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Subunit Arrangement in V-ATPase from Thermus thermophilus

Ken Yokoyama*,‡, Koji Nagata¶, Hiromi Imamura*, Shoji Ohkuma†, MasasukeYoshida.*, £ and Masatada Tamakoshi§

*ATP System Project, ERATO, Japan Science and Technology Corporation, 5800-3Nagatsuta, Midori-ku, Yokohama 226-0026, Japan.¶Department of Applied Biological Chemistry, Graduate School of Agricultural and LifeSciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan†Department of Molecular and Cellular Biology, Faculty of Pharmaceutical Sciences,Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-0934, Japan.£Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta,Midori-ku, Yokohama 226-8503, Japan.§Department of Molecular Biology, Tokyo University of Pharmacy and Life Science,Horinouchi, Hachioji, Tokyo 192-0392, Japan.

Running title; Rotor and Stator Subunits in the V-ATPase.

‡Correspondence address: Fax: +81-45-922-5239, E-mail: kyokoyama-ra@res.titech.ac.jp

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract The VoV1-ATPase of Thermus thermophilus catalyzes ATP synthesis coupled withproton translocation. It consists of an ATPase-active V1 part (ABDF), and a protonchannel Vo part (CLEGI), but to date the arrangement of each subunit is still largelyunknown. Here, we found that acid treatment of VoV1-ATPase induced its dissociationinto two subcomplexes, one with subunit composition ABDFCL and the other with EGI.Exposure of the isolated Vo to acid or 8 M urea also produced two subcomplexes, EGIand CL. Thus, the C subunit (homologue of the d subunit, yeast Vma6p) forms a tightassociation with the L subunit ring, and the E and G subunits constitute a stablecomplex with I (homologue of 100-kDa subunit, yeast Vph1p). Based on theseobservations and our recent demonstration that D, F, and L subunits rotate relative toA3B3 [Imamura et al, (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315, Yokoyamaet al (2003) J. Biol. Chem. 278, 24255-24258], we propose that the C, D, F, and Lsubunits constitute the central rotor shaft and the A, B, E, G and I subunits comprise thesurrounding stator apparatus in the VoV1-ATPase.

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IntroductionVoV1-ATPases (V-ATPases) belong to the ATPase/ATP synthase superfamily which

carry out ATP-dependent proton transport or proton-driven ATP synthesis across themembranes (1-3). They are widely distributed in different types of eukaryotic cellsand some bacteria (2, 4). In eukaryotic cells, VoV1-ATPases exist in both intracellularcompartments and plasma membranes, and are responsible for the acidification ofintracellular compartments, renal acidification, bone resorption, and tumor metastasis(2). On the other hand, most prokaryotic VoV1-ATPases produce ATP using the energyof a transmembrane proton electrochemical gradient, generated by a respiratory chain (4,5).

The overall structure of VoV1-ATPases is similar to that of FoF1-ATPases(a3b3g1d1e1a1b2c10-14), which are responsible for ATP synthesis in mitochondria,chloroplasts and the plasma membranes of eubacteria (3, 6). Both are composed oftwo functional domains, the peripheral catalytic V1 or F1 domain, and a membraneembedded ion translocating Vo or Fo domain.

The structure and subunit arrangements of FoF1-ATPases are well characterized. TheX-ray structure of F1 revealed a hexamer of alternating a and b subunits surrounding acentral cavity containing a mainly a-helical g subunit (7). The g and e subunits makeup a central shaft, which makes direct contact with the c subunit ring in Fo (8). The bsubunit has a hydrophobic N-terminal domain anchored within the membrane, and ahydrophilic C-terminal domain which forms an elongated peripheral stalk that interactswith the F1 domain as a stator (9). The a subunit in Fo, which makes up the statorregion together with b subunits, is situated peripheral to the c subunit ring, and plays acrucial role in proton translocation (3, 10, 11).

Like the FoF1-ATPases, the peripheral V1 domain contains a catalytic core, composedof three copies each of the A and B subunits arranged alternately, forming a hexamericcylinder. The A subunit contains a catalytic site. The D subunit, which fills thecentral cavity of the A3B3 cylinder, makes up a central shaft together with the F subunit(12, 13).

The Vo contains at least two different hydrophobic proteins, proteolipid subunitsand a 100-kDa subunit. The VoV1-ATPases from yeast contains three members of theproteolipid family, predicted to contain at least four transmembrane helices, whichconstitute a hetero-oligomer (14, 15). The 100-kDa subunit has a bipartite structure

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containing a hydrophilic N-terminal domain, and a hydrophobic C-terminal domaincontaining multiple transmembrane helices (16, 17). Although no significant sequencehomology was found between the 100-kDa subunit and the Fo subunit, several lines ofevidence have suggested that the 100-kDa subunit might be a functional equivalent toFo-a subunit (18-20). The d subunit (yeast VMA6 products) has been reported to be apart of the Vo domain (21), although it is a hydrophilic protein. Based on the functional and structural similarity between VoV1-ATPases and FoF1-ATPases, it has been assumed that VoV1-ATPases use a similar rotary mechanism to theFoF1-ATPases (3, 6, 22). The central shaft composed of the g and e subunits of F1 aredirectly associated with the c subunit ring in Fo (8). Thus, the rotation of the centralshaft drives the rotation of the c subunit ring. This movement of the c subunit ringrelative to the Fo-a subunit, which is kept fixed to the a3b3 hexamer by a peripheral stalk,is thought to be directly responsible for proton translocation (3, 6). Recently, wevisualized the rotation of single molecules of V1-ATPase, establishing that VoV1-ATPases functions through a rotary mechanism (12). As with the FoF1-ATPase, V1 andVo are connected by both central and peripheral stalks (2). The subunit composition ofthese stalks has yet to be established. We have previously identified VoV1-ATPase from the thermophilic eubacterium,Thermus thermophilus (23, 24). This VoV1-ATPase is capable of both ATP-drivenproton translocation and proton-driven ATP synthesis, and functions as ATP synthase invivo (5, 23). The T. thermophilus enzyme has a simple domain structure, composed ofnine 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 some of the T.thermophilus subunits are smaller than their eukaryotic counterparts the equivalentsubunits show significant sequence similarity (see Table I). For example, the I subunitshows an overall sequence similarity to eukaryotic Vo-a subunit (100-kDa subunit). Inparticular, A and B subunits are highly conserved among species. The hydrophilic V1 part of T. thermophilus, which is ATPase-active and hence calledV1-ATPase, is made up of four subunits with a stoichiometry of A3B3D1F1 (23). The G,E, and C subunits are also hydrophilic, but these are part of the Vo domain. (23, 24). Here, we report the isolation of several subcomplexes of the VoV1-ATPase from T.thermophilus. The results of this analysis has revealed the subunit arrangement andidentified the rotor/stator subdomain of the VoV1-ATPase from T. thermophilus.

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Materials and MethodsConstruction of a his8 -VoV1-ATPase expressing T. thermophilus strain. ------ A

mutant T. thermophilus strain, AH8, in which the atpA gene was replaced with amodified atpA gene encoding a his8-tagged A subunit, was constructed as follows.First, an atpA-his8 gene was constructed: the cloned atp operon was subjectedmutagenesis by PCR. The mutation primer coding for his8-tag was 5’ AAT GGA GGGACG ATG ATC CAA CAC CAC CAC CAC CAC CAC CAC CAT GGG GTG ATCCAG AAG ATC GCG 3’. pUTpyrE, which carries the pyrE gene cassette, wasconstructed: the XbaI-EcoRI fragment containing the leuB gene of pT8leuB (25) wascloned into pUC119, then the NdeI-EcoRI fragment was replaced with the NdeI-EcoRIfragment containing the pyrE gene of pINV (26). The sequence corresponding to a1550-bp region, upstream of the atpA gene and including the termination code of theatpF gene, was amplified with the primers InteA5/5/Sph (5'-GGGCATGCGAGGTGGTGAGGAAACTGGCCCTG-3'), and InteA5/3/Sal (5'-GGTCGACTACAGCTTGATGTCAAAGCCGATGGTC-3'), followed by SphI and SalIdigestion. The sequence corresponding to a 1750-bp region containing most of theatpA-his gene with its Shine-Dalgarno sequence was amplified with primersInteA/3/5/Eco5 (5'-GATATCTAGAATGGAGGGACGATGATCCAACAC-3') andInteA/3/3/EcoR1 (5'-GAATTCCCCCTTTAGGCCAGCCTTGAAGGCCCC-3'),followed by EcoRV and EcoRI digestion. These two fragments were cloned in theSphI-SalI and the EcoRV-EcoRI sites of pUTpyrE, respectively. T. thermophilus strainTTY1 was genetically transformed with the resultant plasmid as described previously(26), in order to insert the pyrE gene as a selective marker and to replace the originalatpA gene on the chromosome with the modified atpA gene encoding the his-tagged Asubunit (25). Transformants were selected on a minimum-medium plate without uracil.Chromosomal DNA was prepared from a transformed strain, AH8, and integration ofthe pyrE gene into the site between the atpF gene and the atpA gene was confirmed bySouthern blot analysis (data not shown).

Isolation of VoV1-ATPase, V1, and Vo. ---------The recombinant T. thermophilus strainwas grown as described previously (24). The cells (200 g) harvested at log phasegrowth were suspended in 400 ml of 50 mM Tris-Cl (pH 8.0), containing 5 mM MgCl2,and disrupted by sonication. The membranes were separated by centrifugation at 100,

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000 x g for 20 min, and washed with the same buffer twice. The washed membraneswere suspended in 20 mM imidazole-Na (pH 8.0), 0.1 M NaCl, and 10% Triton X-100(w/v), and the suspension sonicated. Debris and insoluble material were removed bycentrifugation at 100, 000 xg for 60 min, and the supernatant was applied onto a Ni-NTA superflow column (QIAGEN, 3 x 10 cm) equilibrated with 20 mM Imidazole-Na(pH 8.0), 0.1 M NaCl, 0.1% Triton X-100. The column was washed with 200 ml ofthe same buffer prior to elution of the bound protein with a linear imidazole gradient (20– 100 mM). The fractions containing the VoV1-ATPases were analyzed withpolyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE), and then combined and dialyzed against 20 mM Tris-Cl (pH 8.0), 0.1 mMEDTA, and 0.05% Triton X-100 for 3 hrs. The dialyzed solution was applied to aResource Q column (6ml, Amersham) equilibrated with 20 mM Tris-HCl (pH 8.0), 0.1mM EDTA, and 0.05% Triton X-100. Protein was eluted with a linear NaCl gradient(0 – 0.5 M). The purity of each fraction was analyzed by SDS-PAGE, and/orpolyacrylamide gel electrophoresis in the presence of alkyl ether sulfate (Softy 12,LION corp., AES-PAGE, ref. 24). Fractions containing VoV1-ATPase, Vo, and V1 werecombined separately and stored at 4˚C until use.

Preparation of CL and IEG subcomplexes from Vo --------- The Vo fraction wasdialyzed overnight against acetate buffer, containing 0.1 M acetate-Na, (pH 4.0), 0.1mM 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 proteinsolution was concentrated by ultrafiltration using a 100-kDa cut-off Centricon filter(Millipore Corp.). The concentrated solution was applied to a Superdex HR-200column (Amersham-Pharmacia) equilibrated with 50 mM Tris-Cl (pH 8.0), 50 mMNaCl, 0.1 mM EDTA, 0.05% Triton X-100 and bound protein eluted with the samebuffer. Each fraction was analyzed by AES-PAGE. Each subcomplex was subjectedto further separation on the Superdex HR-200. The fractions containing eachsubcomplex were combined and stored at 4 ˚C until use.

Preparation of V1-CL subcomplex. ------------- The VoV1-ATPase was dialyzedovernight against acetate buffer containing 0.1 M acetate-Na, (pH 4.0), 0.1 mM EDTA,5 mM DTT, 0.05% Triton X-100. The dialyzed solution was concentrated byultrafiltration, then applied to 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

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analyzed by AES-, and SDS-PAGE. The fractions containing the V1-CL complex werecombined and stored at 4 ˚C until use.

Reconstitution of Vo into liposomes and measurement of proton permeability ofthe liposomes ----- Proteoliposomes containing Vo were reconstituted according to theprocedure by Richard et al. (27). Reconstitution was performed at 25˚C in 25!mMpotassium phosphate buffer (pH 7.3) and 500!mM K2SO4. Unilamellar liposomes wereprepared using phosphatidylcholine (type II, Sigma) by reverse phase evaporation andresuspended at a lipid concentration of 4!mg/ml. Triton X-100 was added to a finalconcentration of 8!mg/ml. Then, 10 µl of Vo solution (5!mg of protein/ml) was addedto 850!µl of the liposome solution. N-Octyl-D-glucopyranoside was added to a finalconcentration of 20!mM, and the mixture was incubated for 5!minutes. Then, pyranine(excitation, 450!nm; emission, 510!nm) was added to the mixture at a finalconcentration of 0.2 µM. The detergent was removed by four successive additions of80!mg/ml washed Bio-beads SM-2 (Bio-Rad). Two milliliters of 25!mM potassiumphosphate buffer (pH 7.3) and 100!mM Na2SO4 were added to 200!µl of the liposomesmixture and incubated for 10 min at 25˚C. Valinomycin was added to the mixture at afinal concentration of 20 µM, then carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) was added to the mixture at a finalconcentration of 0.1 µM.

Others ------Chemicals used were reagent grade and purchased from Sigma orWako Pure Chemicals Ind. ltd. Protein concentrations were determined by the BCAprotein assay (Pierce), using BSA as the standard. Polyacrylamide gel electrophoresisin the presence of SDS or AES was carried out as described previously (24). Theproteins were stained with Coomassie Brilliant Blue.

ResultsPurification of his-tagged VoV1-ATPase.------- To obtain a large amount of

highly purified VoV1-ATPase from T. thermophilus, a his8-tag was introduced at the N-terminal of atpA with a shuttle integration vector system (25, 26, 28). The his-taggedVoV1-ATPase in the membranes was solubilized with Triton X-100, and purified with aNi-NTA agarose column. AES-PAGE analysis revealed that VoV1-ATPase was themajor component in the eluted fractions (Fig. 1a). Typically, ~30 mg of VoV1-ATPasewas obtained from 200 g of the recombinant cells.

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Isolation of Vo with proton channel activity ------- As shown in Fig. 1b, theVoV1-ATPase partially dissociated into V1 and Vo during anion exchange. The peakfractions containing each complex were individually subjected to further purificationwith gel permeation chromatography (Superdex HR-200), and the purified complexesproduced single bands on the AES gel electrophoresis (Fig. 2a, left side). Fig. 2bshows elution profiles of the VoV1-ATPase, V1, and Vo. The molecular size of Vo wasestimated to be 350 kDa. SDS-PAGE analysis revealed that the Vo was composed of I,L, E, G, and C (Fig. 2a, right side, lane 3). The Vo was reconstituted into liposome in order to examine proton channel activity.As shown in Fig. 3, the lumens of the Vo-liposomes were rapidly acidified in responseto a membrane potential imposed by K+ diffusion mediated by valinomycin. Furtherincorporation of protons was induced by the addition of an uncoupler, FCCP. Theprior treatment of the Vo liposomes with dicyclohexylcarbodiimide (DCCD) resulted inloss of proton translocation. No rapid acidification was observed for simple liposomeswithout Vo. The results indicate that the isolated Vo is a functional DCCD sensitiveproton channel.

IEG and CL subcomplexes from Vo --------- The Vo fraction was exposed to anacidic buffer (pH 4.0), and then applied to a gel permeation column. Two new peaksappeared with estimated molecular weights of ~250-kDa and ~130-kDa (Fig. 4a).Each fraction gave a single band on an AES-gel (Fig. 4b, upper panel). SDS-PAGEanalysis revealed that the 250-kDa complex was composed of subunits I, E, and G, andthe 130-kDa complex was composed of subunits C and L (Fig. 4b, lower panel).These complexes were also obtained from Vo by treatment with 8 M urea, suggestingthat the hydrophilic E and G subunits are tightly associated with the hydrophobic Isubunit, and the hydrophilic C subunit with the L subunit ring.

V1-CL subcomplex --------- The VoV1-ATPase was exposed to the low pHacetate buffer and applied to the gel permeation column. As shown in Fig. 4a,following this separation a new peak appeared after the peaks corresponding to VoV1-ATPase and the IEG subcomplex. AES-PAGE and SDS-PAGE analysis revealed thatthe complex in the new peak was composed of the C, L, A, B, D, and F (Fig. 4b, lowerpanel) subunits. The E, G, and I were not present in this complex (Fig. 4b and c).This result indicates that the LC subcomplex binds to the central shaft composed of Dand F subunits.

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DiscussionThe precise arrangement of the subunits in the VoV1-ATPase has to date remained

undetermined. In particular the structure and subunit composition of both the centraland peripheral stalk regions have yet to be clarified. In an attempt to obtain insightinto the subunit arrangement and function, we have studied the T. thermophilus VoV1-ATPase, which has a much simpler subunit composition compared with its eukaryoticcounterpart (Table I). The VoV1-ATPase of T. thermophilus partially dissociates into the Vo and V1 domainsduring anion exchange chromatography, allowing easy isolation. The V1 part of T. thermophilus is made up of four different subunits with a stoichiometry of A3B3D1F1.Cross-linking study have suggested that the D subunit is adjacent to the B subunit at thecentral cavity region of the A3B3 hexamer and the F subunit is associated with the Dsubunit, suggesting the D subunit is rotor (29, 30). In contrast, studies on the VoV1-ATPase from Manduca sexta suggested that subunit E, rather than subunit D, was therotor subunit (31). Electron microscopic study of Na+-pumping VoV1-ATPase fromCaloramator fervidus also suggested that the E subunit was the rotor (32). We recentlydemonstrated rotation of both D and F subunits relative to A3B3 in V1-ATPase from T.thermophilus, establishing that these two subunits constitute the central shaft (12). The Vo moiety of T. thermophilus, which shows proton channel activity, is composedof five different subunits, two typical membrane proteins, subunits I and L, and threehydrophilic subunits, E, G, and C (Fig. 2c). In the rotary mechanism, each subunitshould be classified as part of the rotor or the stator. Subunit I (72-kDa) shows anapparent sequence similarity to yeast VPH1 product, which has been shown to interactwith the proteolipid ring and also plays a critical role in proton translocation (18).Thus, the I subunit is thought to be the functional homologue of Fo-a, and to be part ofthe stator region along with other subunits. The L subunit is a member of a highlyconserved family of hydrophobic proteins, often termed proteolipids, due to theirsolubility in organic solvents (15). The proteolipid subunit, both in FoF1-ATPase andVoV1-ATPase, forms a ring structure and has an essential carboxyl residue involved inproton translocation (2, 8). We have recently demonstrated the rotation of the Lsubunit ring relative to the A3B3 hexamer, indicating that the L subunit is part of therotor region along with at least the D and F subunits (33).

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To address the question of the localization of the C, E, and G subunits in VoV1-ATPase, VoV1-ATPase or Vo were exposed to low pH buffer or 8 M urea to causedissociation into subcomplexes. The Vo part can be divided into two complexes, onecomposed of subunits E, G, and I, and the other composed of subunits L and C. The Esubunit is predicted to be a highly hydrophilic a-helical protein, and a possible F1-gsubunit homologue (31, 32). Our results are consistent with cross-linking studies byArata et al (29, 30), and strongly suggest that the subunit E is a stator subunit, ratherthan a rotor one. The G subunit of T. thermophilus shows significant similarity (~20%identity, overall sequence) to the Fo-b subunit, which constitutes the peripheral stator inFoF1-ATPase. Indeed, secondary structure prediction of the subunit G shows thepresence of a long hydrophilic a-helix at the C-terminal region (data not shown).Tomashek et al indicated that the E (Vma4p) and G (Vma10p) subunits of the yeastconstitute the EG subcomplex in vivo (34, 35). Taken together, it is most likely thatthe hydrophilic E and G subunits are associated with the hydrophobic I subunit andform the stator region, that is, the peripheral stalk. Unlike the T. thermophilus enzyme, both the E and G subunits of the eukaryoticenzyme have been proposed to be components of the V1 domain (1, 2). The differencein localization of E and G subunits between T. thermophilus and eukaryotic enzymesafter dissociation might be due to the affinity of the E and G subunits for A3B3DF and/orthe 100-kDa subunit. It is known that the amount of functional VoV1-ATPase in agiven vacuolar membrane is regulated by reversible dissociation/association of the V1

and Vo domain (1, 2). For instance, the assembly state of the yeast V-ATPase is post-translationally regulated by glucose in vivo (36, 37). The VoV1-ATPase of Manducasexta also shows a similar type of regulation (4, 38). In contrast, the VoV1-ATPase of T.thermophilus functions as an ATP synthase, and no reversible dissociation has beenobserved for this enzyme. The lower affinity of both the E and G subunits for themembrane domain in eukaryote may be important for this reversible dissociation of theV1 and Vo domain. Subunit C, a homologue of Vma6p (or the d subunit) assigned to be Vo part in yeastVoV1-ATPase (21), is also a part of the Vo domain of T. thermophilus. The CLsubcomplex was stable against treatment with 8 M Urea, suggesting a tight interactionbetween the C subunit and the ring of L subunits. Interestingly, the IEG subcomplex iseasily removed from VoV1-ATPase by low pH treatment, leaving ATPase active V1-CL

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subcomplex (illustrated in Fig.5). Based on the electron microscopy studies ofsubcomplexes with different subunit composition, Chaban et al. suggested that the Csubunit of C. fervidus VoV1-ATPase is a component of the central stalk (32). The V1

complex from Methanosarcina mazei has been shown to be made up of five differentsubunits, A, B, C, D and F (39), and each subunit shows an apparent sequencehomology to the counterpart of T. thermophilus. Taken together, we propose that the Csubunit is part of the central stalk of VoV1-ATPase with D and F subunits, and transmitsthe torque generated in A3B3 to the ring of L subunits. Grüber et al. analyzed the low resolution structure of the F1 complex from E. coli, andV1–ATPase from M. mazei by SAXS (40), and identified the stalk structure of eachcomplex. The structure of the stalk of the V1 particle from M. mazei is approximately84 Å long and 60 Å in diameter, whereas the F1 particle from E. coli has a significantshorter stalk, being approximately 40 – 50 Å long and 50-53 Å wide. In the X-raystructure of F1c10 of yeast, the height of the stalk is 50 Å (8). In contrast, themolecular size of subunit D (24-28 kDa) of VoV1-ATPase, which is the functionalhomologue of the F1-g subunit, is smaller than those of the g subunit (31-35 kDa). It islikely that the D subunit might not be in direct contact with the ring of L subunits.

In this study, we demonstrated that VoV1-ATPase of T. thermophilus consists ofthree parts, the V1, which acts as ATP driven motor, the Vo rotor part composed ofsubunits C and L, and the stator part composed of subunits I, E, and G (Fig. 5). Thesesubcomplexes may be a useful alternative to the whole complex for future structuralstudies.

Acknowledgements

We thank C. Ikeda, for culture and enzyme preparation and assays, and Y. Akabane fortechnical advices, and B. Bernadette for critical assessment of the manuscript.

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and Müller, V. (2001) Biochemistry 40, 1890-1896

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40. Grüber, G., Radermacher, M., Ruiz, T., Godovac-Zimmermann, J., Canas, B.,Kleine-Kohlbrecher, D., Huss, M., Harvey, W. R., and Wieczorek, H. (2000)Biochemistry 39, 8609-8616

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Table I. Comparison between VoV1-ATPase subunits in T. thermophilus, Saccharomycescerevisiae and Homo sapience. All sequence data of subunits were cited from the onlinedatabases (SWISS PLOT). Identities of amino acid residue between subunits of T.thermophilusVoV1-ATPase and eukaryotic counterparts were analyzed by PSI- and PHI-BLAST (41).

yeastV-ATPase

Identities betweenTth V and yeast V

T. thermophilusV-ATPase

Identities betweenTth V and human V

HumanV-ATPase

A (70 kDa) 50% A (64 kDa) 50% A (68 kDa)B (58 kDa) 55% B (54 kDa) 55% B (57 kDa)D (28 kDa) 28% D (25 kDa) 28% D (28 kDa)F (13 kDa) 21% F (12 kDa) 16% F (13 kDa)d (40 kDa) 17% C (36 kDa) 16% d (40 kDa)E (26 kDa) 16% E (21 kDa) 20% E (26 kDa)G (13 kDa) 22% G (13 kDa) 13% G (14 kDa)a (96 kDa) 16% I (72 kDa) 16% a (96 kDa)c (16 kDa) 37% L (8 kDa) 37% c (16 kDa)c' (17 kDa) c' (17 kDa)c'' (23 kDa) c'' (23 kDa)H (54 kDa) H (55 kDa)C (44 kDa) C (44 kDa)

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FOOTNOTES

1The recent versions of the nucleotide sequences, and protein sequences of eachsubunit have been submitted to GenBank with accession number D63799. The Xsubunit was termed C subunit in the submitted sequence and in this paper.

The abbreviations used are: SDS-PAGE, polyacrylamide gel electrophoresis in thepresence of sodium dodecylsulfate; AES-PAGE, polyacrylamide gel electrophoresis inthe presence of alkyl ether sulfate (Softy 12, LION); DCCD,dicyclohexylcarbodiimide; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone.

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Figure legends

Fig. 1. Purification of VoV1-ATPase and Vo. a, 6% AES-PAGE of the VoV1-ATPase.

lane 1, 100 µg of solubilized fraction from the washed membranes of recombinant T.thermophilus. lane 2, 20 µg of VoV1-ATPase purified by Ni-NTA columnchromatography. lane 3, 20 µg of VoV1-ATPase from the washed membranes of wildtype T. thermophilus. lane 4, 20 µg of V1 from wild type T. thermophilus. Purificationof the wild type V1 and VoV1-ATPase is described in Ref. 24. b, Elution profile from

ion exchange chrmotagraphy. The eluted complexes were monitored by absorption at280 nm.. Each fraction contained 2 ml of eluate. c, 6% AES-PAGE of each fraction.Each fraction (20 µl) was applied to a 6% acrylamide gel containing AES. Fig. 2. Purified VoV1-ATPase, V1, and Vo. a, left; 6% AES-PAGE. right; 18%

SDS-PAGE. Complexes were visualized by CBB-R staining. lane 1, 20 µg of VoV1-ATPase. lane 2, 20 µg of V1. lane 3, 20 µg of Vo . b, Elution profiles of VoV1-ATPase,V1, and Vo from gel permeation HPLC. Fig. 3. Proton permeability of Vo liposomes. Acidification inside the liposomeswas measured by quenching of fluorescence of pyranin as described in Materials andMethods. 1, reconstituted Vo liposomes. 2, liposomes (control). 3, DCCD (2 µl of 10mM solution in ethanol) was added to 200 µl of Vo liposome mixture, then incubated for10 min at 25 ˚C. Proton permeability of Vo liposomes treated with DCCD wasmeasured by the same method. Further incorporations of protons into the liposomeswere measured by the addition of FCCP.Fig. 4. Subcomplexes from holoenzyme. a, Elution profiles of subcomplexes fromgel permeation FPLC. 1, VoV1-ATPase. 2, Vo. 3, Vo dialyzed with acetate buffer (pH4.0) overnight. 4, VoV1-ATPase dialyzed with acetate buffer (pH 4.0) overnight. b,upper; 6% AES-PAGE. lower; 18% SDS-PAGE. lane 1, 25 µg of VoV1-ATPase.lane 2, 20 µg of V1–CL complex. lane 3, 20 µg of V1. lane 4, 20 µg of Vo. lane 5,10 µg of CL complex. lane 6, 10 µg of IEG complex. c, 12 % SDS-PAGE. lane 1,25 µg of VoV1-ATPase. lane 2, 20 µg of V1. lane 3, 20 µg of V1–CL complex. lane 4,20 µg of Vo. Arrow indicates I subunit.Fig. 5. Structural model of VoV1-ATPase and subcomplexes. The model shows themost probable subunit arrangement in VoV1-ATPase of T. thermophilus. The centralstalk is postulated to include the C, D, and F subunits, whereas the peripheral stalk

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includes the E and G subunits. The VoV1-ATPase (holoenzyme) partially dissociatedinto Vo (proton channel) and V1 (ATP-driven motor) during ion exchange columnchromatography. The Vo dissociated into CL subcomplex (Vo rotor), and IEGsubcomplex (stator) under low pH treatment. The low pH treatment of theholoenzyme also induced dissociation of the stator subcomplex from the holoenzyme(left side).

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Masatada TamakoshiKen Yokoyama, Koji Nagata, Hiromi Imamura, Shoji Ohkuma, Masasuke Yoshida and

Subunit arrangement in V-ATPase from thermus thermophilus

published online August 11, 2003J. Biol. Chem. 

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