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J. Biol. Chem., Vol. 278, Issue 43, 42686-42691, October 24, 2003
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**
From the
ATP System Project, ERATO, Japan Science and Technology Corp., 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, the ¶Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, the ||Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, **Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, and the Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received for publication, June 4, 2003 , and in revised form, July 28, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The overall structure of V0V1-ATPases is similar to that of F0F1-ATPases (
3
3
1
1
1a1b2c10-14), 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 V1 or F1 and a membrane embedded ion translocating domain called V0 or F0.
The structure and subunit arrangements of F0F1-ATPases are well characterized. The x-ray structure of F1 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 F0 (8). The b subunit has a hydrophobic N-terminal domain anchored in the membrane, and a hydrophilic C-terminal domain forms an elongated peripheral stalk that interacts with the F1 moiety as a stator (9). The a subunit in F0, 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 F0F1-ATPases, the peripheral V1 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 A3B3 cylinder, makes up a central shaft with the F subunit (12, 13).
The V0 moiety contains at least two kinds of hydrophobic proteins, proteolipid subunits and 100-kDa subunit. The V0V1-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 F0-a subunit, several lines of evidence have suggested that the 100-kDa subunit might be a functional equivalent to F0-a subunit (18-20). The d subunit (yeast VMA6 products) has been reported as a member of V0 part (21), although it is a hydrophilic protein.
Based on the functional and structural similarity between V0V1-ATPases and F0F1-ATPases, it has been assumed that V0V1-ATPases would use a similar rotary mechanism as the F0F1-ATPases (3, 6, 22). The central shaft composed of and subunits in F1 are directly associated with the c subunit ring in F0 (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 F0-a subunit, which is kept fixed to the 33 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 V1-ATPase, establishing that V0V1-ATPases functions through a rotary mechanism (12). As with the F0F1-ATPase, V1 and V0 are connected by both central and peripheral stalks (2), although the subunit composition of these stalks has not been established.
We have previously identified V0V1-ATPase in a thermophilic eubacterium, Thermus thermophilus (23, 24). The V0V1-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, respectively1 (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 V0-a subunit (100-kDa subunit). Although the molecular size of the L subunit is 
50% of the eukaryotic V0-c subunit (16-kDa proteolipid subunit), the L subunit shows an obvious sequence similarity to the V0-c subunit.
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The hydrophilic V1 part of T. thermophilus, which is ATPase-active and hence called V1-ATPase, is made up of four subunits with a stoichiometry of A3B3D1F1 (23). The G, E, and C subunits are also hydrophilic, but they are not contained in the V1 (23, 24).
Here we report isolation of several subcomplexes of V0V1-ATPases of T. thermophilus, and we propose subunit arrangement as well as rotor/stator identification in the complex.
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MATERIALS AND METHODS |
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Isolation of V0V1-ATPase, V1, and V0—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 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% Triton X-100 (w/v), and then 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 Ni-NTA superflow column (Qiagen, 3 x 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 V0V1-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., AES2-PAGE, Ref. 24). Each fraction containing V0V1-ATPases, V0, and V1 was combined and stored at 4 °C until use.
Preparation of CL and IEG Subcomplexes from V0—The V0 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 the same buffer, and each fraction was analyzed by AES-PAGE. Each subcomplex was subjected to re-chromatography by FPLC with the Superdex HR-200. The fractions containing each subcomplex were combined and stored at 4 °C until use.
Preparation of V1-CL Subcomplex—The V0V1-ATPase 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 X-100. The dialyzed solution was concentrated by ultrafiltration and then subjected to FPLC with the Superdex HR-200 equilibrated with 50 mM Tris-Cl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, and 0.05% Triton X-100. The fractions were analyzed by AES- and SDS-PAGE. The fractions containing the V1-CL complex were combined and stored at 4 °C until use.
Reconstitution of V0 into Liposomes and Measurement of Proton Permeability of the Liposomes—Proteoliposomes containing V0 were reconstituted according to the procedure by Richard et al. (27). Reconstitution was performed at 25 °C in 25 mM potassium phosphate buffer (pH 7.3) and 500 mM K2SO4. Unilamellar liposomes were prepared by reverse phase evaporation using phosphatidylcholine (type II, Sigma) and resuspended at a lipid concentration of 4 mg/ml. Triton X-100 was added to a final concentration of 8 mg/ml. Then 10 µl of V0 solution (5 mg of protein/ml) was added to 850 µl of the liposome solution. N-Octyl-D-glucopyranoside was added to a final concentration of 20 mM, and the mixture was incubated for 5 min. Then pyranine (excitation, 450 nm; emission, 510 nm) was added to the mixture at a final concentration of 0.2 µM. The detergent was removed by four successive additions of 80 mg/ml washed Bio-Beads SM-2 (Bio-Rad). Two milliliters of 25 mM potassium phosphate buffer (pH 7.3) and 100 mM Na2SO4 were added to 200 µl of liposome solutions and incubated for 10 min at 25 °C. Valinomycin was added to the mixture at a final concentration of 20 µM, and then carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to the mixture at a final concentration of 0.1 µM.
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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30 mg of V0V1-ATPase was obtained from 200 g of the recombinant cells.
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Isolation of V0 Proton Channel Activity—As shown in Fig. 1b, the V0V1-ATPase partially dissociated into V1 and V0 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 V0V1-ATPase, V1, and V0. The molecular size of V0 was estimated to be 350 kDa. SDS-PAGE analysis revealed that the V0 was composed of I, L, E, G, and C (Fig. 2a, right, lane 3).
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The V0 was reconstituted into liposome in order to examine proton channel activity. As shown in Fig. 3, the lumens of V0 liposome were rapidly acidified in response to a membrane potential imposed by K+ diffusion mediated by valinomycin. Further incorporation of protons was induced by the addition of an uncoupler, FCCP. The prior treatment of the V0 liposomes with dicyclohexylcarbodiimide (DCCD) resulted in loss of proton translocation. No rapid acidification was observed for simple liposomes without V0. The results indicate that the isolated V0 is a functional DCCD-sensitive proton channel.
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IEG and CL Subcomplexes from V0—The V0 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 250 and 130 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 V0 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.
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V1-CL Subcomplex—The V0V1-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 V0V1-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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The V0V1-ATPase of T. thermophilus partially dissociated into V0 and V1 during the ion exchange column chromatography, and they were easily isolated. The V1 part of T. thermophilus is made up of four different subunits with a stoichiometry of A3B3D1F1. The D subunit had been the most probable candidate of rotor subunit in V1 portion (2). Cross-linking studies have suggested that the D subunit was adjacent to B subunit at central cavity region of A3B3 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+-pumping V0V1-ATPase from Caloramator fervidus also suggested that the E subunit was the rotor (32). We recently demonstrated rotation of both D and F subunits relative to A3B3 in V1-ATPase from T. thermophilus, and we established that these two subunits constitute the central shaft (12).
The V0 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 F0-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 F0F1-ATPase and V0V1-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 A3B3 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 V0V1-ATPase, V0V1-ATPase or V0 was exposed to low pH buffer or with 8 M urea to dissociate them into subcomplexes. The V0 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 F1- 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 (20% identity, overall sequence) to the F1-b subunits in F0, which constitutes the peripheral stator in F0F1-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 V1 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 A3B3DF and/or 100-kDa subunit. It is known that the amount of functional V0V1-ATPase in a given vacuolar membrane is regulated by reversible dissociation/association of the V1 and V0 domain (1, 2). For instance, the assembly state of the yeast V-ATPase is post-translationally regulated by glucose in vivo (36, 37). The V0V1-ATPase of M. sexta also shows a similar type of regulation (4, 38). In contrast, the V0V1-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 V1 and V0 domain.
Subunit C, a homologue of Vma6p (or the d subunit) assigned to be the V0 part in yeast V0V1-ATPase (21), was also a member of the V0 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 V0V1-ATPase by low pH treatment, leaving an ATPase active V1-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 V0V1-ATPase is a component of the central stalk. The V1 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 V0V1-ATPase with D and F subunits and transmits the torque generated in A3B3 to the ring of the L subunits.
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Grüber et al. (40) analyzed the low resolution structure of the F1 complex from Escherichia coli and V1-ATPase from M. mazei by SAXS, and identified the stalk structure of each complex. The structure of the stalk of the V1 particle from M. mazei is 84 Å long and 60 Å in diameter, whereas the F1 particle from E. coli has a significant shorter stalk, being 40-50 Å long and 50-53 Å wide. In the x-ray structure of F1c10 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 V0V1-ATPase, which is the functional homologue of the F1- 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 V0V1-ATPase of T. thermophilus consists of three parts, the V1, which acts as an ATP-driven motor, the V0 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.
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FOOTNOTES |
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* 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 recent versions of the nucleotide sequences, and protein sequences of each subunit have been submitted to GenBankTM. 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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