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J. Biol. Chem., Vol. 278, Issue 26, 23714-23719, June 27, 2003

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Subunit Rotation of Vacuolar-type Proton Pumping ATPase

RELATIVE ROTATION OF THE G AND c SUBUNITS^*,">

Tomoyuki Hirata  , Atsuko Iwamoto-Kihara ¶, Ge-Hong Sun-Wada , Toshihide Okajima , Yoh Wada  and Masamitsu Futai   ||

From the Division of Biological Sciences, Institute of Scientific and Industrial Research, Osaka University, Osaka 567-0047, the Nanoscience and Nanotechnology Center, Institute of Scientific and Industrial Research, Osaka University and Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Corporation, Ibaraki, Osaka 567-0047, and ^¶Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo 153-8902, Japan

Received for publication, March 18, 2003 , and in revised form, April 1, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vacuolar-type ATPases V₁V₀ (V-ATPases) are found ubiquitously in the endomembrane organelles of eukaryotic cells. In this study, we genetically introduced a His tag and a biotin tag onto the c and G subunits, respectively, of Saccharomyces cerevisiae V-ATPase. Using this engineered enzyme, we observed directly the continuous counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the c subunit. V-ATPase generated essentially the same torque as the F-ATPase (ATP synthase). The rotation was inhibited by concanamycin and nitrate but not by azide. These results demonstrated that the V- and F-ATPase carry out a common rotational catalysis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vacuolar-type ATPase (V-ATPase)¹ pumps protons into the lumens of endomembrane organelles such as vacuoles, lysosomes, endosomes, and synaptic vesicles (1–4). The same enzyme is localized in the plasma membranes of specialized mammalian cells including osteoclasts and kidney intercalated cells (1–4). V-ATPase is a multisubunit complex composed of two functional sectors, V₁ and V₀; membrane peripheral V₁ is responsible for ATP hydrolysis and integral V₀ for proton translocation. In yeast, V₁ and V₀ are composed of eight (A, B, C, D, E, F, G, and H) and five (a, c, c', c'', and d) subunits, respectively (5). The structure and function of the yeast enzyme are conserved by those of animals because yeast mutants are complemented by the animal counterparts including those of human (6), mouse (7–9), and nematode (10).

The general structure of V-ATPase is similar to that of F-ATPase (ATP synthase), which consists of the F₁ (, , , , and ) and F₀ (a, b, and c) sectors. The structure, function, and mechanism of F-ATPase have been studied extensively (11–15). The binding change mechanism suggests that the three catalytic sites of F-ATPase alternate during ATP synthesis and that the ATP releasing step requires energy (16). Protons are transported through a transmembrane pathway formed from the single a subunit and a ring of multiple c subunits. The F-ATPase can also hydrolyze ATP and forms an electrochemical proton gradient. We (13) and Junge and co-workers (17) showed continuous rotation of a complex of and the c ring (c₁₀₁₄) of purified F-ATPase when the ₃₃ hexamer was immobilized (13, 17). On the contrary, the ₃₃ hexamer could rotate when the c ring, either purified (18) or embedded in membranes (14), was immobilized. Finally, rotation of the a subunit relative to the c ring has been shown in membranes (14).

V-ATPase has five more subunits than the basic F-ATPase of bacteria. On the basis of the results of chemical cross-linking studies (19–21) and limited sequence homology (A to , B to , G to b, and c to c, V- to F-ATPase), the V-ATPase subunits correspond to those of the F-ATPase (1, 4). Although the structure is conserved between A and and B and , there are still significant differences between the two enzymes: G does not have the hydrophobic segment that b has, and the V-ATPase c and c' subunits are the duplicated form of F-ATPase c (22); V₀ has three hydrophobic proteolipid subunits (c, c', and c'') (23), whereas F₀ has only one (c). Negatively stained images of bovine coated vesicle V-ATPase revealed a more complicated structure of the secondary stalk connecting V₁ and V₀ (24), possibly consistent with the presence of V-ATPase subunits for which homologues were not found in F-ATPase. V-ATPase shows lower catalytic cooperativity (25) and a lower ratio of H⁺ transported per ATP hydrolyzed (26, 27) when compared with F-ATPase. Thus, the two proton pumps should have unique structures and mechanisms for their distinct physiological roles.

Although the physiological role and structure of the two enzymes are different, it has been proposed that V-ATPase has a rotary mechanism similar to that of F-ATPase (1). To examine this possibility, we first focused on the V-ATPase G subunit because various lines of evidence suggested that the G subunit is located at the peripheral stalk region of the enzyme. The G subunit exhibits homology (24% identity) with F-ATPase b (28), which rotates relative to the c ring. Recently, it has been reported that the G subunit may correspond to the subunit of F-ATPase (20, 21). Assuming that the G subunit rotates relative to the c ring of V-ATPase, we introduced a His tag and a biotin-binding domain to c and G, respectively, of the yeast enzyme. Upon ATP hydrolysis, we observed continuous counter-clockwise rotation of an actin filament connected to the G subunit of V-ATPase.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains, Epitope Tagging, and Culture Conditions—All strains used were derivatives of YPH499 (29), as shown in Table I. The details of the methods used for mutant construction can be found in the supplemental material (http://www.jbc.org). Strain TH60-4C used in rotation observation carries the chromosomal genes for the c subunit with His₆ at the carboxyl terminus and the G subunit fused with a biotin tag (Lys-20 to Leu-124 of the biotin-binding domain of transcarboxylase) (30) at the carboxyl terminus. Cells were grown at 30 °C in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) containing 50 mM succinate and 50 mM potassium phosphate adjusted to pH 5.0 with KOH (31).

Preparation of Vacuolar Vesicles and Solubilization of V-ATPase— Yeast vacuoles, obtained by the method of Takeshige et al. (32), were converted into vesicles in 10 mM MES-Tris, pH 6.9, containing 25 mM KCl and 5 mM MgCl₂. After centrifugation, the vesicles were suspended in the same buffer with 20% glycerol and stored at –80 °C. For solubilization of V-ATPase, the vacuolar vesicles were suspended in buffer (10 mM Tris-HCl, pH 7.5, containing 10% glycerol, 5 mM -mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride) and then centrifuged at 100,000 x g for 10 min. Zwitterionic detergent ZW 3-14 was slowly added to the vacuolar vesicle suspension (5 mg protein/ml) to give a final concentration of 14 mM. After a 10-min incubation, the mixture was centrifuged at 100,000 x g for 60 min, and then the supernatant was rapidly frozen with liquid nitrogen and stored at –80 °C until use.

Western Blot Analysis of the Tagged Subunits—Solubilized vacuolar proteins were denatured at 70 °C for 35 min in 10 mM Tris-HCl, pH 6.8, containing 8 M urea, 5% SDS, 5% -mercaptoethanol, and 1 mM EDTA and then applied to a 0 –20% polyacrylamide gradient gel (for detection of the His-tagged c subunit, a 15% polyacrylamide gel was used) in the presence of SDS. After electrophoresis and blotting onto a nitrocellulose membrane, the following antibodies were used for immunological detection: anti-H⁺-ATPase 69-kDa subunit mouse monoclonal antibody clone 8B1 (1:100 dilution, obtained from Molecular Probe); mouse monoclonal anti-polyhistidine antibody clone His-1 (1:100 dilution, Sigma); and horseradish peroxidase-conjugated goat anti-mouse IgG (1:5000 dilution, Jackson ImmnoResearch Laboratories). Antibodies were used after dilution in TBST (100 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, and 0.05% Tween 20) containing 5% skim milk, except that TBST containing 5% bovine serum albumin was used for the anti-polyhistidine antibody. Signals were detected with an ECL detection system (Amersham Biosciences). The biotin-tagged G subunit was probed with streptavidin-conjugated alkaline phosphatase (Novagen).

Measurement of ATPase Activities and Proton Translocation— ATPase activity and protein concentrations were determined as described previously (7). The formation of an electrochemical proton gradient in vacuolar vesicles (3 µg of protein) was assayed in the buffer used for ATPase with 1 µM 9-amino-6-chloro-2-methoxyacridine as a fluorescence probe (excitation, 410 nm; emission, 490 nm).

Observation of Subunit Rotation—For rotation assay, the solubilized V-ATPase (5 µl of 3 mg/ml protein) was diluted 4-fold with Buffer A (10 mM HEPES-NaOH, pH 7.2, containing 25 mM KCl, 6 mM MgCl₂, and 10 mg/ml bovine serum albumin) and then introduced into a Ni²⁺-nitrilotriacetate-coated glass flow cell at 25 °C (14). After a 5-min incubation, a fluorescent (tetramethyl-rhodamine labeled) actin filament was attached to the G subunit through a biotin tag and streptavidin (14). Immediately after introducing the reaction mixture (5 mM ATP, 0.1 mg/ml asolectin, 0.01% ZW3–14, 25 mM glucose, 1 µM biotin, 1% -mercaptoethanol, 216 µg/ml glucose oxidase, and 36 µg/ml catalase in Buffer A), an 0.6-mm² area was scanned under a Zeiss Axiovert 135 equipped with a cooled, intensified, charge-coupled device camera (CCD; PentaMax-512EFT, Princeton Instruments) (33).

Digital images (0.142 x 0.142 µm/pixel) of an actin filament were acquired at a 10-ms resolution and then subjected to centroid analysis using Winview (Roper Scientific). The rotation angle of a filament (degree) and rotational rate (revolution/s) were calculated from the centroid of the actin filament (34). The length of the filament was taken as the average value obtained from 10 independent images processed with Metamorph (Universal Imaging Corp.). Viscous drag was calculated as (4/3) x x L³/[ln(L/r) – 0.447], where is the viscosity of the medium (1.0 x 10^–9 pN·s·nm^–2), L, the length of the actin filament (700 –1100 nm), and r the radius of the filament (5 nm). Frictional torque (N) was calculated as n = (angular velocity of the filament) x (viscous drag). Angular velocity is equal to 2 x (rotational rate). Continuously rotating filaments, of which the ends were attached to V-ATPase, were used for the torque calculation.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Expression of Epitope-tagged Copies of the c and G Subunits in Yeast—The system we used to examine the rotation of V-ATPase is shown schematically in Fig. 1A. We introduced sequences for the His tag and the biotin-binding domain to the yeast chromosomal genes of the c and G subunits, respectively. The biotin-binding domain-containing proteins could be biotinylated in vivo when expressed in yeast cells (30). It has been shown that the c or G subunit with an epitope tag at the carboxyl terminus can form a functional V-ATPase (28, 35). Consistent with the previous findings, the strain expressing the V-ATPase with the His-tagged c and the biotin-binding domain-fused G could grow at neutral pH similar to the wild type, whereas a null mutant, vma3 (TH57-20A), could not form a colony (Fig. 1B).

View larger version (29K): [in this window] [in a new window]   FIG. 1. Observation system for V-ATPase rotation. A, observation system for V-ATPase rotational catalysis. An actin filament was connected to the G subunit of V-ATPase, which was immobilized on a glass surface through a His tag introduced into the c subunit. Ni-NTA, nickel-nitrilotriacetic acid. B, growth phenotype of the strain expressing the tagged subunits. Growth at pH 7.5 or 5.0 was examined for three strains: TH60-4C, expressing the VMA3-His tag and VMA10-biotin tag (for rotation); TH45-2A, wild type; and TH57-20A, vma mutant with vma3. C, expression of tagged V-ATPase subunits in strain TH60-4C. Solubilized vacuolar proteins were obtained from TH60-4C (lane 1, VMA3-His tag/VMA10-biotin tag) and TH45-2A (lane 2, wild type). The vacuolar proteins (2 µg for the A subunit and biotin tag, or 20 µg for the His tag) were subjected to gel electrophoresis and immobilized on polyvinylidene difluoride (A subunit and biotin tag) or nitrocellulose (His tag) membranes. Proteins were probed with anti-A (Vma1p) V-ATPase subunit antibodies, alkaline-phosphatase (ALP)-conjugated streptavidin, or anti-His tag antibodies.

The stable expression of the two epitope-tagged subunits was examined by Western blot analysis. Solubilized vacuolar proteins were analyzed with anti-His tag antibodies and streptavidin (Fig. 1C). The G subunit with the biotin-binding domain was detected at the position corresponding to a molecular mass of 31 kDa, this value being larger than the predicted value (24 kDa), probably because of the high contents of positively charged residues in the engineered subunit (lysine and arginine, 16.5% of total residues). A signal corresponding to a molecular mass of 18 kDa was detected with anti-His tag antibody, consistent with values for the c subunit connected with the His tag. No signals were detected with the solubilized vacuolar vesicles from wild-type cells (TH45-2A). Expression of the A subunit was confirmed in both strains. These results indicate that a fully active V-ATPase complex with tagged subunits was present in the vacuolar membrane.

Properties of V-ATPase with Tagged Subunits—We found that the amount of the A subunit in the engineered strain (TH60-4C) was about 20% of that observed in wild-type vacuolar membranes (determined by densitometry (data not shown); similar results were obtained with solubilized vacuolar proteins as shown in Fig. 1C), suggesting that the expression level of engineered V-ATPase was lower than that of the wild type. The ATPase activity of the engineered V-ATPase in vacuolar vesicles was about 20% of that observed for the wild type (Table II). Thus, the engineered cells expressed a smaller number of V-ATPase with wild-type activity. The initial rate of proton pumping (fluorescence quenching) was 11% of that of the wild type, and the coupling efficiency (proton pumping activity/ATPase) was slightly lower for the engineered vesicles (Table II). These results suggest that the engineered V-ATPase does not show severe defects in turnover rate or energy coupling, although its expression level in vacuolar membranes is low.

G Subunit Rotation Relative to the c Proteolipid—We observed the rotation of an actin filament connected to the membrane-embedded F-ATPase previously (14). The method used provided an ideal system for studying rotary catalysis in membrane enzyme because it is free from any effect during solubilization and/or purification with a detergent. We examined V-ATPase rotation using vacuolar membrane fragments with or without further sonication. However, the occurrence of rotating filament was significantly low, possibly because of the absence of planar membranes in the preparation. Therefore, we examined the rotation of V-ATPase right after solubilization to avoid possible loss of any subunit(s) from the complex during purification (36, 37).

V-ATPase was introduced into a flow cell and then immobilized on a nickel-nitrilotriacetic acid-coated glass surface through the His tag. An actin filament was attached to the G subunit through the biotin tag and streptavidin. Upon ATP addition, we could readily observe counter-clockwise rotation of the filaments (Fig. 2, A and B). However, no rotating filaments were observed when V-ATPase without the His tag (from strain TH50-5D) or the biotin-binding domain (strain TH44-5D) was introduced into the flow cell (Fig. 2C).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Rotation of an actin filament connected to the G subunit of V-ATPase. A, rotation of an actin filament (1.0 µm) was recorded at a 10-ms resolution time. Images are shown with arrows that indicate schematically the orientation of the filament. A video showing the rotation of an actin filament (30 frames/s) is available in supplemental material (video A). B, rotation of filaments of different lengths connected to the G subunit. Rotation of the filaments (0.8, 1.0, and 1.3 µm) connected to the G subunit was recorded immediately after the addition of 5 mM ATP. C, absence of a rotating filament when V-ATPase without a His tag or biotin tag was used. V-ATPase without the His tag (TH50-5D) or the biotin-binding domain (TH44-5D) was introduced into a flow cell and then examined for rotation. Immediately after the addition of ATP, an 0.6-mm2 area was scanned rapidly to find rotating filaments. The experiments were repeated 12 times in each case, and the total number of rotating filaments is shown.

Inhibition of Rotation by V-ATPase-specific Inhibitors—V-ATPase shows unique anion sensitivities; nitrate is inhibitory, but chloride has no effect (36), whereas both ions have no effect on F-ATPase. The numbers of rotating filaments were calculated in the presence of these ions; the number of rotating filaments decreased with an increase in the nitrate concentration, but chloride had no effect (Fig. 3A). ATPase activity was inhibited similarly with increases in nitrate concentration (Fig. 3B).

View larger version (14K): [in this window] [in a new window]   FIG. 3. Effects of nitrate and chloride on the rotation of an actin filament connected to the V-ATPase G subunit. A, effects of KNO3 and KCl on the rotation of an actin filament connected to the G subunit. The rotation of an actin filament was recorded in the presence of varying concentrations of KNO3 or KCl. KCl was omitted from the assay solution, and 6 mM magnesium gluconate was used instead of MgCl2. The number of rotating filaments was determined as described in Fig. 2C. B, effects of KNO3 and KCl on ATPase activity. V-ATPase activities were assayed in the presence of varying concentration of KNO3 or KCl, and the results are shown as activity relative to ATPase activity without the addition of KNO3 or KCl.

Azide and concanamycin are widely known specific inhibitors of F- and V-ATPases, respectively (34, 38). As expected, the rotation of an actin filament connected to the V-ATPase G subunit was not affected by azide (Fig. 4A), but no rotating filament was observed in the presence of concanamycin (Fig. 4B). The actin filament also stopped rotating after the addition of concanamycin (Fig. 4C). The V-ATPase rotation was inhibited possibly because the antibiotic bound to the V₀ sector (25, 39, 40) or the a subunit (41).

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effects of azide and concanamycin A on V-ATPase rotation. A, effect of azide on the rotation of an actin filament connected to the G subunit. The rotation of actin filaments (1.0 and 1.3 µm) was observed in the presence of 0.5 mM azide. B, rotating filaments in the presence of concanamycin A or azide. Rotating filaments (in the presence of 0.5 mM azide or 0.1 µM concanamycin A) were counted as described in the legend to Fig. 2C. The experiments were repeated 12 times, and the total number of rotating filaments is shown. C, effect of concanamycin A on the rotation of actin filaments (0.8 –1.2 µm). The rotation of actin filaments was followed on the subsecond scale, and 50 µl of the assay mixture containing 0.1 µM concanamycin A was slowly introduced into the flow cell (volume 10 µl) (arrowhead). After standing for 30 s, the rotation was recorded again (red lines). As controls, reaction mixtures without the antibiotic were introduced (blue line).

The lag between rotational steps of each filament was variable, as shown by an expanded time scale (Fig. 5A). Thus, we selected linear segments (Fig. 5A, highlighted sections), and average rates were plotted against viscous drag applied to the filaments. Through these calculations, we estimated that V-ATPase rotation generated a frictional torque of 36 ± 4 pN·nm (Fig. 5B). This value is essentially the same as the torque generated in F₀F₁ (Table III) (13, 14, 18). However, detailed analysis, using a probe other than an actin filament, will be necessary to reach a definite conclusion.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Rotational torque generated by V-ATPase. A, examples of rotational rate estimation. The time courses of filament (0.8 µm) rotation were expanded, and the rates were obtained from the linear segments, shown by green lines. B, effect of viscous drag on the rotational rates of actin filaments connected to the G subunit. The rotation of each filament was recorded, and the average rate for 20 linear segments of the time course was plotted against viscous drag. Colored lines represent the calculated rotational rates of filaments assuming constant torque of 30 (red), 40 (green), and 50 (blue)pN·nm. Other details including the viscosity drag calculation have been described in the text or elsewhere (33).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have demonstrated that V-ATPase carries out rotational catalysis. Using a genetically engineered enzyme with a His-tagged proteolipid c subunit and a biotin-binding domain connected to the G subunit, we could observe directly the counter-clockwise rotation of an actin filament attached to the G subunit when the enzyme was immobilized on a glass surface through the His tag. Thus, the rotary mechanism is well conserved in the two enzymes, although V-ATPase shows significant structural and functional differences from F-ATPase including: 1) a distinct subunit composition (3); 2) lower catalytic cooperativity (25); and 3) a lower ratio of H⁺ transported per ATP hydrolyzed (26, 27).

We introduced a biotin tag to the G subunit for our rotation assay. Subunit G exhibits homology to F-ATPase subunit b (28), which forms a stator (peripheral stalk) together with subunit a, preventing the ₃₃ hexamer from rotating when the c₁₀₁₄ complex is rotating. During the study of V-ATPase rotation, Forgac and co-workers (20, 21) have shown that the G subunit is cross-linked to the residues located at the top portion of the B subunit. Their results suggest that subunits G and E form a peripheral stalk connecting the V₁ and V₀ sectors and that the G subunit is located near the top of V₁ farthest away from the membrane. In F-ATPase, the peripheral stalk contains the subunit, which is located near the top of the entire complex (42). Thus, the G subunit may correspond to the subunit of F₁ and the E subunit to the extra membrane domain of the b subunit. Although the x-ray structure of V-ATPase needs to be determined, various lines of evidence have suggested that the G subunit is located at the peripheral stalk of the enzyme. Therefore, it is reasonable that we could observe rotation of the G subunit relative to the c ring by introducing a probe to this subunit.

A striking difference between V-ATPase and F-ATPase is the presence of three different proteolipid subunits (Vma3p, Vma11p, and Vma16p for c, c', and c'', respectively) within the V₀ sector compared with a single subunit c in F₀. All three proteolipids are required for functional yeast V-ATPase because loss or mutation of any one of them results in a complete loss of V-ATPase activity (23). The specific glutamate residues in the three subunits are essential for proton translocation, similar to Asp-61 of the F-ATPase subunit c (23, 43). It has been shown that each V-ATPase complex contains single copies of both Vma11p and Vma16p and multiple copies of Vma3p (35). The rotation of an actin filament attached to the V-ATPase was similar to one attached to F-ATPase, and no effect of multiple proteolipids was observed. However, it is possible that the difference between the V₀ and F₀ was not observed in the rotation because an actin filament was used as a probe in the assay. The rotational rate observed for an actin filament was significantly lower than that estimated from the enzyme without a probe (36). Thus, the examination V-ATPase rotation with different probes, including gold particles (44), is of interest.

Several lines of evidence have indicated that V-ATPase interacts with microfilaments in mammalian cells (45–47). We have observed that the distribution of the V-ATPase in plasma membrane of osteoclasts was inhibited by cytochalasin D, which is known to depolymerize actin filaments (58). The B1 and B2 isoforms of the B subunit of V-ATPase contain a microfilament binding site in their amino-terminal domain (46). In yeast, it has been shown that actin participates in several intracellular trafficking pathways, and Eitzen et al. (48) have found that actin, bound to the surface of purified yeast vacuoles in the absence of cytosol or cytoskeleton, regulates the last compartment-mixing stage of homotypic vacuole fusion. Thus, we can assume that the actin filament may be an anchor of the A₃B₃ hexamer and that the V₀ c ring rotates together with the central stalk relative to the A₃B₃ hexamer.

Recently, multiple isoforms of V-ATPase subunits in higher eukaryotes have been identified (7–9, 49–52). Because many of them complemented the yeast counterpart, it will be of interest to examine their roles in the rotary mechanism using the present experimental system. Furthermore, the rotation may be affected by mutations causing defective proton translocation (23, 43) or ATPase activity (53, 54). Our rotation assay also constitutes an efficient tool for addressing the arrangement and functions of individual subunits in V-ATPase through characterization of the rotatory mechanism with probes attached to different subunits.

Most recently, Yokoyama and co-workers (55) showed the rotation of an isolated peripheral membrane sector of Thermus thermophilus H⁺-ATPase responsible for ATP synthesis. The bacterial enzyme has been classified into A-type ATPases found in the plasma membranes of most archea and some kinds of eubacteria (for review, see Ref. 56). A-type ATPase is related to V-ATPase but is more similar to F-ATPase (57), although the three ATPases are believed to have the same ancestor. Taken together, our present studies on V-ATPase as well as those on F-ATPase (12–14, 18) and A-ATPase (55) have clearly demonstrated that these three distantly related proton-translocating ATPases carry out common rotational catalysis.

	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.

^"> The on-line version of this article (available at http://www.jbc.org) contains supplemental material.

^|| To whom correspondence should be addressed. Tel.: 81-6-6879-8480; Fax: 81-6-6875–5724; E-mail: m-futai{at}sanken.osaka-u.ac.jp.

¹ The abbreviations used are: V-ATPase, vacuolar H⁺-ATPase; MES, 2-morpholinoethanesulfonic acid; pN, piconewton.

	ACKNOWLEDGMENTS

 
We thank Dr. K. Nishio for helpful discussions and for carrying out the blind test on the rotation analysis. We are also grateful to S. Shimamura, M. Nakajima, and Y. Iko for their assistance.

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