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J. Biol. Chem., Vol. 278, Issue 27, 24255-24258, July 4, 2003

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Rotation of the Proteolipid Ring in the V-ATPase^*

Ken Yokoyama  , Masahiro Nakano ¶, Hiromi Imamura , Masasuke Yoshida  ¶ and Masatada Tamakoshi ||

From the ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan, ^¶Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan, and ^||Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan

Received for publication, March 26, 2003 , and in revised form, April 18, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
V₀V₁-ATPase catalyzes the interconversion of the energy of proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1–3). 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 (1). 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 (4, 5). 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 (1). 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 (5). 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 (6). The A subunit contains a catalytic site, and the A and B subunits are arranged alternately to form a hexameric cylinder (7). The D subunit fills the central cavity of the cylinder and, together with the F subunit, forms a central stalk (7–9). 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 (2, 10, 11). 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 (4, 7). 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 (12).

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 (1, 4, 13). 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 (14). ATP hydrolysis in catalytic sites of F₁ drives rotation of the central stalk subunits, and (15, 16), and this rotation in turn drives the rotation of the F₀c-ring (16–18) that is thought to be directly responsible for proton translocation (19). 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 (1, 20). 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 (21). Here, we show the visual demonstration of ATP-dependent rotation of the proteolipid ring in V₀V₁-ATPase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Purification of Mutant V₀V₁-ATPase—A mutated V₀V₁-ATPase (AHis₈ tags/A-S232A/A-T235S/L-E23C) was constructed by integration vector system (22–24). 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 (24). 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 (22). 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 (25), 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 (6, 26). 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 x 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 x g for 60 min, and the supernatant was applied onto a Ni²⁺-NTA¹-Superflow column (3 x 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 x 36 mm²; top, 18 x 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 ( = 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 (5). 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).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
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 (22–25). T. thermophilus is absolutely aerobic (27), 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 (5). 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 K_m value of 450 ± 80 µM and a k_cat 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 (13, 16, 28). 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 (18). 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.

View larger version (8K): [in this window] [in a new window]   FIG. 1. DCCD sensitivity of the isolated V0V1-ATPase. A, effect of DCCD on ATPase activity. The isolated V0V1-ATPase was incubated with 100 µM DCCD at 25 °C. Open circle, V0V1-ATPase isolated in C12E8; closed circle, V0V1-ATPase isolated in Triton X-100. B, effect of DCCD on ATP-driven H+ translocation. H+ translocation was monitored by fluorescence quenching of ACMA. The addition of ATP (4 mM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (1 µg/ml) is indicated by arrows. Trace 1, V0V1-ATPase vesicles without DCCD treatment; trace 2, V0V1-ATPase vesicles pretreated with 100 µM DCCD for 30 min. Other experimental details are described under "Material and Methods."

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 k_cat 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).

View larger version (43K): [in this window] [in a new window]   FIG. 2. A, schematic illustration of the experimental setup to observe rotation of V0V1-ATPase. A, the V0V1-ATPase was fixed on the glass surface with N-terminal His8 tags of the A subunits. A bead was attached to the proteolipid (L subunit) through biotin-streptavidin linkage. Rotation of an obliquely attached bead was observed. The arrow indicates the observed direction of rotation. Arrangement of some subunits is hypothetic. B, biotinylation of the proteolipid (L subunit). The biotinylated V0V1-ATPases were analyzed with 15% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Left (lanes 1 and 2), protein staining by Coomassie Brilliant Blue; right (lanes 3 and 4), Western blotting stained by alkaline phosphatase-streptavidin conjugate; lanes 1 and 3, biotinylated V0V1-ATPase; lanes 2 and 4; molecular size standards (250, 150, 100, 75, 50, 37, 25, 15, and 10 kDa).

View larger version (22K): [in this window] [in a new window]   FIG. 3. Time 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.

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.

View larger version (10K): [in this window] [in a new window]   FIG. 4. Effect of DCCD on rotation. The V0V1- or V1-ATPase was incubated with 100 µM DCCD for 30 min in 20 mM Tris-Cl (pH 8.0), 100 mM NaCl, 1 mM MgCl2, and 0.05% C12E8, and rotation was examined. After the addition of 4 mM ATP, 0.2-mm2 area was scanned within 5 min to count the number of rotating beads. In control experiments, V0V1- or V1-ATPase was treated in the same procedures with the exception that DCCD was not included. Under these conditions, 2000 beads were found in the area irrespective of samples with or without DCCD treatment. Open bar, V0V1- or V1-ATPase without DCCD treatment; filled bar, V0V1- or V1-ATPase with DCCD treatment.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
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 (16, 18, 29) is avoided. Together with a previous report (21), 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.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Fax: 81-45-922-5239; E-mail: kyokoyama-ra{at}res.titech.ac.jp.

¹ The abbreviations used are: NTA, nitrilotriacetic acid; DCCD, N'N'-dicyclohexylcarbodiimide; C₁₂E₈, octaethylene glycol monododecyl ether; ACMA, 9-amino-6-chloro-2-methoxyacridine.

	ACKNOWLEDGMENTS

 
We thank C. Ikeda for enzyme preparation and assays and H. Ueno and T. Suzuki for critical discussions and technical advice.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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