using single mutants (bL2C or cS2C) as controls indicated that the b-c cross-link was responsible for these defects. Thus, rotation of the c-oligomer ring relative to subunit b is obligatory for proton translocation; if there is no rotation of the c-ring there is no proton flow through F0. ATP synthase from a thermophilic Bacillus strain PS3 (recombinant TF0F1 investigated in the present study has a histidine tag of 10 residues at the N terminus of the β subunit) N,N′-dicyclohexylcarbodiimide lauryldimethylamine oxide dithiothreitol 9-amino-6-chloro-2-methoxyacridine polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate with prior reducing procedures (termed as reducing SDS-PAGE when necessary for clarification) SDS-PAGE without prior reducing treatment ATP synthases catalyze ATP synthesis/hydrolysis coupled with a transmembrane H+ (proton) translocation in bacteria, chloroplasts, and mitochondria (1.Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1639) Google Scholar, 2.Senior A.E. Physiol. Rev. 1988; 68: 177-231Crossref PubMed Scopus (498) Google Scholar, 3.Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. 2001; 2: 669-677Crossref Scopus (739) Google Scholar). The enzyme is composed of two portions, a water-soluble F1, which has catalytic sites for ATP synthesis/hydrolysis (4.Ko Y.H. Pedersen P.L. J. Biol. Chem. 1999; 274: 28853-28856Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and a membrane-integrated F0, which mediates H+ translocation. The bacterial enzyme has the simplest subunit structure, α3β3γ1δ1ε1for F1 and a1b2c10–11(?)for F0. F1 is easily and reversibly detached from F0 by removal of Mg2+ in a low ionic strength solution. F1 is by itself a rotary motor driven by ATP hydrolysis (5.Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (2005) Google Scholar, 6.Ren H. Allison W.S. Biochim. Biophys. Acta. 2000; 1458: 221-233Crossref PubMed Scopus (48) Google Scholar) in which a central stalk made of γ and ε subunits rotates relative to the surrounding α3β3 hexamer ring (7.Aggeler R. Ogilvie I. Capaldi R.A. J. Biol. Chem. 1997; 272: 19621-19624Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 8.Schulenberg B. Wellmer F. Lill H. Junge W. Engelbrecht S. Eur. J. Biochem. 1997; 249: 134-141Crossref PubMed Scopus (46) Google Scholar). Remaining F0 sector in the membrane acts as a proton channel that mediates passive proton translocation across the membrane. F0 in ATP synthase is thought to work as a rotary motor driven by the energy of proton translocation down the electrochemical potential. Structural studies on F0 with electron microscopy (9.Birkenhager R. Hoppert M. Deckers-Hebestreit G. Mayer F. Altendorf K. Eur. J. Biochem. 1995; 230: 58-67Crossref PubMed Scopus (129) Google Scholar) and atomic force microscopy (10.Takeyasu K. Omote H. Nettikadan S. Tokumasu F. Iwamoto-Kihara A. Futai M. FEBS Lett. 1996; 392: 110-113Crossref PubMed Scopus (117) Google Scholar, 11.Singh S. Turina P. Bustamante C.J. Keller D.J. Capaldi R.A. FEBS Lett. 1996; 397: 30-34Crossref PubMed Scopus (105) Google Scholar, 12.Seelert H. Poetsch A. Dencher N.A. Engel A. Stahlberg H. Muller D.J. Nature. 2000; 405: 418-419Crossref PubMed Scopus (423) Google Scholar) have suggested that subunits a and b2 are peripherally located outside of a ring of subunit coligomers (c-ring). A low resolution crystal structure of an F1+c10 subcomplex from yeast mitochondria revealed a tight interaction between γε subunits of F1 and the c-ring of F0 (13.Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1110) Google Scholar). The cross-links were readily made between introduced cysteines of subunit c and γε subunits of F1 without losing functional coupling between F1 and F0 (14.Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Crossref PubMed Scopus (138) Google Scholar). ATP facilitated the movement of subunit c relative to subunit a that was assessed by the a-c cross-link (15.Hutcheon M.L. Duncan T.M. Ngai H. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8519-8524Crossref PubMed Scopus (64) Google Scholar). A side stalk, made of b2 and δ, connects the stator of F0 and that of F1 and prevents the stators from being dragged by rotation of the central stalk (16.Weber J. Senior A.E. Biochim. Biophys. Acta. 1997; 1319: 19-58Crossref PubMed Scopus (397) Google Scholar). From these and other observations, it is generally accepted that the c-ring rotates relative to stator subunits ab2 (17.Junge W. Lill H. Engelbrecht S. Trends Biochem. Sci. 1997; 22: 420-423Abstract Full Text PDF PubMed Scopus (450) Google Scholar, 18.Elston T. Wang H. Oster G. Nature. 1998; 391: 510-513Crossref PubMed Scopus (452) Google Scholar, 19.Cross R.L. Biochim. Biophys. Acta. 2000; 1458: 270-275Crossref PubMed Scopus (63) Google Scholar). Thus, proton influx into the cytoplasm through F0 (in the case of mitochondria, into the matrix) would cause rotation of the c-ring and hence the central γε stalk, which then enforces each catalytic site in F1 to synthesize ATP. As a reverse reaction, ATP hydrolysis in F1 drives reverse rotation of the γε stalk and c-ring, which causes proton efflux through F0. To explore the mechanism of proton flow through F0 and its coupling with rotation, measurement of the proton flow together with the c-ring rotation is absolutely required. The study has been impeded by the unstable nature of ATP synthases; the structural and functional integrity of the enzyme is easily damaged during experimental procedures (20.Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Crossref PubMed Scopus (72) Google Scholar, 21.Jones P.C. Hermolin J. Jiang W. Fillingame R.H. J. Biol. Chem. 2000; 275: 31340-31346Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). For example, the cross-linking between subunits b and c in Escherichia coli ATP synthase impairs coupling between proton pumping and ATPase activity (21.Jones P.C. Hermolin J. Jiang W. Fillingame R.H. J. Biol. Chem. 2000; 275: 31340-31346Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To overcome this difficulty, we have established an expression system for a stable ATP synthase of thermophilic Bacillus PS3 (TF0F1)1in E. coli cells. Cysteine residues introduced into subunits b and c of TF0F1 were readily cross-linked in the presence of CuCl2. The resultant enzyme lost both proton pumping and ATPase activity, an indication of retaining tight coupling after the cross-linking. The passive proton translocation through F0was also disabled by this cross-link. Thus, the c-ring must rotate for protons to pass through F0. DNA manipulations were carried out by following the methods of the literature (22.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). A plasmid pTR-kε, which is an expression vector for α3β3γε subcomplex of Bacillus PS3 F1-ATPase (23.Suzuki T. Suzuki J. Mitome N. Ueno H. Yoshida M. J. Biol. Chem. 2000; 275: 37902-37906Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), was used as a start material. A 4.4-kilobase pair DNA fragment containing Bacillus PS3 uncBEFHA′ genes (coding for a, c, b, δ, and α subunits of TF0F1) was amplified by PCR from the plasmid pUC119/TF0F1 (24.Ohta S. Yohda M. Ishizuka M. Hirata H. Hamamoto T. Otawara-Hamamoto Y. Matsuda K. Kagawa Y. Biochim. Biophys. Acta. 1988; 933: 141-155Crossref PubMed Scopus (128) Google Scholar) using primers 5′-CCGCGGGAATTCTAAGAAGGAGATATACATATGGAGCATAAAGCGCCGCTTGTCG-3′ and 5′-GGCCGATCGGTACCAGCGCGTCGATCGCTTTAATCC-3′ (underlined bases correspond to EcoRI and KpnI sites, respectively). The amplified fragment was digested with EcoRI and KpnI and ligated into the pTR-kε2 previously digested with both restriction enzymes. For convenience, a SpeI restriction site was introduced just downstream of the uncE gene (c) by the method of Kunkel and Roberts (25.Kunkel T.A. Roberts J.D. Methods Enzymol. 1987; 154: 367-382Crossref PubMed Scopus (4868) Google Scholar) using a synthetic oligonucleotide, 5′-TTTACTTAGGTCGATAACTAGTCGATATGGAAAGTGA-3′. The resultant plasmid was named pTR19-ASDS. Mutated TF0F1s were constructed by the Mega-primer method (26.Landt O. Grunert H.P. Hahn U. Gene (Amst.). 1990; 96: 125-128Crossref PubMed Scopus (642) Google Scholar), using the following mutation primers: 5′-CGGTGCGATACTAGTCGAAAGGAGTGAAACGCGGTGTGCTGGAAGGCAAACCTATGGGCGCTCG-3′ (bLeu2Cys) or 5′-CAATCGCAGCTGCAAGTACACCCAAGCACATGTTGATAGATCCTCCTTCACC-3′ (cSer2Cys). In the mutants, the region amplified by PCR was verified by nucleotide sequencing. E. coli DK8 (23.Suzuki T. Suzuki J. Mitome N. Ueno H. Yoshida M. J. Biol. Chem. 2000; 275: 37902-37906Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) harboring pTR19-ASDS was aerobically cultivated in 2× YT medium (22.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) containing ampicillin (100 μg/ml) at 37 °C for 20 h. Cells (40 g, wet weight) harvested from the culture were dissolved in 280 ml of buffer PA3 (10 mm HEPES/KOH, pH 7.5, containing 5 mm MgCl2 and 10% glycerol) and disrupted by a French press (1200 millibars, twice). After removing the cell debris, the membrane fraction was collected by centrifugation (200,000 ×g, 1 h, 4 °C). The membrane fraction obtained was washed, precipitated, and then suspended in 40 ml of buffer PA3. This suspension was used as the inverted membrane vesicles in this study. After washing the inverted vesicles with a 2-fold volume of buffer PA3 by centrifugation, the vesicles were dissolved in 120 ml of buffer PA3 containing 2% Triton X-100 and 1% sodium cholate and subjected to sonification for 2 min at room temperature. The solution containing solubilized TF0F1 was obtained by a centrifugation (140,000 × g, 15 min, 25 °C). The solution was diluted 3-fold with buffer M (20 mm potassium phosphate buffer, pH 7.5, containing 100 mm KCl) containing 20 mm imidazole and applied to a Ni-nitrilotriacetic acid column (Qiagen, Germany). After washing with 10 volumes of buffer MT (buffer M supplemented with 0.5% Triton X-100), TF0F1 was eluted with buffer MT containing 200 mm imidazole. Then the eluted fraction was precipitated by ammonium sulfate in the presence of 1% sodium cholate (27.Sone N. Yoshida M. Hirata H. Kagawa Y. J. Biol. Chem. 1975; 250: 7917-7923Abstract Full Text PDF PubMed Google Scholar). The precipitate was dissolved in a small volume of 50 mmTris/HCl, pH 8.0. About 10 mg of the purified TF0F1 were routinely obtained from a 1-liter culture. The homogeneity of the purified TF0F1was judged by a SDS-PAGE analysis (see Fig. 1A). Oxidation of cysteine residues in F0 was performed as follows. Recombinant cells expressing TF0F1 (10 g) were washed and suspended in 40 ml of buffer PA3 containing 200 μg/ml CuCl2 and incubated at 25 °C for 30 min. The recombinant TF0F1 has one cysteine residue at position 27 of subunit a, but this residue has no reactivity under these oxidizing conditions. After adding 10 mm EDTA to the solution, the cells were washed with 40 ml of buffer PA3 twice, and inverted membrane vesicles were prepared by the procedures described above. Reconstitution of proteoliposomes was performed by the method of Kaim and Dimroth (28.Kaim G. Dimroth P. FEBS Lett. 1998; 434: 57-60Crossref PubMed Scopus (47) Google Scholar) with some modifications. Phospholipid (soybean l-α-phosphatidylcholine, type II-S, Sigma) was suspended in buffer PA3 at the concentration of 44 mg/ml and sonicated for 3 min on ice water to prepare liposomes. Either TF0F1 (0.5 mg) or TF0 (0.2 mg) was added to 200 μl of the liposome solutions. The solution was frozen with liquid nitrogen and thawed at room temperature. After adding an equal volume of distilled water, the liposomes were briefly sonicated for 15 s, collected by centrifugation (200,000 ×g, 20 min, 4 °C), and resuspended in 200 μl of buffer PA3. F1 was removed from the inverted vesicles by washing with 0.2 mm EDTA (29.Yoshida M. J. Biol. Chem. 1975; 250: 7910-7916Abstract Full Text PDF PubMed Google Scholar), and the resultant F1-stripped inverted vesicles were used for analysis of proton efflux through F0. Isolation of pure F0 complexes and the analysis of proton influx through F0 were performed as described previously (30.Okamoto H. Sone N. Hirata H. Yoshida M. Kagawa Y. J. Biol. Chem. 1977; 252: 6125-6131Abstract Full Text PDF PubMed Google Scholar). ATPase activity was measured at 37 °C using an ATP-regenerating system (23.Suzuki T. Suzuki J. Mitome N. Ueno H. Yoshida M. J. Biol. Chem. 2000; 275: 37902-37906Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The activity that hydrolyzes 1 μmol of ATP per min is defined as 1 unit. DCCD sensitivity was measured as follows. Either the inverted vesicles (10 mg of protein/ml) or proteoliposomes (prepared as above) were added to an equal volume of 100 mm Tris-HCl (pH 8.8) containing 100 μm DCCD. These conditions were chosen to avoid undesired DCCD labeling of the catalytic residue (Glu-190) of β subunits of F1. After an incubation at 25 °C for 30 min, the vesicles were diluted 100-fold and subjected to the measurement of ATPase activity. ATP-driven H+-pumping activity of the vesicles was measured by the method of Aggeler et al. (31.Aggeler R. Weinreich F. Capaldi R.A. Biochim. Biophys. Acta. 1995; 1230: 62-68Crossref PubMed Scopus (43) Google Scholar) using a Jasco model 720 spectrofluorometer at 42 °C at 480 nm using an excitation wavelength of 410 nm. Authentic Bacillus PS3 F0F1 was purified from Bacillus PS3 cells as described previously (27.Sone N. Yoshida M. Hirata H. Kagawa Y. J. Biol. Chem. 1975; 250: 7917-7923Abstract Full Text PDF PubMed Google Scholar). Two-dimensional SDS-PAGE was carried out as performed previously (23.Suzuki T. Suzuki J. Mitome N. Ueno H. Yoshida M. J. Biol. Chem. 2000; 275: 37902-37906Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Protein concentrations were determined using the BCA protein assay kit from Pierce, with bovine serum albumin as a standard. An operon structure containing unc genes coding for TF0F1 was introduced into a downstream of a strong promoter, trc, and then expressed in a F0F1-deficient E. coli strain, DK8. The resultant recombinant strain (DK8/pTR19-ASDS) acquired an ability to form colonies on succinate minimum medium plates within 2 days (the diameter is about 2 mm), which is almost the same growth rate of a wild-type E. coli strain (JM109), indicating that the recombinant TF0F1 is functional as ATP synthase in the E. coli cell. TF0F1consisting of eight subunits was constitutively expressed in the plasma membranes, which amounted to ∼20% of the whole membrane proteins (Fig. 1A, lane 1). The purified TF0F1 was comprised of eight kinds of subunits, the same as authentic TF0F1 purified from the original Bacillus PS3 cells 2On the gel of Fig. 1A, a band of recombinant b subunit (lanes 1 and 2) moved slightly slower than the counterpart of the TF0F1 purified from Bacillus PS3 cells (lane 3). N-terminal sequencing of the recombinant subunit revealed that an N-terminal segment comprising 11 residues is not processed in the E. coli cells, which is different from the authentic enzyme (23.Suzuki T. Suzuki J. Mitome N. Ueno H. Yoshida M. J. Biol. Chem. 2000; 275: 37902-37906Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). The low mobility of recombinant β subunit is ascribed to 10 residues of a histidine tag introduced at the N terminus.(Fig. 1A, lanes 2and 3). The membranes of expressing E. coliexhibited 1.0 unit/mg of protein (at 37 °C) of ATPase activity. More than 80% of the activity was inactivated by a 50-min incubation with 50 μm DCCD or by a 20-min incubation with 100 μm DCCD (Fig. 1B). This inhibition is comparable or slightly more efficient than that (75%) observed for the authentic TF0F1 purified from Bacillus PS3 cells (27.Sone N. Yoshida M. Hirata H. Kagawa Y. J. Biol. Chem. 1975; 250: 7917-7923Abstract Full Text PDF PubMed Google Scholar). This inactivation is due to labeling subunit c but not labeling catalytic glutamic acid (Glu-190) in the β subunit of F1 because ATPase activity was unaffected by DCCD treatment when measured in the presence of lauryldimethylamine oxide (LDAO), which unleashes ATPase activity of F1 from F0. The membrane ATPase was inhibited almost completely by 5 mm azide, an inhibitor of ATPase activity of F1 and ATP synthase. Two cysteine residues were introduced into TF0F1 by substituting Leu-2 of subunit b and Ser-2 of subunit c to obtain a mutant TF0F1,bL2C/cS2C. To use as controls, we also made two single mutants, bL2C or cS2C, that had one substituted cysteine in subunit b or c. These mutants were expressed in E. coli DK8 cells. N-terminal regions of subunits b and c are located in the periplasmic surface of plasma membrane (21.Jones P.C. Hermolin J. Jiang W. Fillingame R.H. J. Biol. Chem. 2000; 275: 31340-31346Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and as expected, the introduced cysteine residues in bL2C/cS2C formed a disulfide cross-link by treating expressing E. coli cells with an oxidant, 200 μm CuCl2. After careful removal of the oxidant, inverted membrane vesicles were prepared from the cells. In the presence of LDAO, the vesicles from the mutants showed ATPase activities similar to that of the wild type, indicating that the wild type and mutants were expressed at comparable levels (wild type, 6.6 ± 0.1 units/mg; bL2C, 5.8 ± 0.1 units/mg; cS2C, 5.3 ± 0.1 units/mg;bL2C/cS2C, 6.8 ± 0.2 units/mg of membrane protein). Mutant TF0F1s were purified from the vesicles and analyzed by SDS-PAGE after incubation with or without 50 mm DTT at 25 °C for 1 h (Fig. 2A). The mutant TF0F1s incubated with DTT showed eight bands, the same as that of wild type (lanes 1–4). However, those not exposed to DTT had an additional one (bL2C and cS2C, lanes 6 and 7) or three band(s) (bL2C/cS2C, lane 8). Based on the N-terminal peptide sequences and estimated molecular sizes of the bands, these new bands were identified as disulfide cross-linked products of b-b, b-c, and c-c as indicated by arrows in Fig. 2A. Cross-link yields in bL2C/cS2C were analyzed by two-dimensional SDS-PAGE, first in non-reducing and second in reducing conditions (Fig. 2B). The three bands were separated in the second electrophoresis into spot(s) corresponding to monomeric subunit b and/or c. In bacterial ATP synthase, subunit b has been known to exist as a homodimer (32.Dmitriev O. Jones P.C. Jiang W. Fillingame R.H. J. Biol. Chem. 1999; 274: 15598-15604Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and only one of two copies of subunit b is assumed to lie adjacent to the c-ring that is able to form a cross-link (21.Jones P.C. Hermolin J. Jiang W. Fillingame R.H. J. Biol. Chem. 2000; 275: 31340-31346Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Taking this into account, the yields of b-b and b-c cross-links in bL2C/cS2C were estimated from the densities of spots to be 16 ± 5 and 68 ± 7%, respectively. A further increase of the cross-links of b-b or b-c was not observed in bL2C/cS2C even though either 500 μmCuCl2 or 1 mm copper/phenanthroline was used as an oxidant. It is worth mentioning that a significant amount of b-c cross-link was formed (yield ∼40%) even in the membranes that were not treated with CuCl2 (data not shown). 3The membrane vesicles without CuCl2treatment showed levels of ATPase activity and proton pumping intermediate between the +DTT and −DTT level. It appears evident that there is close proximity between the N-terminal ends of subunit b(s) and one (or more) of subunit c(s). The inverted vesicles prepared from the cells oxidized by CuCl2 treatment were incubated with or without 50 mm DTT for 1 h, and H+-pumping activity was analyzed. DTT-treated vesicles of all mutants showed substantial ATP-driven H+-pumping activities, comparable with that of the wild type (Fig. 3A, left panel). Therefore, the introduced cysteine residue(s) at position 2 of subunit b and that of subunit c, alone or together, do not significantly affect the F0 function. This was also the case for the vesicles of mutants bL2C and cS2C that were not reduced by DTT. However, the vesicles of a mutant bL2C/cS2C (without DTT treatment) had drastically decreased H+-pumping activity (Fig. 3A,right panel). Membranes of the oxidized vesicles from bL2C/cS2C are capable of holding the electrochemical potential of protons generated by NADH oxidation, as described later, and proton leak cannot be a reason for the apparent loss of H+ pumping. The inactivation of bL2C/cS2C by oxidation was also observed for ATPase activity. Oxidized vesicles from bL2C/cS2C without DTT treatment retained only 37% of the ATPase activity of that of the DTT-treated ones whereas activities of the vesicles from the single mutants (and wild type) were hardly affected by oxidation-reduction treatment (Fig. 3B). The inhibition of ATPase was completely recovered by adding 0.1% LDAO in the ATPase assay mixture (data not shown), confirming that the failure was not in F1 but in F0. The same experiments were repeated for the proteoliposomes reconstituted from purified TF0F1 and soybean phospholipids (Fig. 3,C and D). As observed for the inverted vesicles, inactivation of H+-pumping and ATPase activities was evident only for the oxidized bL2C/cS2C. To summarize the results, only oxidized TF0F1containing double mutations bL2C/cS2C has a defect in ATP hydrolysis and in H+ pumping. This defect is caused by a b-c cross-link and cannot be ascribed to the b-b and c-c cross-links. This is because (as shown in Fig. 2A) the amount of b-b and c-c cross-links produced in the oxidized bL2C/cS2C was too little to account for the observed inactivation. Furthermore, the oxidized single mutants,bL2C and cS2C, contained more b-b and c-c cross-links, respectively, than those in the oxidized bL2C/cS2C as shown in Fig. 2A but still their activities were not inactivated significantly. On the contrary, the amount of b-c cross-link in oxidized bL2C/cS2C (∼68%) agrees fairly well with the degree of inactivation (∼60% for ATPase activity). Thus, prevention of movement of the c-ring relative to subunit bis fatal for the catalytic function of ATP synthase with proper coupling. Inverted vesicles that were prepared from CuCl2-oxidized cells were washed with 0.2 mmEDTA to obtain F1-stripped inverted vesicles. The electrochemical potential of protons was generated across the membrane of inverted F0 vesicles by the respiratory chain on the vesicles using NADH as a substrate, and the downhill proton efflux through F0 was assessed by monitoring fluorescence quenching of ACMA. Without F1, protons taken up in vesicles by respiration easily diffused out through F0, and only a small fluorescence quenching was maintained at steady state as a balance between activities of respiration and proton flow through F0 (Fig. 4A,wild type). Prior reducing treatment of the vesicles by DTT did not change the result significantly. Inverted F0vesicles prepared from single mutants, bL2C and cS2C, behaved similarly; the quenching was small and the effect of DTT treatment was minor (Fig. 4A, bL2C and cS2C). Also the extent of fluorescence quenching was small for the DTT-treated inverted F0 vesicles of bL2C/cS2C (Fig. 4A,bL2C/cS2C). However, when the same inverted F0 vesicles of bL2C/cS2C were subjected to the test without prior DTT treatment, remarkable fluorescence quenching was induced in response to the addition of NADH. The magnitude of quenching by NADH matched well the one observed for the inverted vesicles, without F1-stripping treatment, prepared from cells expressing wild-type F0F1 (data not shown). This result clearly indicates that b-c cross-link blocks proton efflux through F0. It also implies that b-c cross-link does not make F0 leak protons. To confirm further the above contention, F0 was isolated from purified TF0F1 and reconstituted into proteoliposomes. F0 proteoliposomes were incubated with or without DTT and then loaded with 0.5 m KCl. Membrane potential (inside negative) was generated by the addition of valinomycin, and downhill proton influx through F0 was assessed by monitoring fluorescence quenching of ACMA (Fig. 4B). In the case of F0 proteoliposomes of the wild type, irrespective of whether they were treated with DTT or not, valinomycin induced significant fluorescence quenching that reflected proton flow through F0. F0 proteoliposomes of bL2C/cS2C with prior DTT treatment displayed a similar extent of fluorescence quenching. On the contrary, the quenching was greatly suppressed when F0 proteoliposomes of bL2C/cS2C without prior DTT treatment were examined. In any case, preincubation of F0proteoliposomes with DCCD resulted in complete abolishment of valinomycin-induced quenching. These results, together with those of the inverted F0 vesicles, led to the conclusion that proton efflux and influx through F0 are blocked by b-ccross-link. The major message of this report concerns the relation between the c-ring rotation and the proton flow through F0. As illustrated in Fig. 5, the F0 with a disulfide cross-link between subunits b and c was unable to mediate proton translocation (Fig. 4). With prevented proton translocation, ATP hydrolysis was also prevented, suggesting the retention of tight coupling between F0 and F1 in the cross-link containing TF0F1 (Fig. 3). Regardless of the directions of proton translocation, either the periplasmic side to the cytoplasmic side or the cytoplasmic side to the periplasmic side, translocations were equally blocked by the cross-linking. The inactivation is reversible; reduction of the disulfide restored the proton translocation by F0 and ATP-driven proton pumping by TF0F1. These results strongly indicate that protons cannot pass through F0 without rotation of the c-ring, or conversely, rotation of the c-ring must accompany proton translocation. Cross-linking of b and c subunits caused neither a proton leak nor the unleashing of activation of ATPase of F1. Thus the possibility that the cross-link itself disrupts the function of F0 is minimal, if not null. In our experimental setups, proton translocations across membranes down the Δμ̃H+ were measured. Previous data from other laboratories indicate that the coupling of proton translocation and c-ring rotation is maintained even in the absence of Δμ̃H+. Dimroth's group has detected 22Na+/Na+ exchange across proteoliposome membranes in the absence of Δμ̃H+through F0 isolated from Na+-transporting ATP synthase of Propionigenium modestum (33.Kaim G. Dimroth P. EMBO J. 1998; 17: 5887-5895Crossref PubMed Scopus (72) Google Scholar). They did not examine the movement of the c-ring but assumed that the back-and-forth thermal rotary motion of the c-ring in F0 was responsible for the exchange. Fillingame and his colleagues (21.Jones P.C. Hermolin J. Jiang W. Fillingame R.H. J. Biol. Chem. 2000; 275: 31340-31346Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) demonstrated by using the b-ccross-linking of uncoupled E. coli ATP synthase that the subunit c adjacent to subunit b is mobile and exchanges with subunits c that initially occupied other positions. This exchange occurs independently of ATP, suggesting thermal motion of the c-ring. Although not tested by their experiments, it is natural to assume that this thermal motion accompanies proton translocation. Taken together, it is safe to conclude that isolated F0 is a rotary proton channel in which the "friction" between rotor and stator is small enough to allow free fluctuating rotary motion even by environmental thermal energy. Nevertheless, this friction may differentiate the F0 channel from other open ion channels; the ion conductance of the former is several orders lower than that of the latter (F0, ∼7 s−1 (33.Kaim G. Dimroth P. EMBO J. 1998; 17: 5887-5895Crossref PubMed Scopus (72) Google Scholar); potassium channel, >106 s−1 (34.Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5848) Google Scholar); aquapolin-1, >109s−1 (35.Murata K. Mitsuoka K. Hirai T. Walz T. Agre P. Heymann J.B. Engel A. Fujiyoshi Y. Nature. 2000; 407: 599-605Crossref PubMed Scopus (1477) Google Scholar)). Once Δμ̃H+ is applied, the F0 rotary channel starts to rotate unidirectionally and protons are transported also unidirectionally across membranes. We thank our colleagues, Drs. E. Muneyuki and T. Hisabori, for helpful discussions.
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