Subunit arrangement in V-ATPase from Thermus thermophilus.

The V0V1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled with proton translocation. It consists of an ATPase-active V1 part (ABDF) and a proton channel V0 part (CLEGI), but the arrangement of each subunit is still largely unknown. Here we found that acid treatment of V0V1-ATPase induced its dissociation into two subcomplexes, one with subunit composition ABDFCL and the other with EGI. Exposure of the isolated V0 to acid or 8 m urea also produced two subcomplexes, EGI and CL. Thus, the C subunit (homologue of d subunit, yeast Vma6p) associates with the L subunit ring tightly, and I (homologue of 100-kDa subunit, yeast Vph1p), E, and G subunits constitute a stable complex. Based on these observations and our recent demonstration that D, F, and L subunits rotate relative to A3B3 (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; Yokoyama, K., Nakano, M., Imamura, H., Yoshida, M., and Tamakoshi, M. (2003) J. Biol. Chem. 278, 24255-24258), we propose that C, D, F, and L subunits constitute the central rotor shaft and A, B, E, G, and I subunits comprise the surrounding stator apparatus in the V0V1-ATPase.

V 0 V 1 -ATPases (V-ATPases) are the ATPase/ATP synthase superfamily that catalyzes the exchange of the energy between proton translocation across membranes and the energy of ATP hydrolysis/synthesis (1)(2)(3). They are widely distributed in different types of eukaryotic cells and some bacteria (2,4). In eukaryotic cells, V 0 V 1 -ATPases exist in both intracellular compartments and plasma membranes, and are responsible for the acidification of intracellular compartments, renal acidification, born resorption, and tumor metastasis (2). On the other hand, most prokaryotic V 0 V 1 -ATPases produce ATP using the energy of a transmembrane proton electrochemical gradient that is generated by a respiratory chain (4,5).
The overall structure of V 0 V 1 -ATPases is similar to that of F 0 F 1 -ATPases (␣ 3 ␤ 3 ␥ 1 ␦ 1 ⑀ 1 a 1 b 2 c 10Ϫ14 ), which are responsible for ATP synthesis in mitochondria, chloroplast, and plasma membranes of eubacteria (3,6). Both are composed of two functional domains, the peripheral catalytic V 1 or F 1 and a membrane embedded ion translocating domain called V 0 or F 0 .
The structure and subunit arrangements of F 0 F 1 -ATPases are well characterized. The x-ray structure of F 1 revealed a hexamer of alternating ␣ and ␤ surrounding a central cavity containing a highly ␣-helical ␥ subunit (7). The ␥ and ⑀ subunit constituted a central shaft, which directly contacted with the c subunit ring in F 0 (8). The b subunit has a hydrophobic Nterminal domain anchored in the membrane, and a hydrophilic C-terminal domain forms an elongated peripheral stalk that interacts with the F 1 moiety as a stator (9). The a subunit in F 0 , which consists of a stator part together with b subunits, is situated peripherally to the c subunit ring and plays a crucial role in the proton translocation (3,10,11).
Like the F 0 F 1 -ATPases, the peripheral V 1 part contains a catalytic core, which is composed of three copies each of A and B subunits. The A subunit contains a catalytic site, and the A and B subunits are arranged alternately, forming a hexameric cylinder. The D subunit, which fills the central cavity of the A 3 B 3 cylinder, makes up a central shaft with the F subunit (12,13).
The V 0 moiety contains at least two kinds of hydrophobic proteins, proteolipid subunits and 100-kDa subunit. The V 0 V 1 -ATPases from yeast contains three members of the proteolipid family, which are predicted to contain at least four transmembrane helices, and they constitute a hetero-oligomer (14,15). The 100-kDa subunit has a bipartite structure containing a hydrophilic N-terminal domain and a hydrophobic C-terminal domain containing multiple transmembrane helices (16,17). Although no significant sequence homology was found between the 100-kDa subunit and F 0 -a subunit, several lines of evidence have suggested that the 100-kDa subunit might be a functional equivalent to F 0 -a subunit (18 -20). The d subunit (yeast VMA6 products) has been reported as a member of V 0 part (21), although it is a hydrophilic protein.
Based on the functional and structural similarity between V 0 V 1 -ATPases and F 0 F 1 -ATPases, it has been assumed that V 0 V 1 -ATPases would use a similar rotary mechanism as the F 0 F 1 -ATPases (3,6,22). The central shaft composed of ␥ and ⑀ subunits in F 1 are directly associated with the c subunit ring in F 0 (8). Thus, the rotation of the central shaft in turn drives the rotation of the c subunit ring. This movement of the c subunit ring relative to F 0 -a subunit, which is kept fixed to the ␣ 3 ␤ 3 hexamer by a peripheral stalk, is thought to be directly responsible for proton translocation (3,6). Recently, we visualized the rotation of single molecules of V 1 -ATPase, establishing that V 0 V 1 -ATPases functions through a rotary mechanism (12). As with the F 0 F 1 -ATPase, V 1 and V 0 are connected by both central * 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 nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) D63799. and peripheral stalks (2), although the subunit composition of these stalks has not been established.
We have previously identified V 0 V 1 -ATPase in a thermophilic eubacterium, Thermus thermophilus (23,24). The V 0 V 1 -ATPase is capable of both ATP-driven proton translocation and proton-driven ATP synthesis and functions as ATP synthase in vivo (5,23). The T. thermophilus has a simple subunit structure, composed of nine different subunits, A, B, D, F, C, E, G, I, and L, with molecular sizes of 64, 54, 25,12,36,21,13,72, and 8 kDa, respectively 1 (Table I). Although the molecular size of some subunits of T. thermophilus is smaller than that of eukaryotic counterparts, each subunit of T. thermophilus shows a sequence similarity to its eukaryotic counterpart (see Table I). For instance, the I subunit shows an overall sequence similarity to eukaryotic V 0 -a subunit (100-kDa subunit). Although the molecular size of the L subunit is ϳ50% of the eukaryotic V 0 -c subunit (16-kDa proteolipid subunit), the L subunit shows an obvious sequence similarity to the V 0 -c subunit.
The hydrophilic V 1 part of T. thermophilus, which is ATPaseactive and hence called V 1 -ATPase, is made up of four subunits with a stoichiometry of A 3 B 3 D 1 F 1 (23). The G, E, and C subunits are also hydrophilic, but they are not contained in the V 1 (23,24).
Here we report isolation of several subcomplexes of V 0 V 1 -ATPases of T. thermophilus, and we propose subunit arrangement as well as rotor/stator identification in the complex.

Construction of a His 8 -V 0 V 1 -ATPase, V 1 , Expressing T. thermophilus
Strain-A mutant T. thermophilus strain, AH8, in which the atpA gene was replaced with a modified atpA gene encoding a His 8 -tagged A subunit, was constructed as follows. At first, an atpA-his 8 gene was constructed; the cloned atp operon was subjected to a PCR mutagenesis. The mutation primer was 5Ј AAT GGA GGG ACG ATG ATC CAA CAC CAC CAC CAC CAC CAC CAC CAT GGG GTG ATC CAG AAG ATC GCG 3Ј. pUTpyrE, which carries the pyrE gene cassette, was constructed; the XbaI-EcoRI fragment containing the leuB gene of pT8leuB (25) was cloned in pUC119, and then the NdeI-EcoRI fragment was replaced with the NdeI-EcoRI fragment containing the pyrE gene of pINV (26). The sequence corresponding to a 1550-bp region, which is upstream of the atpA gene and includes the termination code of the atpF gene, was amplified with primers InteA5/5/Sph (5Ј-GGGCATGC-GAGGTGGTGAGGAAACTGGCCCTG-3Ј), and InteA5/3/Sal (5Ј-GGTC-GACTACAGCTTGATGTCAAAGCCGATGGTC-3Ј), followed by SphI and SalI digestion. The sequence corresponding to a 1750-bp region containing most parts of the atpA-his gene with its Shine-Dalgarno sequence was amplified with primers InteA/3/5/Eco5 (5Ј-GATATCTA-GAATGGAGGGACGATGATCCAACAC-3Ј) and InteA/3/3/EcoR1 (5Ј-GAATTCCCCCTTTAGGCCAGCCTTGAAGGCCCC-3Ј), followed by EcoRV and EcoRI digestion. These two fragments were cloned in the SphI-SalI and the EcoRV-EcoRI sites of pUTpyrE, respectively. The resultant plasmid pyrE strain, T. thermophilus TTY1 (26), was genetically transformed as described previously in order to insert the pyrE gene as a selective marker and to replace the original atpA gene on the chromosome with the modified atpA gene encoding the His-tagged A subunit (25). Transformants were selected on a minimum-medium plate without uracil. Chromosomal DNA was prepared from a transformed strain, AH8, and integration of the pyrE gene into the site between the atpF gene and the atpA gene was confirmed by Southern blot analysis (data not shown).
Isolation of V 0 V 1 -ATPase, V 1 , and V 0 -The recombinant T. thermophilus was grown as described previously (24). 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 MgCl 2 , and disrupted by sonication. The membranes were precipitated by centrifugation at 100,000 ϫ 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% Triton X-100 (w/v), and then the suspension was sonicated. The debris and insoluble materials were removed by centrifugation at 100,000 ϫ g for 60 min, and the supernatant was applied onto a Ni-NTA superflow column (Qiagen, 3 ϫ 10 cm) equilibrated with 20 mM sodium imidazole (pH 8.0), 0.1 M NaCl, 0.1% Triton X-100. 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 V 0 V 1 -ATPases were analyzed with PAGE in the presence of SDS-PAGE, and then were combined and dialyzed against 20 mM Tris-Cl (pH 8.0), 0.1 mM EDTA, and 0.05% Triton X-100 for 3 h. The dialyzed solution was applied to a Resource Q column (6 ml, Amersham Biosciences) equilibrated with 20 mM Tris-Cl (pH 8.0), 0.1 mM EDTA, and 0.05% Triton X-100. The proteins were eluted with a linear NaCl gradient (0 -0.5 M). The purity of each fraction was analyzed by SDS-PAGE and/or PAGE in the presence of alkyl ether sulfate (Softy 12, LION corp., AES 2 -PAGE, Ref. 24). Each fraction containing V 0 V 1 -ATPases, V 0 , and V 1 was combined and stored at 4°C until use.
Preparation of CL and IEG Subcomplexes from V 0 -The V 0 fraction was dialyzed overnight against acetate buffer, containing 0.1 M sodium acetate (pH 4.0), 0.1 mM EDTA, 5 mM DTT, 0.05% Triton, or urea buffer, containing 8 M urea, 10 mM Tris-Cl (pH 8.0), 0.1 mM EDTA, 5 mM DTT, 0.05% Triton X-100. The dialyzed solution was concentrated with ultrafiltration using Centricon (Millipore Corp.). The concentrated solution was subjected to FPLC with a Superdex HR-200 column (Amersham Biosciences) equilibrated with 50 mM Tris-Cl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 0.05% Triton X-100. The proteins were eluted with 1 The recent versions of the nucleotide sequences, and protein sequences of each subunit have been submitted to GenBank TM . The X subunit was termed as C subunit in that version and in this paper. 2 The abbreviations used are: AES, alkyl ether sulfate (Softy 12, LION); DCCD, dicyclohexylcarbodiimide; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; FPLC, fast protein liquid chromatography; Ni-NTA, nickel-nitrilotriacetic acid; DTT, dithiothreitol. Others-Chemicals used were reagent-grade and purchased from Sigma or Wako Pure Chemicals. Protein concentrations were determined by BCA protein assay (Pierce), and bovine serum albumin was used as the standard. PAGE in the presence of SDS or AES was carried out as described previously (24). The proteins were stained with Coomassie Brilliant Blue.

RESULTS
Purification of His-tagged V 0 V 1 -ATPase-To obtain a large amount of highly purified V 0 V 1 -ATPase from T. thermophilus, His 8 tag was introduced at the N-terminal of atpA with a shuttle integration vector system (25,26,28). The His-tagged V 0 V 1 -ATPase in the membranes was solubilized with Triton X-100 and purified with a Ni-NTA-agarose column. The AES-PAGE analysis revealed that V 0 V 1 -ATPase was the major component in the eluted fractions (Fig. 1a). Typically, ϳ30 mg of V 0 V 1 -ATPase was obtained from 200 g of the recombinant cells. Fig. 1b, the V 0 V 1 -ATPase partially dissociated into V 1 and V 0 during the anion exchange column chromatography. The peak fractions containing each complex were individually subjected to further purification with gel permeation chromatography (Superdex HR-200), and the purified complexes produced single bands on the AES gel electrophoresis (Fig. 2a, left). Fig. 2b shows elution profiles of the V 0 V 1 -ATPase, V 1 , and V 0 . The molecular size of V 0 was estimated to be 350 kDa. SDS-PAGE analysis revealed that the V 0 was composed of I, L, E, G, and C (Fig. 2a, right, lane 3).

Isolation of V 0 Proton Channel Activity-As shown in
The V 0 was reconstituted into liposome in order to examine proton channel activity. As shown in Fig. 3, the lumens of V 0 liposome were rapidly acidified in response to a membrane potential imposed by K ϩ diffusion mediated by valinomycin. Further incorporation of protons was induced by the addition of an uncoupler, FCCP. The prior treatment of the V 0 liposomes with dicyclohexylcarbodiimide (DCCD) resulted in loss of proton translocation. No rapid acidification was observed for simple liposomes without V 0 . The results indicate that the isolated V 0 is a functional DCCD-sensitive proton channel.
IEG and CL Subcomplexes from V 0 -The V 0 fraction was exposed to acidic buffer (pH 4.0) and then applied to gel permeation chromatography. Two new peaks appeared with estimated molecular mass of ϳ250 and ϳ130 kDa (Fig. 4a). Each fraction showed a single band in the AES gel (Fig. 4b, upper  panel). The SDS-PAGE analysis revealed that the 250-kDa complex was composed of subunits I, E, and G, and the 130-kDa complex was composed of subunits C and L (Fig. 4b, lower  panel). These complexes were also obtained from V 0 by treatment with 8 M urea, suggesting that hydrophilic E and G subunits are associated tightly with hydrophobic I subunit and hydrophilic C subunit with the L subunit ring. V 1 -CL Subcomplex-The V 0 V 1 -ATPase was exposed to the low pH acetate buffer and applied to gel permeation chromatography. As shown in Fig. 4a, following this separation, a new peak appeared after the peaks corresponding to V 0 V 1 -ATPase and the IEG subcomplex. AES-PAGE and SDS-PAGE analysis revealed that the complex in the new peak was composed of C, L, A, B, D, and F (Fig. 4b, lower panel). The E, G, and I were not present in the complex (Fig. 4, b and c). This result indicates that the LC subcomplex binds to the central shaft composed of D and F subunits. DISCUSSION The precise arrangement of the subunits in the V 0 V 1 -ATPase remains an important, unclarified issue. In particular, the structure and subunit composition of both central and peripheral stalk have yet to be clarified. In an attempt to obtain insight into the subunit arrangement and function, we have studied the T. thermophilus V 0 V 1 -ATPase, which has a much simpler subunit composition compared with eukaryotic counterpart (Table I).
The V 0 V 1 -ATPase of T. thermophilus partially dissociated into V 0 and V 1 during the ion exchange column chromatography, and they were easily isolated. The V 1 part of T. thermophilus is made up of four different subunits with a stoichiometry of A 3 B 3 D 1 F 1 . The D subunit had been the most probable candidate of rotor subunit in V 1 portion (2). Cross-linking studies have suggested that the D subunit was adjacent to B subunit at central cavity region of A 3 B 3 hexamer, and the F subunit was associated with the D subunit (29,30). In contrast, studies on the V-ATPase from Manduca sexta suggested that subunit E, rather than subunit D, was the rotor subunit (31). Electron microscopic study of Na ϩ -pumping V 0 V 1 -ATPase from Caloramator fervidus also suggested that the E subunit was the Arrow indicates I subunit. The conditions of gel permeation high pressure liquid chromatography, low pH treatment against V 0 or V 0 V 1 -ATPase, and purification of each subcomplex from V 0 V 1 -ATPase were described under "Materials and Methods." rotor (32). We recently demonstrated rotation of both D and F subunits relative to A 3 B 3 in V 1 -ATPase from T. thermophilus, and we established that these two subunits constitute the central shaft (12).
The V 0 moiety of T. thermophilus, which shows proton channel activity, is composed of five different subunits, two typical membrane proteins, subunits I and L, and three hydrophilic subunits, E, G, and C (Fig. 2c). In the rotary mechanism, each subunit should be classified as part of the rotor or the stator part. Subunit I (72 kDa) shows an apparent sequence similarity to yeast VHP1 product, which interacts with the proteolipid ring and also plays a critical role in proton translocation (18). Thus, the I subunit is thought to be functional homologue of F 0 -a, and to constitute the stator part with other subunits. The L subunit is a member of a highly conserved family of hydrophobic subunits, often termed as proteolipid due to their solubility in organic solvents (15). The proteolipid subunit, both in F 0 F 1 -ATPase and V 0 V 1 -ATPase, forms a ring structure and has an essential carboxyl residue involved in proton translocation (2,8). We have demonstrated recently (33) the rotation of L subunit ring relative to A 3 B 3 hexamer, indicating that the L subunit is part of the rotor region along with the D and F subunits.
To address the question of the localization of C, E, and G subunits in V 0 V 1 -ATPase, V 0 V 1 -ATPase or V 0 was exposed to low pH buffer or with 8 M urea to dissociate them into subcom-plexes. The V 0 part can be divided into two complexes, one is composed of subunits E, G, and I, and the other is composed of subunits L and C. The E subunit was predicted to be a highly hydrophilic ␣-helical protein and one of candidates of F 1 -␥ subunit homologue (31,32). Our results are consistent with the cross-linking studies by Arata et al. (29,30) and strongly suggest that the subunit E is a stator subunit, rather than a rotor subunit. The G subunit of T. thermophilus shows significant similarity (ϳ20% identity, overall sequence) to the F 1 -b subunits in F 0 , which constitutes the peripheral stator in F 0 F 1 -ATPase. Indeed, the secondary structure prediction of the subunit G shows the presence of a long hydrophilic ␣-helix at the C-terminal region (data not shown). Tomashek et al. (34,35) indicated that the E (Vma4p) and G (Vma10p) subunits of yeast constitute the EG subcomplex in vivo. Taken together, it is most likely that hydrophilic E and G subunits were associated with hydrophobic I subunit and form the stator part, that is the peripheral stalk.
Unlike T. thermophilus enzyme, both E and G subunits of eukaryote have been proposed to be components of V 1 moiety (1,2). The difference in localization of E and G subunits between T. thermophilus and eukaryotic enzymes after the dissociation procedure might be due to the affinity of E and G subunits to A 3 B 3 DF and/or 100-kDa subunit. It is known that the amount of functional V 0 V 1 -ATPase in a given vacuolar membrane is regulated by reversible dissociation/association of The central stalk is postulated to include C, D, and F subunits, whereas the peripheral stalk includes E and G subunits. The V 0 V 1 -ATPase (holoenzyme) partially dissociated into V 0 (proton channel) and V 1 (ATP-driven motor) during ion exchange column chromatography. The V 0 dissociated into CL subcomplex (V 0 rotor) and IEG subcomplex (stator) by the low pH treatment. The low pH treatment of holoenzyme also induced dissociation of the stator subcomplex from the holoenzyme (left side).

TABLE I
Comparison between V 0 V 1 -ATPase subunits in T. thermophilus, Saccharomyces cerevisiae, and Homo sapience All sequence data of subunits were cited from the on line data bases (Swiss Prot). Identities of amino acid residue between subunits of T. thermophilus V 0 V 1 -ATPase and eukaryotic counterparts were analyzed by PSI-and PHI-BLAST (41 the V 1 and V 0 domain (1,2). For instance, the assembly state of the yeast V-ATPase is post-translationally regulated by glucose in vivo (36,37). The V 0 V 1 -ATPase of M. sexta also shows a similar type of regulation (4,38). In contrast, the V 0 V 1 -ATPase of T. thermophilus functions as an ATP synthase, and no reversible dissociation has been observed for this enzyme. The lower affinity of both E and G subunits to the membrane domain in eukaryotes may be important in the reversible dissociation of the V 1 and V 0 domain. Subunit C, a homologue of Vma6p (or the d subunit) assigned to be the V 0 part in yeast V 0 V 1 -ATPase (21), was also a member of the V 0 part of T. thermophilus. The CL subcomplex was stable against the treatment with 8 M urea, suggesting that the C subunit tightly binds to the L subunit ring. Interestingly, the IEG subcomplex is easily removed from V 0 V 1 -ATPase by low pH treatment, leaving an ATPase active V 1 -CL subcomplex (illustrated in Fig. 5). Based on the electron microscopy studies of subcomplexes with different subunit composition, Chaban et al. (32) suggested that the C subunit of C. fervidus V 0 V 1 -ATPase is a component of the central stalk. The V 1 complex from Methanosarcina mazei was made up from five different subunits, A-D and F (39), and each subunit shows an apparent sequence homology to counterpart of T. thermophilus. Taken together, we propose that the C subunit is part of the central stalk of V 0 V 1 -ATPase with D and F subunits and transmits the torque generated in A 3 B 3 to the ring of the L subunits.
Grü ber et al. (40) analyzed the low resolution structure of the F 1 complex from Escherichia coli and V 1 -ATPase from M. mazei by SAXS, and identified the stalk structure of each complex. The structure of the stalk of the V 1 particle from M. mazei is ϳ84 Å long and 60 Å in diameter, whereas the F 1 particle from E. coli has a significant shorter stalk, being ϳ40 -50 Å long and 50 -53 Å wide. In the x-ray structure of F 1 c 10 of yeast, the height of the stalk is 50 Å (8). On the other hand, the molecular size of subunit D (24 -28 kDa) in V 0 V 1 -ATPase, which is the functional homologue of the F 1 -␥ subunit, is smaller than those of the ␥ subunits (31-35 kDa). It is likely that the D subunit might not be in direct contact with the ring of the L subunits.
In this study, we demonstrated that V 0 V 1 -ATPase of T. thermophilus consists of three parts, the V 1 , which acts as an ATP-driven motor, the V 0 rotor part composed of subunits C and L, and the stator part composed of subunits I, E, and G (Fig. 5). These subcomplexes would also be useful for future structural studies.