Rotation of the Proteolipid Ring in the V-ATPase
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m303104200
ISSN1083-351X
AutoresKen Yokoyama, Masahiro Nakano, Hiromi Imamura, Masasuke Yoshida, Masatada Tamakoshi,
Tópico(s)Metalloenzymes and iron-sulfur proteins
ResumoV0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312–2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N′,N′-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at ∼3.0 rev/s in average at 4 mm ATP and was abolished by N′,N′-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase. V0V1-ATPase is a proton-translocating ATPase responsible for acidification of eukaryotic intracellular compartments and for ATP synthesis in archaea and some eubacteria. We demonstrated recently the rotation of the central stalk subunits in V1, a catalytic sector of V0V1-ATPase (Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312–2315), but the rotation of the proteolipid ring, a predicted counterpart rotor in the membrane V0 sector, has remained to be proven. V0V1-ATPase that retained sensitivity to N′,N′-dicyclohexylcarbodiimide was isolated from Thermus thermophilus, immobilized onto a glass surface through the N termini of the A subunits of V1, and decorated with a bead attached to a proteolipid subunit of V0. Rotation of beads was observed in the presence of ATP, and direction of rotation was always counterclockwise viewed from the membrane side. The rotation proceeded at ∼3.0 rev/s in average at 4 mm ATP and was abolished by N′,N′-dicyclohexylcarbodiimide treatment. Thus, the rotation of the central stalk in V1 accompanies rotation of a proteolipid ring of V0 in the functioning V0V1-ATPase. V0V1-ATPase catalyzes the interconversion of the energy of proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Google Scholar, 2Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Google Scholar, 3Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Google Scholar). Eukaryotic cells adopt V0V1-ATPase as an ATP hydrolysis-driven proton pump that carries out acidification of cellular compartments such as lysosomes and extracellular fluid in the case of renal acidification, bone resorption, and tumor metastasis (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Google Scholar). On the contrary, in archaea and some eubacteria, a major role of V0V1-ATPase is to produce ATP that is driven by downhill proton flow across membranes (4Gruber G. Wieczorek H. Harvey W.R. Muller V. J. Exp. Biol. 2001; 204: 2597-2605Google Scholar, 5Yokoyama K. Muneyuki E. Amano T. Mizutani S. Yoshida M. Ishida M. Ohkuma S. J. Biol. Chem. 1998; 273: 20504-20510Google Scholar). V0V1-ATPase is a complicated protein complex with a molecular mass of ∼800 kDa composed of many different subunits arranged into two sectors, V0 and V1 (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Google Scholar). We have studied V0V1-ATPase from a thermophilic eubacterium, Thermus thermophilus, a stable enzyme that allows experimental procedures difficult for enzymes from other sources, and developed the expression systems of subcomplexes and subunits in Escherichia coli (5Yokoyama K. Muneyuki E. Amano T. Mizutani S. Yoshida M. Ishida M. Ohkuma S. J. Biol. Chem. 1998; 273: 20504-20510Google Scholar). The V1 sector of T. thermophilus, which has ATP hydrolyzing activity by itself, is made up of four different subunits, A (63 kDa), B (53 kDa), D (25 kDa), and F (12 kDa) with a stoichiometry of A3B3D1F1 (6Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Google Scholar). The A subunit contains a catalytic site, and the A and B subunits are arranged alternately to form a hexameric cylinder (7Yokoyama K. Ohkuma S. Taguchi H. Yasunaga T. Wakabayashi T. Yoshida M. J. Biol. Chem. 2000; 275: 13955-13961Google Scholar). The D subunit fills the central cavity of the cylinder and, together with the F subunit, forms a central stalk (7Yokoyama K. Ohkuma S. Taguchi H. Yasunaga T. Wakabayashi T. Yoshida M. J. Biol. Chem. 2000; 275: 13955-13961Google Scholar, 8Arata Y. Baleja J.D. Forgac M. Biochemistry. 2002; 41: 11301-11307Google Scholar, 9Xu T. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 28909-28915Google Scholar). The V0 sector is responsible for proton translocation across membranes, and the principal components involved in proton translocation are a highly conserved family of hydrophobic subunits, often termed as proteolipid because of their solubility in organic solvents. The eukaryotic V0 sector contains three similar but different proteolipid species, which are predicted to contain at least four transmembrane helices, arranged in a ring-like structure (2Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Google Scholar, 10Wilkens S. Forgac M. J. Biol. Chem. 2001; 276: 44064-44068Google Scholar, 11Powell B. Graham L.A. Stevens T.H. J. Biol. Chem. 2000; 275: 23654-23660Google Scholar). On the contrary, single proteolipid proteins with two transmembrane helices, termed L subunit, make a ring-like structure in V0 sector of T. thermophilus V0V1-ATPase (4Gruber G. Wieczorek H. Harvey W.R. Muller V. J. Exp. Biol. 2001; 204: 2597-2605Google Scholar, 7Yokoyama K. Ohkuma S. Taguchi H. Yasunaga T. Wakabayashi T. Yoshida M. J. Biol. Chem. 2000; 275: 13955-13961Google Scholar). The V0 sector of T. thermophilus contains another membrane protein, I subunit (71 kDa), a homolog of yeast Vph1p, that interacts with the proteolipid ring and plays also a critical role in proton translocation (12Kawasaki-Nishi S. Nishi T. Forgac M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12397-12402Google Scholar). V0V1-ATPase is known to be structurally and evolutionary related to F0F1-ATP synthase, which is responsible for ATP production in mitochondria, chloroplasts, and many eubacteria (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Google Scholar, 4Gruber G. Wieczorek H. Harvey W.R. Muller V. J. Exp. Biol. 2001; 204: 2597-2605Google Scholar, 13Boyer P.D. Annu. Rev. Biochem. 1997; 66: 714-749Google Scholar). F0F1-ATP synthase is also composed of two sectors, F0 and F1. The c subunit in F0 is a proteolipid protein composed of two transmembrane helices and forms a ring structure (14Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Google Scholar). ATP hydrolysis in catalytic sites of F1 drives rotation of the central stalk subunits, γ and ϵ (15Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Google Scholar, 16Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Google Scholar), and this rotation in turn drives the rotation of the F0c-ring (16Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Google Scholar, 17Nishio K. Iwamoto-Kihara A. Yamamoto A. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13448-13452Google Scholar, 18Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Google Scholar) that is thought to be directly responsible for proton translocation (19Oster G. Wang H. Biochim. Biophys. Acta. 2000; 1458: 482-510Google Scholar). Because of the functional and structural similarity between V0V1-ATPase and F0F1-ATP synthase, it has been assumed that V0V1-ATPase would use a similar rotary mechanism in catalysis (1Nishi T. Forgac M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 94-103Google Scholar, 20Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell. Biol. 2001; 2: 669-677Google Scholar). In fact, the rotation of the D and F subunits relative to the A and B subunits in V1-ATPase from T. thermophilus was recently proven (21Imamura H. Nakano M. Noji H. Muneyuki E. Ohkuma S. Yoshida M. Yokoyama K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2312-2315Google Scholar). Here, we show the visual demonstration of ATP-dependent rotation of the proteolipid ring in V0V1-ATPase. Purification of Mutant V0V1-ATPase—A mutated V0V1-ATPase (AHis8 tags/A-S232A/A-T235S/L-E23C) was constructed by integration vector system (22Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Google Scholar, 23Tamakoshi M. Yamagishi A. Oshima T. Gene (Amst.). 1998; 222: 125-132Google Scholar, 24Tamakoshi M. Uchida M. Tanabe K. Fukuyama S. Yamagishi A. Oshima T. J. Bacteriol. 1997; 179: 4811-4844Google Scholar). pUTpyrE, which carries the pyrE gene cassette, was constructed. The XbaI-EcoRI fragment containing the leuB gene of pT8leuB was cloned in pUC119, and then the NdeI-EcoRI fragment was replaced with the NdeI-EcoRI fragment containing the pyrE gene of pINV (24Tamakoshi M. Uchida M. Tanabe K. Fukuyama S. Yamagishi A. Oshima T. J. Bacteriol. 1997; 179: 4811-4844Google Scholar). The sequence corresponding to a 1550-bp region, which is upstream of the atpA gene, containing the termination codon of the atpF gene and the sequence corresponding to a 1750-bp region containing the mutated atpA gene (A-His8 tags/A-S232A/A-T235S) with its Shine-Dalgarno sequence were cloned in the SphI-SalI and EcoRV-EcoRI sites of pUTpyrE, respectively. A pyrE strain, T. thermophilus TTY1, was genetically transformed with the resultant plasmid as described previously (22Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Google Scholar). Transformants were selected on a minimum-medium plate without uracil. To introduce cysteine residue to L subunit, another integration vector was then constructed. The sequence corresponding to a 1250-bp region, which is upstream of the atpL gene, containing the termination codon of the atpG gene and the sequence corresponding to a 1350-bp region containing the mutated atpL gene (l-E23C) with its Shine-Dalgarno sequence were cloned in the SphI-SalI and EcoRV-EcoRI sites of pBHTK1 (25Nureki O. Shirouzu M. Hashimoto K. Ishitani R. Terada T. Tamakoshi M. Oshima T. Chijimatsu M. Takio K. Vassylyev D.G. Shibata T. Inoue Y. Kuramitsu S. Yokoyama S. Acta Crystallogr. Sec. D. 2002; 58: 1129-1137Google Scholar), respectively. The transformant involved in atpA mutation was transformed with the resultant plasmid containing mutated atpL gene. The transformant was selected on a nutrient-medium plate containing 0.1 mm kanamycin. Chromosomal DNA was prepared from a transformed strain, and mutations were confirmed by sequencing with a ABI 310 sequencer. The recombinant T. thermophilus was grown as described previously (6Yokoyama K. Oshima T. Yoshida M. J. Biol. Chem. 1990; 265: 21946-21950Google Scholar, 26Yokoyama K. Akabane Y. Ishii N. Yoshida M. J. Biol. Chem. 1994; 269: 12248-12253Google Scholar). The cells (200 g) harvested at log-phase growth were suspended in 400 ml of 50 mm Tris-Cl (pH 8.0), containing 5 mm MgCl2 and disrupted by sonication. The membranes were precipitated by centrifugation at 100,000 × g for 20 min and washed with the same buffer twice. The washed membranes were suspended in 20 mm sodium imidazole (pH 8.0), 0.1 m NaCl, and 10% (w/v) octaethylene glycol monododecyl ether (C12E8), and the suspension was sonicated. The debris and insoluble materials were removed by centrifugation at 100,000 × g for 60 min, and the supernatant was applied onto a Ni2+-NTA 1The abbreviations used are: NTA, nitrilotriacetic acid; DCCD, N′N′-dicyclohexylcarbodiimide; C12E8, octaethylene glycol monododecyl ether; ACMA, 9-amino-6-chloro-2-methoxyacridine. -Superflow column (3 × 10 cm, Qiagen) equilibrated with 20 mm sodium imidazole (pH 8.0), 0.1 m NaCl, and 0.1% C12E8. The column was washed with 200 ml of the same buffer. The protein was eluted with a linear imidazole gradient (20–100 mm). The fractions containing the V0V1-ATPase were combined and dialyzed against 20 mm Tris-Cl (pH 8.0), 0.1 mm EDTA, and 0.05% C12E8 for 2 h. The dialyzed solution was applied to a Resource Q column (Amersham Biosciences) equilibrated with 20 mm Tris-Cl (pH 8.0), 0.1 mm EDTA, and 0.05% C12E8. The proteins were eluted with a linear NaCl gradient (0–0.5 m). The above purification procedures were carried out at 4 °C and completed within 8 h. The purified V0V1-ATPase was immediately biotinylated with a 10-molar excess of 6-{N′-[2-(N-maleimido)ethyl]-N-piperazinylamido}-hexyl-d-biotinamide (biotin-PEAC5-maleimide, Dojindo) in 20 mm Tris-Cl (pH 8.0), 0.1 mm EDTA, 100 mm NaCl, and 0.05% C12E8. After a 15-min incubation at 25 °C, proteins were separated from unbound reagents with a PD10 column (Amersham Biosciences). The biotinylated V0V1-ATPases were kept on ice and used for experiments within a day. Specific biotinylation of the L subunit was checked by Western blotting using streptavidin-alkaline phosphatase conjugate (Amersham Biosciences). Rotation Experiments—A flow cell (5 μl) was made of two coverslips (bottom, 24 × 36 mm2; top, 18 × 18 mm2) separated by two spacers of 50-μm thickness. The glass surface of the bottom coverslip was coated with Ni2+-NTA. The biotinylated V0V1-ATPase (0.1–1 μm) in buffer A (50 mm Tris-HCl (pH 8.0), 100 mm KCl, 2 mm MgCl2, 0.05% C12E8, and 0.5% (w/v) bovine serum albumin) was applied to the flow cell and was washed with 20 μl of buffer A. When sensitivity to inhibition by N′,N′-dicyclohexylcarbodiimide (DCCD) was examined, the biotinylated enzyme (1 μm) was incubated for 30 min at 25 °C in buffer A containing 100 μm DCCD prior to infusing into the flow cell. The suspension (10 μl) of 0.1% (w/v) streptavidin-coated beads (ϕ = 0.56 μm, Bangs Laboratories Inc.) in buffer A was infused into the flow cell, and unbound beads were washed out with 40 μl of buffer A. Observation of rotation was started after infusion of 10 μl of buffer A supplemented with indicated concentrations of ATP and an ATP-regenerating system (0.2 mg/ml creatine kinase and 2.5 mm creatine phosphate). Rotation of beads was observed with a bright-field microscope (IX70, Olympus) at a magnification of 1000. Images were video-recorded (30 frames/s) with a CCD camera. All of these procedures and observations were carried out at 25 °C. Other Assays—ATPase activity was measured at 25 °C with an enzyme-coupled ATP-regenerating system (5Yokoyama K. Muneyuki E. Amano T. Mizutani S. Yoshida M. Ishida M. Ohkuma S. J. Biol. Chem. 1998; 273: 20504-20510Google Scholar). The ATPase assay solution contained 50 mm Tris-HCl (pH 8.0), 100 mm KCl, 2 mm MgCl2, 4 mm ATP-Mg, 2 mm phosphoenolpyruvate, 100 μg/ml lactate dehydrogenase, 100 μg/ml of pyruvate kinase, 0.2 mm NADH, and 0.05% C12E8. When DCCD sensitivity of ATPase activity was examined, the enzyme (1 μm) was incubated at 25 °C with 100 μm DCCD in 20 mm Tris-Cl (pH 8.0), 1 mm MgCl2, 0.1 m NaCl, and 0.05% C12E8. The aliquots were taken out at indicated times, and residual ATPase activity was assayed. In the case when DCCD sensitivity of V0V1-ATPase isolated from membranes with Triton X-100 was examined, the reaction mixture and assay mixture contained 0.05% Triton X-100 instead of 0.05% C12E8. ATP-driven H+ translocation by V0V1-ATPase was monitored at 25 °C by fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) (excitation at 410 nm; emission at 480 nm). Reconstituted vesicles containing purified V0V1-ATPase were suspended at 10 μg of protein/ml in 10 mm HEPES/KOH (pH 8.0), 100 mm KCl, 5 mm MgCl2, and 0.3 μg/ml ACMA, and the reaction was initiated by adding 4 mm ATP. At indicate times, 1 μg/ml carbonyl cyanide p-trifluoromethoxyphenylhydrazone was added. Protein concentrations were determined by BCA protein assay (Pierce). Properties of Mutated V0V1-ATPase for Rotation Assay—To prepare mutant V0V1-ATPase (A-His8 tags/A-S232A/A-T235S/l-E23C) of T. thermophilus for the rotation experiment, we have been using the shuttle integration vector system (22Tamakoshi M. Yaoi T. Oshima T. Yamagishi A. FEMS Microbiol. Lett. 1999; 173: 431-437Google Scholar, 23Tamakoshi M. Yamagishi A. Oshima T. Gene (Amst.). 1998; 222: 125-132Google Scholar, 24Tamakoshi M. Uchida M. Tanabe K. Fukuyama S. Yamagishi A. Oshima T. J. Bacteriol. 1997; 179: 4811-4844Google Scholar, 25Nureki O. Shirouzu M. Hashimoto K. Ishitani R. Terada T. Tamakoshi M. Oshima T. Chijimatsu M. Takio K. Vassylyev D.G. Shibata T. Inoue Y. Kuramitsu S. Yokoyama S. Acta Crystallogr. Sec. D. 2002; 58: 1129-1137Google Scholar). T. thermophilus is absolutely aerobic (27Oshima T. Imahori K. J. Biochem. (Tokyo). 1974; 75: 179-183Google Scholar), and the membranes of recombinant T. thermophilus cells must contain the functional mutated ATP synthase. The His8 tags were added to the N termini of the A subunits to immobilize the enzyme to the Ni2+-NTA-coated glass surface. Glutamic acid 23 of the L subunit was replaced with cysteine for biotinylation. Turnover rate of wild type V0V1-ATPase rapidly decays because of entrapping inhibitory Mg-ADP in a catalytic site (5Yokoyama K. Muneyuki E. Amano T. Mizutani S. Yoshida M. Ishida M. Ohkuma S. J. Biol. Chem. 1998; 273: 20504-20510Google Scholar). The active time interval is too short to find and analyze the rotating molecules, and therefore, the S232A/T235S double substitution in the A subunit, which was found to suppress the Mg-ADP inhibition, was introduced. The mutated V0V1-ATPase was solubilized and purified to homogeneity in the presence of C12E8. The ATPase activity of isolated V0V1-ATPase lasted for at least 10 min after adding ATP. The V0V1-ATPase exhibited simple Michaelis-Menten kinetics with a Km value of 450 ± 80 μm and a kcat value of ∼9.5 ± 1.0 s–1. Fig. 1A shows sensitivity of V0V1-ATPase to inactivation by DCCD, a specific inhibitor that modifies a critical carboxylate in proteolipid subunit. DCCD has been generally used as a marker to show that the F0F1-ATPase is intact (13Boyer P.D. Annu. Rev. Biochem. 1997; 66: 714-749Google Scholar, 16Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Google Scholar, 28Kaim G. Matthey U. Dimroth P. EMBO J. 1998; 17: 688-695Google Scholar). If proton translocation and ATP hydrolysis is uncoupled because of damage of the functional connection between F0 and F1, ATPase activity is no longer sensitive to DCCD inhibition. This contention is also the case for V0V1-ATPase, and we looked for the procedures to isolate the DCCD-sensitive enzyme. Among the detergents we tested, C12E8 gave the best results. The ATPase activity of V0V1-ATPase solubilized and purified in C12E8 was inhibited by DCCD and was nearly completely lost after a 30-min incubation (Fig. 1A). The isolated V1-ATPase was not inhibited by DCCD (data not shown). On the contrary, Triton X-100 has a deteriorating effect on V0V1-ATPase as was observed for F0F1-ATPase (18Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Google Scholar). The enzyme solubilized and purified in Triton X-100 lost the sensitivity to DCCD inhibition (Fig. 1A). The isolated V0V1-ATPase reconstituted into phospholipid liposomes showed an ATP-dependent proton translocation activity, and this activity was also completely lost by DCCD treatment (Fig. 1B). Thus, our preparation of V0V1-ATPase retained intact activity that coupled proton translocation and ATP hydrolysis. Rotation of the Proteolipid Ring—The observation system of V0V1-ATPase rotation is similar to that used for V1-ATPase (Fig. 2A). Rotation was visualized by a bead obliquely attached to the L subunit, which was illuminated as a bright-field image under optical microscopic field. Specificity of biotinylation of the L subunits in V0V1-ATPase was confirmed by protein immunoblotting with streptavidin (Fig. 2B). The biotinylation of V0V1-ATPase did not affect the enzymatic properties, turnover rate, and DCCD sensitivity (data not shown). The His8 tags of the enzymes were immobilized to the Ni2+-NTA-coated glass surface. We found rotating beads attached to the L subunit in V0V1-ATPase when the flow cell was infused by a buffer containing ATP (Fig. 3A). 10–20 rotating beads were usually found in 0.2-mm2 area of a single flow cell where a total of 2000–3000 beads were found. Rotations were unidirectional and, similar to V1-ATPase, directions were always counterclockwise when viewed from the membrane side (V0 side). Azide, which has been known as specific inhibitor of F1-ATPase, did not affect the observed rotation of V0V1-ATPase at 4 mm ATP (Fig. 3B). The average rotation rate at 4 mm ATP was calculated to be ∼3.0 rev/s for the beads showing apparently uninhibited rotation that continued for >20 s without pause longer than 2 s. One revolution consumes three ATP molecules, and therefore, the above rotation rate may correspond to a kcat value of ∼10 s–1 that agrees well to the value obtained from the bulk phase kinetics. Rotations at 1 mm ATP (not shown) appeared very similar to those observed at 4 mm ATP, and the average rotation rate was ∼2.7 rev/s. At 0.2 mm ATP, the substrate ATP binding to the enzyme becomes the rate-limiting step in the whole catalytic cycle and the rotation was slowed significantly (Fig. 3C).Fig. 3Time courses of rotation of beads attached to the L subunit.A, rotation at 4 mm ATP. B, rotation at 4 mm ATP in the presence of 0.5 mm sodium azide. C, rotation at 0.2 mm ATP.View Large Image Figure ViewerDownload (PPT) To address inhibitory effect of DCCD on rotation of V0V1-ATPase, the enzyme was incubated with DCCD for 30 min before rotation assay. The number of rotating beads decreased to ∼5% compared with non-treated enzyme (Fig. 4). The inhibitory effect of DCCD on rotation is comparable with that on ATPase activity of the enzyme in the bulk solution. Rotation of the beads attached to V1-ATPase was not affected with DCCD treatment. These results indicate that the observed rotations were those of the functional V0V1-ATPase with coupling entity. We show here the rotation of the proteolipid ring of V0V1-ATPase. The rotation is DCCD-sensitive, and therefore, such controversy over the intactness of the enzyme as was raised on the DCCD-insensitive rotation of F0F1-ATPase in Triton X-100 (16Tsunoda S.P. Aggeler R. Yoshida M. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 898-902Google Scholar, 18Tsunoda S.P. Aggeler R. Noji H. Kinosita Jr., K. Yoshida M. Capaldi R.A. FEBS Lett. 2000; 470: 244-248Google Scholar, 29Sambongi Y. Iko Y. Tanabe M. Omote H. Iwamoto-Kihara A. Ueda I. Yanagida T. Wada Y. Futai M. Science. 1999; 286: 1722-1724Google Scholar) is avoided. Together with a previous report (21Imamura H. Nakano M. Noji H. Muneyuki E. Ohkuma S. Yoshida M. Yokoyama K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2312-2315Google Scholar), it is established that rotor apparatus of the V0V1-ATPase contains at least three kinds of subunits, i.e. D, F, and proteolipid. Whether other subunits also contribute to make up the rotor awaits further study.
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