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Originally published In Press as doi:10.1074/jbc.M608253200 on October 18, 2006

J. Biol. Chem., Vol. 281, Issue 50, 38582-38591, December 15, 2006
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Reconstitution in Vitro of V1 Complex of Thermus thermophilus V-ATPase Revealed That ATP Binding to the A Subunit Is Crucial for V1 Formation*

Hiromi Imamura{ddagger}1, Saeko Funamoto{ddagger}, Masasuke Yoshida{ddagger}§, and Ken Yokoyama{ddagger}§2

From the {ddagger}ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Agency (JST), 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan and the §Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan

Received for publication, August 29, 2006 , and in revised form, October 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar-type H+-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V1 complex, responsible for ATP hydrolysis, and a membrane-embedded Vo complex, responsible for proton translocation. The V1 complex of Thermus thermophilus V-ATPase has the subunit composition of A3B3DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V1 complex by reconstituting the V1 complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V1 complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A1B1 and A3B3 subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V1 complex. Kinetic analysis of assembly of the A and B monomers into the A1B1 heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acidification of intracellular acidic compartments of eukaryotic cell, such as lysosomes, endosomes, or synaptic vesicles is important for various cellular processes, including receptor-mediated endocytosis and secondary transport, and is accomplished by ATP-driven proton translocation of the vacuolar-type H+-ATPases (V-ATPases),3 which is localized in the membranes of these compartments (1-3). V-ATPases are also found in the plasma membrane of some specialized cells, such as macrophages (4), renal intercalated cells (5), or osteoclasts (6), and transport proton from the cytosol to the extracellular space. It is known that the V-ATPases in the plasma membrane is also responsible for various cellular functions such as acidification of extracellular space or homeostasis of pH in the cytosol (1-3). V-ATPases are large protein complexes consisting of two major parts, a peripheral V1 complex and a membrane-embedded Vo complex. V1 and Vo are responsible for ATP hydrolysis and proton translocation, respectively. Proton translocation at Vo is accompanied by rotation of the membrane-spanning c-ring, which is driven by ATP hydrolysis at V1 (7-10). In addition to the role in proton translocation, it was recently suggested that Vo complex is directly involved in membrane fusion events (11, 12). Homologues of eukaryotic V-ATPases have also been found in the plasma membranes of some bacteria (10, 13, 14). Although the bacterial V-ATPases have a simpler subunit composition and function as an ATP synthase or Na+ pump rather than a proton pump, all subunits have significant homology with eukaryotic counterparts (10, 15).

It is known that most of the V-ATPase subunits are essential for formation of a functional VoV1 complex in vivo (1). When any of the genes encoding V-ATPase subunits, except H subunit, are deleted, formation of V1,Vo, and/or VoV1 complexes is inhibited. Assembly of V-ATPase subunits also requires other proteins or compounds. For example in yeast, it is known that Vma12p/Vma21p/Vma22p proteins, which are not final components of the V-ATPase, aid the assembly of Vo subunits in the endoplasmic reticulum (16, 17), and that a protein complex, RAVE, which binds to the free cytosolic V1 complex, plays a role in association of V1 and Vo (18, 19). In addition, it has been suggested that under both in vitro (20) and in vivo (21) conditions, binding of ATP to the A subunit is important for assembly. Another important feature of assembly of V-ATPase components is that proton translocation by V-ATPase is regulated by reversible association/dissociation of V1 and Vo complexes (22). The proton translocation activity of the V-ATPase is completely abolished by the dissociation of the V1 complex from the Vo complex.

Here, we report in vitro reconstitution of the V1 complex of Thermus thermophilus from its components. The V1 complex of T. thermophilus V-ATPase is composed of 8 subunits with a subunit composition of A3B3DF (15). The A and B subunits form a hexameric A3B3 ring, in which A and B are arranged alternately, and the D and F subunits constitute a central rotor shaft, which penetrates into the cavity of the A3B3 ring. We investigated the role of ATP in V1 formation in detail, and found that ATP binding to the A subunit is a crucial step for assembly of the V1 components into the complex. Furthermore, potential intermediate subcomplexes were identified. We also describe a model for the assembly of the V1 complex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—ATP, ADP, and bovine serum albumin were products of Sigma. AMP-PNP and restriction endonucleases were purchased from Roche. N-Methylanthraniloyl ADP (mant-ADP) was purchased from Invitrogen. Cy3-maleimide and Cy5-maleimide were from GE Healthcare. Other reagents were from Wako Pure Chemicals (Osaka, Japan), unless otherwise stated.

Expression and Purification of Proteins—Expression of the T. thermophilus V1 complex (A3B3DF) in Escherichia coli containing a His10 tag at the N terminus of the A subunit was performed as described previously (23). The E. coli cells were suspended in 0.1 M sodium phosphate, pH 8.0, 20 mM imidazole-HCl, 0.3 M NaCl and disrupted by sonication, followed by heat treatment at 65 °C for 30 min. After removal of denatured E. coli proteins by centrifugation at 19,000 x g for 60 min, the supernatant was applied to a nickel-nitrilotriacetic acid Superflow column (Qiagen), which was then washed thoroughly and eluted with 0.1 M sodium phosphate, pH 8.0, 0.2 M imidazole-HCl, 0.3 M NaCl. The buffer was switched to 20 mM Tris-HCl, pH 8.0, containing 1 mM EDTA by ultrafiltration (AmiconUltra, Millipore) and the solution was applied to a UNO-Q column (Bio-Rad). Proteins were eluted with a linear gradient of NaCl, from 0 to 400 mM. V1 and the A subunit were eluted in different fractions and were stored at 4 °C. The B subunit of T. thermophilus was expressed in E. coli strain BL21(DE3)-CodonPlus-RP (Stratagene) harboring a plasmid pHis-B, in which genes for the B subunits are inserted between the NdeI and EcoRI sites of pET17b (Novagen). A His6 tag was introduced at the N terminus of the B subunit. The B subunit was purified basically by the same procedure used for V1 and stored at 4 °C. The DF subcomplex of T. thermophilus was expressed in E. coli strain BL21-CodonPlus-RP harboring a plasmid pUC-DF, in which genes for the D and F subunits were inserted tandemly between the EcoRI and SalI sites of pUC18. A His6 tag was fused to the C terminus of the F subunit. The E. coli cells were suspended in 0.1 M sodium phosphate, pH 8.0, 20 mM imidazole-HCl, 0.3 M NaCl and disrupted by sonication, followed by centrifugation at 27,000 x g for 90 min. The supernatant was applied to a nickel-nitrilotriacetic acid Superflow column, which was then washed thoroughly with 0.1 M sodium phosphate, pH 8.0, 60 mM imidazole-HCl, 0.3 M NaCl, 0.05% dodecyl maltoside and eluted with 0.1 M sodium phosphate, pH 8.0, 0.2 M imidazole-HCl, 0.3 M NaCl, 0.05% dodecyl maltoside. Fractions containing DF were pooled and stored at 4 °C. Protein concentration of the V1 complex, the A and B subunits, were determined from UV absorbance calibrated by quantitative amino acid analysis using molar extinction coefficient of 360,000, 82,000, and 42,000 M-1 cm-1, respectively. The protein concentration of the A3B3 subcomplex was determined by the same method assuming that it has the same extinction coefficient as V1. Protein concentrations of the DF subcomplex were estimated with SDS-PAGE by comparing density of D subunit with that of known concentrations of V1.

Gel-permeation Chromatography—Gel-permeation chromatography (Superdex HR 200, Amersham Biosciences) was used to analyze assembly and disassembly, and to purify the A3B3 subcomplex. The column was equilibrated with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl. The flow rate was 0.5 ml/min. Molecular weight standards were purchased from Sigma.

Fluorescent Labeling of Proteins—To label the A and B subunits with a fluorescent dye, Glu114 or Glu374 of the A subunit and Thr107 or Lys304 of the B subunit were replaced with cysteine. All intrinsic cysteine residues were changed to serine or alanine (A-Cys28, A-Cys508, and B-Cys268 to Ser, and A-Cys255 to Ala). The A and B subunits, each containing the single introduced cysteine, were purified, and then incubated with 10 mM dithiothreitol for 30 min to reduce the sulfhydryl group of cysteine. After removal of dithiothreitol by gel permeation chromatography equilibrated with 20 mM MOPS-NaOH, pH 7.0, 150 mM NaCl, proteins were incubated with 5-fold molar excess of Cy3-maleimide (A subunit) or Cy5-maleimide (B subunit) at room temperature for 2 h. Non-reacted fluorescent dye was removed with NAP-10 (GE Healthcare) and Superdex HR200. The labeling ratios of dye to protein were 0.85, 0.89, 0.69, and 0.72, respectively, for AE114C-Cy3, AE374C-Cy3, BT107C-Cy5, and BK304C-Cy5 as estimated by spectrophotometry. The molar extinction coefficients used were 150,000 M-1 cm-1 at 550 nm (Cy3) and 250,000 M-1 cm-1 at 650 nm (Cy5).

FRET Measurement between A and B Subunits—FRET from the donor (Cy3) in the A subunit to the acceptor (Cy5) in the B subunit was monitored with a fluorescence spectrometer (FP-6500, JASCO). To acquire fluorescent spectra, the donor was selectively excited with light at 532 nm (bandwidth 10 nm) and the fluorescence intensity from 550 to 700 nm was scanned (bandwidth 3 nm). For time course measurements, the donor was excited with light at 532 nm (bandwidth 5 nm) and the fluorescence intensity was measured at 565 nm (bandwidth 5 nm). All measurements were performed in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1 mg/ml bovine serum albumin at 37 °C.

FRET Measurement between mant-ADP and Proteins—To monitor FRET from the tryptophan residue in protein to bound mant-ADP, tryptophan was excited with light at 285 nm (bandwidth 3 nm). For time course measurements, the fluorescence intensity was measured at 435 nm (bandwidth 3 nm). All measurements were carried out in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM MgCl2 at 37 °C, with a fluorometer (FP-6500).

ATPase Assay—ATPase activity was measured at 25 °C, as previously described (24). Briefly, protein was added to a solution comprised of 50 mM Tris-HCl, pH 8.0, 100 mM KCl, 2 mM MgCl2, MgATP, and an ATP regenerating system (0.1 mg/ml pyruvate kinase, 0.1 mg/ml lactate dehydrogenase, 2 mM phospho-enol-pyruvate, and 0.2 mg/ml NADH), and the decrease in absorbance at 340 nm was monitored with a spectrometer (V-550, JASCO).


Figure 1
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  		FIGURE 1.
Reconstitution of T. thermophilus V1 complex. A, isolation of V1 components. Isolated V1 components were analyzed by SDS-PAGE; V1 complex (lanes 1 and 5), A3B3 subcomplex (lane 2), A subunit (lane 3), B subunit (lane 4), and DF subcomplex (lane 6). B, reconstitution of V1 from A and B subunits and DF subcomplex. The A and B subunits (13.3 µM) were mixed with DF subcomplex (2.1 µM). The mixtures were incubated without nucleotide (lane 3) or with 1 mM MgATP (lanes 1, 2, and 4), or with MgADP (lane 5)in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl (buffer A, total volume of 15 µl) for 1 h at 37 °C, then applied to 7.5% Native PAGE, followed by staining with Coomassie Brilliant Blue. Lane 6, V1 complex. C, reconstitution of V1 using nucleotide analog or mutant A subunit. The mixtures of 13.3 µM B subunit and 2.1 µM DF subcomplex were incubated with 13.3 µM each of the wild-type A (lanes 1-3), AE257A (lane 4), or AKTAA (lane 5) with the indicated nucleotides in buffer A (total volume of 15 µl) for 1 h at 37 °C. The mixtures were applied to 7.5% Native PAGE, followed by staining with Coomassie Brilliant Blue. Lane 6, V1 complex. D, formation of subcomplexes of V1. The mixtures of 10 µM each of A and B subunit were incubated with 0 (lane 1), 0.8 (lane 2), 1.6 (lane 3), 2.4 (lane 4), and 3.2 µM (lane 5) of DF subcomplex in the presence of 1 mM MgATP, followed by analysis on Native PAGE. Lane 6, V1 complex. E, subunit composition of subcomplexes of V1. Each band of Native PAGE shown in D was excised, followed by separation with SDS-PAGE. Lane 1, V1; lane 2, the band indicated by the asterisk in D; lane 3, A3B3. F, reconstitution of V1 from A3B3 and DF subcomplexes. The mixtures of 0.4 µM A3B3 and 1.1 µM DF subcomplexes were incubated with (lane 4) or without (lane 1-3) 1 mM MgATP at 37 °C for 1 h, followed by analysis on Native PAGE. The gels were stained with Coomassie Brilliant Blue.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Requirement of ATP for Reconstitution of the V1 Complex in Vitro—The subunit composition of the V1 complex of T. thermophilus is A3B3DF. It is an ATP-driven rotary motor, in which D and F subunits rotate relative to A3B3 hexamer (7, 9). There are three catalytic sites in one V1 complex. Most of the residues that participate in catalysis are located in the A subunit (2). To analyze the assembly process of V1 subunits, the A and B subunits, and DF subcomplex of T. thermophilus were expressed in E. coli and purified to near homogeneity (Fig. 1A). They were mixed together in the absence and presence of ATP4 and submitted to native polyacrylamide gel electrophoresis (Native PAGE) to determine whether the V1 complex and/or small subcomplex is formed. In the absence of ATP, no V1 formation was observed (Fig. 1B, lane 3). In contrast, A, B, and DF were assembled into the V1 complex in the presence of ATP (Fig. 1B, lane 4). This is consistent with the previous observation that ATP is required for the in vitro reconstitution of yeast V1 complex from yeast cell extract (20). A small band that was observed just below the V1 complex is the A3B3 subcomplex (Fig. 1, B and C), which we describe later. To determine whether both ATP binding and hydrolysis are required or only ATP binding is necessary for V1 formation, we carried out reconstitution experiments using mutant A subunits and an ATP analog. As shown in Fig. 1C, the use of a non-hydrolyzable ATP analog, AMP-PNP, instead of ATP supported assembly of subunits into the V1 complex. We also assessed whether the AE257A mutant could be incorporated into the V1 complex. Glu257 in the A subunit is equivalent to Glu190 in the beta subunit of Bacillus PS3 F1-ATPase, which has been suggested to be an essential residue for hydrolysis, but not for binding ATP (25, 26). When AE257A was mixed with the B subunit, the DF subcomplex, and ATP, the V1 complex was successfully reconstituted (Fig. 1C, lane 4). These results clearly suggest that hydrolysis of ATP is not necessary for V1 formation. Moreover, A, B, and DF assembled into the V1 complex when incubated with ADP (Fig. 1B, lane 5). Next, we assessed assembly using the AK234A/T235A mutant (hereafter AKTAA mutant), in which the conserved Lys234 and Thr235 within the Walker motif A (GXXXXGKT) of the A subunit were replaced with alanine. It has been reported that the AAA+ protein ClpB with this mutation in the Walker motif has greatly reduced capacity to bind nucleotide (27). Indeed, the AKTAA mutant lost the ability to bind the fluorescent nucleotide, mant-ADP as described below. As expected, no assembly was observed when the AKTAA mutant was examined (Fig. 1C, lane 5). These results indicate that binding of nucleotide to the catalytic sites is necessary for assembly of the subunits into the V1 complex, and that hydrolysis of ATP is not necessary.

Nucleotide-dependent Formation of Subcomplexes—Because T. thermophilus V1 is an assembly of 8 subunits, there should be several intermediate subcomplexes when V1 subunits assemble to form the complex. Existence of intermediates in assembly is consistent with the observation that yeast strains lacking one or more genes encoding V1 subunits accumulate various subcomplexes (28, 29). During reconstitution of the V1 complex, we observed two bands on Native PAGE (Fig. 1D), which migrated slightly faster than the V1 complex. This suggested formation of two large subcomplexes during the assembly process. One subcomplex migrated slower than the V1 complex on Native PAGE even in the absence of DF and contained only A and B subunits (Fig. 1, B, lane 1; D, lane 1; E, lane 3). Thus, this subcomplex should be the A3B3 subcomplex (also see below). The amount of A3B3 assembled from the A and B subunits decreased as the amount of DF subcomplex increased (Fig. 1D). On the other hand, another subcomplex was observed when the amount of DF increased (Fig. 1, D, lane 4 and 5), and contained A, B, and DF (Fig. 1E, lane 2). Because this subcomplex contained 77% of the B subunit compared with V1 (as determined by densitometer and normalized by the amount of D subunit) observed on SDS-PAGE, its subunit composition is thought to be A2B2DF.

Next, we purified the A3B3 subcomplex using gel permeation chromatography, and examined whether it can accommodate the DF subcomplex to form the V1 complex. Interestingly, the A3B3 and DF subcomplexes assembled into the V1 complex even in the absence of ATP (Fig. 1F, lane 3). This suggests that the A3B3 subcomplex might be a precursor of the V1 complex, and that ATP, which is obligatory for V1 formation, is not used for the assembly step in which the DF subcomplex is incorporated into the cavity of the A3B3 subcomplex. Although A3B3 and DF assembled into V1 complex in the presence of ATP, formation of the A2B2DF subcomplex was also observed under these conditions (Fig. 1F, lane 4). The A2B2DF subcomplex might assemble from the DF and A1B1 heterodimer, as the A3B3 disassembles in the presence of ATP (see below). It is also likely that A2B2DF is a direct precursor of the V1 complex. However, because of the instability of this subcomplex, this could not be demonstrated.

We next investigated the assembly of the A and B subunits in the absence of DF in detail. When the A and B subunits were incubated with ATP, formation of the A3B3 subcomplex was observed as shown in Fig. 2A. The A3B3 subcomplexes were also formed when AMP-PNP or AE257A was used, suggesting that hydrolysis of ATP is unnecessary (Fig. 2B, lanes 2 and 3), although the amount of A3B3 formed was decreased significantly compared with the case where wild-type A subunit and ATP were used. The decrease in the amount of A3B3 observed might suggest that A3B3 is stabilized by hydrolysis of bound ATP. We also analyzed the assembly of the A and B subunits using gel-permeation chromatography. As shown in Fig. 2C, only one peak with an estimated molecular mass of 62 kDa was observed in the absence of ATP. This peak should be a mixture of the monomeric A and B subunits, which have calculated molecular masses of 64 and 53 kDa, respectively. In contrast, additional 309- and 122-kDa peaks were observed in the presence of ATP (Fig. 2C), indicating the formation of the A1B1 heterodimer in addition to the A3B3 subcomplex. Both peaks were also observed when AE257A mutant was used instead of the wild-type A subunit (Fig. 2C), or when AMP-PNP was used instead of ATP (data not shown). The A1B1 heterodimer did not clearly resolve from the B subunit by Native PAGE (Fig. 2B, asterisks). On the other hand, when AKTAA mutant was used neither A3B3 nor A1B1 was observed (Fig. 2C). This indicates that binding of ATP to the A subunit induces or stabilizes interaction between A and B subunits and subsequent formation of A1B1, which would then assemble into the A3B3 complex. Given that the V1 complex did not assemble when ATP was absent or AKTAA was used, the formation of the A1B1 heterodimer induced by ATP binding is most likely the primary step of V1 complex formation.


Figure 2
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  		FIGURE 2.
Assembly of A and B subunits in the absence of DF subcomplex. A, analysis of assembly on Native PAGE. The mixture of 20 µM each of A or B subunits was incubated without nucleotides (lanes 1, 3, and 5) or with 1 mM MgATP (lanes 2, 4, and 6) or MgADP (lane 7) in buffer A (total volume of 15 µl) for 1 h at 37°C. The mixtures were applied to 7.5% Native PAGE, followed by staining with Coomassie Brilliant Blue. Lane 8, A3B3; lane 9, V1 complex. B, analysis of assembly on Native PAGE using nucleotide analog or mutant A subunit. The mixtures of 20 µM each of the B subunit and/or wild-type A and/or the AE257A and/or the AKTAA mutant were incubated with 1 mM MgATP (lanes 1, 3, and 4) or MgAMP-PNP (lane 2) in buffer A (total volume of 15 µl) for 1 h at 37 °C. The mixtures were applied to 7.5% Native PAGE, followed by staining with Coomassie Brilliant Blue. Lane 5, A3B3. C, analysis of assembly with gel-permeation chromatography. The mixtures of 20 µM each of the wild-type or mutant A and B subunits were incubated at 37 °C for 1 h in the presence or absence of 1 mM MgATP, followed by analysis using Superdex-200 gel-permeation chromatography equilibrated with buffer A. The elution volumes and molecular mass of marker proteins are indicated at the top of the panel. D, analysis of assembly at various concentrations of monomers. Various concentrations of A and B subunits were incubated with 1 mM MgATP at 37 °C for 1 h, followed by analysis on Native PAGE. Lane 1, 0.5 µM; lane 2, 1 µM; lane 3, 2 µM; lanes 4 and 5 µM; lane 5, 10 µM; lane 6, 20 µM (each).

 


Figure 3
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  		FIGURE 3.
Disassembly of A3B3 subcomplex. A, analysis of disassembly of A3B3 on Native PAGE. A3B3 (0.8 µM) were incubated without nucleotide (lane 1), or with 1 mM MgATP (lane 2), MgAMP-PNP (lane 3), or MgADP (lane 4) at 37 °C for 1 h, followed by separation by Native PAGE. Lane 5, A subunit; lane 6, B subunit. B, analysis of disassembly with gel-permeation chromatography. A solution (60 µl) containing 0.8 µM A3B3 was incubated at 37 °C for 1 h in the presence or absence of 1 mM MgATP or MgAMP-PNP, followed by separation with Superdex-200 gel-permeation chromatography equilibrated with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl.

 
The amount of A3B3 formed was dependent on the concentration of monomeric subunits (Fig. 2D). The A and B subunits must be at or above the critical concentration, which seems to be a few micromolar, for assembly into A3B3.

Nucleotide-dependent Disassembly of A3B3 Subcomplex—In the absence of nucleotides, the purified A3B3 subcomplex was stable at 4 °C for a few days (data not shown), and appeared as a single band on Native PAGE (Fig. 3A, lane 1) and eluted as a single peak in gel-permeation chromatography (Fig. 3B). However, when A3B3 was incubated with ATP, a band representing the A3B3 subcomplex disappeared and a new band appeared on Native PAGE (Fig. 3A, lane 2). Gel-permeation chromatography also showed disappearance of A3B3 and emergence of a 115-kDa complex (Fig. 3B). This indicates that ATP induced disassembly of the A3B3 subcomplex into A1B1 heterodimer. Disassembly of A3B3 was also observed when incubated with AMP-PNP or ADP (Fig. 3, A, lanes 3 and 4, and B), except that a trace amount of A3B3 was still present after treatment with ADP. Although A3B3 was completely disassembled by ATP under the conditions shown in the figure, a substantial amount of A3B3 remained after treatment with ATP when the concentration of A3B3 was much higher (data not shown). This apparent resistance of A3B3 to nucleotides might be caused by reassembly of A1B1 to form A3B3. Because the amount of assembled A3B3 is highly dependent on the concentration of the components (Fig. 2D), it does not appear that reassembly occurs at low concentrations. It is not clear why dissociation of A3B3 complex by addition of ATP yielded only trace amounts of the monomers (Fig. 3B), whereas addition of ATP to the monomeric A and B subunits gave a substantial amount of monomers (Fig. 2C). One possible reason for this inconsistency is that the isolated subunits used for assembly experiments contain inactive species that are partially denatured and cannot assemble. In contrast to the A3B3 subcomplex, nucleotide-induced disassembly was not observed for V1 complex (data not shown). The presence of the central stalk must prevent nucleotide-induced conformational changes that lead to disassembly of the A3B3 ring.

Asymmetric Binding of the A Subunit with B Subunits—In the V1 complex, one A subunit interacts with two B subunits, and vice versa, because the A and B subunits are arranged alternately (Fig. 4A). One of the two interfaces between A and B subunits includes a catalytic site, whereas the other does not. Thus, two A1B1 species might exist, i.e. A-B and B-A. To determine whether A1B1 assembled from the A and B monomers contains one or two A1B1 complexes, we tried to estimate the relative distance between two positions within the A1B1 subcomplex using FRET. FRET is a powerful tool for estimating distances within the complex and for investigating dynamics of various biological processes, including protein interaction and ligand binding (30). Glu114 or Glu374 of the A subunit, and Thr107 or Lys304 of the B subunit were replaced by cysteine, and the intrinsic cysteine residues were replaced by substitution. Glu114 of the A subunit and Lys304 of the B subunit are close to the catalytic site, whereas Glu374 of the A subunit and Thr107 of the B subunit are distant from the catalytic site (Fig. 4A). These unique cysteine residues were selectively labeled by Cy3 (A subunit) or Cy5 (B subunit) dyes. If the distance between Cy3 (donor) and Cy5 (acceptor) dyes are close enough, decrease in the donor fluorescent intensity and increase in the acceptor fluorescent intensity associated with interaction of the Cy3-labeled A and the Cy5-labeled B subunits was expected. First, we examined whether FRET is observed between the Cy3-labeled A and the Cy5-labeled B subunits. Donor intensity from the Cy3-labeled A subunit did not change upon addition of Cy5-labeled B subunit to a solution containing Cy3-labeled A subunit. However, when ATP was added to the mixture of Cy3-labeled A and Cy5-labeled B subunits, the donor intensity decreased gradually (Fig. 4B). This is consistent with the result of Native PAGE and gel-permeation analyses, which indicate that the A and B subunits assemble in an ATP-dependent manner. In the absence of the B subunit, donor intensity from Cy3-labeled A subunit also decreased upon addition of ATP, but very rapidly (within a minute) and to a much smaller (about 5% for 1 mM ATP) extent (data not shown). Thus, the decrease in donor emission most likely reflects interaction between A and B subunits. Because the amount of A3B3 assembled from low concentrations of the A and B subunits is quite low (Fig. 2D), the fluorescence change noted above must reflect formation of the A1B1 heterodimer, not the A3B3, under the conditions of this experiment. Next, we measured the fluorescence spectrum of a mixture of the Cy3-labeled A subunit and the Cy5-labeled B subunit at 0 and 60 min after addition of ATP. Fig. 4C shows four sets (A114C-Cy3 and B107C-Cy5, A114C-Cy3 and B304C-Cy5, A374C-Cy3 and B107C-Cy5, and A374C-Cy3 and B304C-Cy5) of fluorescence spectra. The highest FRET induced by ATP was seen between A114C-Cy3 and B304C-Cy5, whereas the lowest FRET was between A374C-Cy3 and B107C-Cy5. If the A subunits interact with the B subunit via a non-catalytic interface, higher FRET between A374C-Cy3 and B107C-Cy5 should be observed. Thus, this result strongly suggests that the A subunit interacts with the B subunit via the interface containing the catalytic site in the assembled A1B1.


Figure 4
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  		FIGURE 4.
Interaction between A and B subunits probed by FRET. A, structural model of A3B3. The model has been constructed by Shäfer et al. (32) by fitting the crystal structures of the A subunit of Pyrococcus horikoshii (47) and the B subunit of Methanosarcina mazei Gö1 (32) to the electron microscopic density of T. thermophilus V-ATPase (48). The A and B subunits were shown in magenta and green, respectively. Cysteine-introduced residues were indicated by cyan spheres and arrows. Three catalytic sites were indicated by arrowheads. B, ATP-dependent change in donor fluorescent intensity. Time course of fluorescent intensity of AE114C-Cy3 (100 nM) was monitored at 565 nm, and BK304C-Cy5 (100 nM) and MgATP (1 mM) were added at 20 and 40 min, respectively. C, fluorescent spectral change after addition of ATP. The mixture containing Cy3-labeled A subunit (100 nM) and Cy5-labeled B subunit (100 nM) was incubated at 37 °C, and then MgATP (1 mM) was added. Spectra were measured at 0 (dotted red line) and 60 min (solid blue line) after addition of MgATP. Measurements were carried out for four pairs of dye-labeled subunits; AE114C-Cy3 and BT107C-Cy5 (top left), AE114C-Cy3 and BK304C-Cy5 (top right), AE374C-Cy3 and BT107C-Cy5 (bottom left), and AE374C-Cy3 and BK304C-Cy5 (bottom right).

 
Binding of the A Subunit with ATP Precedes Binding with the B Subunit—We next asked how A, B, and ATP assemble into the A1B1 heterodimer. Kinetic analyses of real-time donor fluorescent changes associated with dimerization of A114C-Cy3 and B304C-Cy5 were carried out. In this experiment, concentrations of the B subunit and ATP varied and were kept much higher than that of the A subunit, which was fixed at 4 nM to maintain the changes in the concentration of free ATP and the monomeric B subunit at negligible levels. Fig. 5A represents a typical time course of donor fluorescence change, which was well fitted with a single exponential equation, indicating that there is only one rate-limiting step in interaction of the A and B subunits. Thus, there are five possible models for A1B1 formation: the A subunit interacts: 1) simultaneously with the B subunit and ATP to form A1B1; 2) very rapidly with the B subunit first, then slowly with ATP to stabilize the complex;, 3) slowly with the B subunit first, then very rapidly with ATP; 4) very rapidly with ATP first, then slowly with the B subunit; or 5) slowly with ATP first, then very rapidly with the B subunit. However, the gradual decrease in fluorescence observed clearly rules out model 2. The apparent rate constants increased linearly with B subunit concentration (Fig. 5, B and C), consistent with models 1, 3, and 4. On the other hand, the apparent rate constants increased with ATP concentration, but were saturated at high ATP concentrations (Fig. 5, D and E). This result clearly rules out models 1, 3, and 5, because models 1 and 5 predict that apparent rate constants increase linearly with ATP concentration, and model 3 predicts that apparent rate constants decrease with ATP concentration. In contrast, B subunit concentration and ATP concentration dependence of the apparent rate constants were well fitted with the equation derived from model 4 (Fig. 5, C and E). In conclusion, the A subunit and ATP are in rapid equilibrium with A·ATP (Formula = 0.28 mM), and A·ATP and the B subunit are in slow equilibrium with A1B1 (Formula = 1.7 x 104 M-1 s-1, Formula = 2.0 x 10-4 s-1).

Rapid Binding of Mant-ADP to the A Subunit—Next, we investigated the binding of nucleotide to the isolated subunits using a fluorescent nucleotide analog, mant-ADP. Mant-nucleotides have been used to study interaction between nucleotides and a number of ATPases (31). If mant-nucleotide is bound to a protein, excitation of a tryptophan residue in the protein leads to enhancement of fluorescence from mant-ADP due to FRET from the tryptophan residue to the mant-dye. When the A subunit was added to the mant-ADP solution, fluorescence around 435 nm, which is emitted from mant-ADP, was increased (Fig. 6A, top left panel). Excess free ATP suppressed the increase in fluorescence (Fig. 6A, top right panel). In addition, the extent of the fluorescence increase was dependent on the amount of the A subunit (Fig. 6B). These data clearly indicate that the monomeric A subunit can bind nucleotide. On the contrary, a similar fluorescence increase was not observed with the AKTAA mutant (Fig. 6A, bottom left panel), indicating that affinity for nucleotide was significantly decreased by the mutation. Fluorescence increase was not observed with the B subunit (Fig. 6A, bottom right panel). This observation is not consistent with previous reports that the isolated B subunit can bind nucleotide (32). The monomeric B subunit might have low affinity for mant-ADP that might not to be detected using this method. We next monitored real-time changes in emission from the acceptor molecule upon addition of the A subunit. As shown in Fig. 6C, the acceptor fluorescence change was completed within 1 min at 37 °C after adding the A subunit. This is much faster than formation of the A1B1 subcomplex observed using FRET (Fig. 5), and supports our conclusion that binding of ATP to the A subunit precedes binding of the B subunit.


Figure 5
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  		FIGURE 5.
Kinetic analysis of FRET between A and B subunits. Donor fluorescent intensity at 565 nm of the mixture containing AE114C-Cy3 and BK304C-Cy5 was monitored at 37 °C. AE114C-Cy3 was fixed at 4 nM in this experiment. A, estimation of apparent rate constants of donor fluorescent change. Typical fluorescent change after addition of ATP is shown. An apparent rate constant of the fluorescent decrease was estimated by fitting donor fluorescent intensity (gray line) with a single exponential function (red line). ATP was added at time 0, and data between 0 and 20 s were excluded from analysis. B, donor fluorescent change at various concentrations of an acceptor. Donor fluorescent intensity was monitored at a fixed ATP concentration (1 mM) and at various B subunit concentrations (red, 50 nM; orange, 100 nM; light green, 150 nM; blue, 200 nM; purple, 250 nM). C, apparent rate constants at various concentrations of the B subunit. Apparent rate constants estimated from measurements in B were plotted against concentration of B subunit. From the y intercept of the fits with a linear segments (black line) Formula = 2.0 x 10-4s-1 was deduced. D, donor fluorescent change at various concentrations of ATP. Donor fluorescent intensity was monitored at fixed [B subunit] (200 nM) and at various ATP concentrations (red, 0.1 mM; orange, 0.2 mM; light green, 0.4 mM; green, 0.6 mM; blue, 1.0 mM; purple, 2.0 mM). E, apparent rate constants at various concentrations of ATP. Apparent rate constants estimated from measurements in D were plotted against concentration of ATP. Fits with an equation, kapp = Formula + {[B subunit][ATP]/(Formula + [ATP])} x Formula, are shown as the black line, where Formula = 1.7 x 104 M-1 s-1, Formula = 2.0 x 10-4 s-1 and Formula = 0.28 mM (see text for details).

 


Figure 6
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  		FIGURE 6.
Mant-ADP binding to the A subunit. A, spectral change upon addition of mant-ADP. Fluorescent spectra of the solution containing mant-ADP (50 µM) in the absence (top left, bottom left, and bottom right) and presence (top right) of MgATP (500 µM) before (blue dotted line) and after (green dotted line) addition of 200 nM wild-type A subunit (top left and top right), AKTAA mutant (bottom left), or B subunit (bottom right). Difference fluorescent spectra shown as the red line were obtained by subtracting the blue dotted line from the green dotted line. Increase in fluorescence of mant-dye was shown by the arrow. All spectra were normalized to the fluorescent intensity at 433 nm before adding protein. B, protein-dependent increase of fluorescence from mant-dye. The wild-type A subunit was added to the solution containing 50 µM mant-ADP. Difference fluorescent spectra, which was obtained as in A, at different concentrations of the A subunit (red, 100 nM; light green, 200 nM; blue, 300 nM) are shown. C, time course of increase in emission from mant-dye. Fluorescent intensity at 435 nm of the solution containing mant-ADP (50 µM) was monitored. The reactions were initiated by adding the A subunit (200 nM) as indicated by the arrow.

 


Figure 7
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  		FIGURE 7.
Kinetic analysis of ATP hydrolysis by TSSA mutant V1 and A3B3. A, the time course of ATP hydrolysis by V1(TSSA) at 1000 (black line), 500 (green line), and 200 µM ATP (red line). Reaction was started by adding 20µlof1µM V1(TSSA) into 2 ml of reaction mixture. The reactions were monitored 4 s after addition of the enzyme to ensure the enzyme was completely mixed in the reaction mixture in our system. B, the time course of ATP hydrolysis by A3B3(TSSA) at 200 (black line), 40 (red line), 10 (green line), and 2 µM ATP (blue line). Reaction was started by adding 20 µlof5.9 µM A3B3(TSSA) into 2 ml of the reaction mixture. C, the initial ATPase activities of A3B3(TSSA) at various ATP concentrations. Initial ATPase activity at the various ATP concentrations were analyzed using the Michaelis-Menten equation obtaining Vmax = 13.5 s-1 and Km = 14.3 µM (red solid line). An initial ATPase activity of A3B3(TSSA) was estimated from fitting the function of ATPase activity (see D and text) at time = 0 s. Inset, the ATPase activity of V1(TSSA) at the rapid hydrolysis phase (see text for details) analyzed by using the Michaelis-Menten equation obtaining Vmax = 77.8 s-1 and Km = 534 µM (blue solid line). D, inactivation of ATPase activity of A3B3(TSSA) at 40 µM ATP (gray line), a derivative of the red line in B, was fitted with a single exponential function (red line, see text for details). E, dependence of inactivation (blue) and activation (red) rates of ATPase activity of A3B3(TSSA) at various ATP concentrations. Values were estimated by fitting the inactivation of ATPase activity as in D (see text for details). F, average number of hydrolyzed ATP molecules per single A3B3(TSSA) before inactivation. Values were given by an initial ATPase activity x 1/ka-i.

 
Analysis of Continuous ATP Hydrolysis of A3B3 Subcomplex—We have previously demonstrated that the A3B3D subcomplex is the minimum unit for rotation and that the F subunit is not absolutely required for either ATP hydrolysis or rotation (23). However, it is still unclear whether the D subunit is required for ATP hydrolysis. The ATPase activity of the A3B3 subcomplex, which assembled from wild-type A and B subunits and ATP, was measured by a coupling assay using an ATP regeneration system, but there was no detectable ATPase activity (data not shown). The A3B3 subcomplex, however, contained about 1.5 mol of ADP per mol of A3B3, suggesting that A3B3 can hydrolyze ATP but is inhibited by bound ADP. The inhibitory effect of ADP on the V1 complex was shown earlier (24). The V1 complex with the A-S232A/A-T235S mutations (we call it TSSA mutations) is much less susceptible to ADP inhibition than wild-type (7). We then measured ATPase activity of the TSSA mutant of the A3B3 subcomplex. Fig. 7, A and B, shows the time course of ATP hydrolysis by V1(TSSA) and A3B3(TSSA), respectively. Hydrolysis of ATP by V1(TSSA) proceeds in three distinct phases. After addition of V1(TSSA) to the assay mixture, an apparent initial lag is observed, followed by a second phase with a high rate hydrolysis. Then the rate of hydrolysis decelerates slowly, probably due to ADP inhibition (this phase is not apparent in the figure). The A3B3(TSSA), unlike wild-type, showed an apparent ATPase activity, but both the turnover rate and hydrolysis profile were considerably different from that of the V1(TSSA) (Fig. 7B). In contrast to the V1(TSSA), the A3B3(TSSA) did not show an initial lag, and moreover, the ATPase activity of A3B3(TSSA) decelerated rapidly, especially at high ATP concentrations. Initial ATPase activity of the A3B3 subcomplex fits well with a Michaelis-Menten equation (Fig. 7C) with Km and Vmax values estimated as 14.3 ± 4.9 µM and 13.5 ± 1.1 s-1, respectively. Both values are much smaller than those for the V1(TSSA) (Km = 534 ± 24 µM and Vmax = 77.8 ± 1.1 s-1, inset in Fig. 7C). The results indicate that the central shaft composed of the D and F subunits is not required for ATPase activity, but that the incorporation of the shaft dramatically changes the catalytic properties of A3B3 subcomplex.

The deceleration of the ATPase activity of the A3B3(TSSA) provided some information on disassembly of A3B3 subcomplex, although A3B3(TSSA) is slightly different from wild-type A3B3 in that A3B3(TSSA) disassembles in the presence of ATP into monomeric A and B subunits (data not shown), whereas wild-type disassembles into A1B1. Fig. 7D shows the time-dependent change of the rate of hydrolysis of 40 µM ATP by A3B3(TSSA), indicating that the ATPase activity decelerated exponentially during hydrolysis and reached a steady-state rate within a few minutes. This time course suggests that the initial rapid hydrolysis is catalyzed by the A3B3(TSSA) free from degradation components, and that A3B3(TSSA) gradually disassembled, causing the observed decrease in activity. Finally, the steady-state hydrolysis is catalyzed by the A3B3(TSSA) where disassembly and reassembly is in equilibrium. The apparent rate constants for conversion from the active to the inactive form (ka-i) and from the inactive to the active form (ki-a) at initial A3B3 concentrations of 59 nM, which were estimated from the rate of deceleration (ka-i + ki-a) and the ratio of the steady state activity to initial activity (ki-a/[ka-i + ki-a]), are shown in Fig. 7, D and E. The ka-i and ki-a values most likely correspond to disassembly and reassembly rates, respectively. According to this assumption, disassembly of the A3B3 and reassembly into the A3B3 takes {approx}57 and {approx}1200 s, respectively, at 40 µM ATP. In addition, ka-i and ki-a increased with the concentration of ATP (Fig. 7E), further supporting the fact that ATP is critical for both the assembly and disassembly.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although assembly of V-ATPase subunits has been extensively investigated both in vivo (16, 17, 33) and in vitro (20, 34-37), how subunits assemble to form the V1 complex has been ambiguous. In this study, we investigated the assembly pathway for the V1 complex by reconstituting the T. thermophilus V1 complex from its components in vitro. One of the most important findings of this study is that the binding of nucleotide to the catalytic site of the A subunit is essential for interaction of the A and B subunits to form the V1 complex. This is consistent with previous reports that ATP is required for assembly of yeast V1 in vitro (20) and that mutations in the Walker A motif of the A subunit leads to defective formation of a functional V-ATPase in vivo (21). Furthermore, based on both the kinetics of FRET change between A and B subunits and the mant-ADP binding assay, we found that binding of nucleotide to the monomeric A subunit precedes assembly of the A and B subunits. It was also suggested that only one of the two interfaces is involved in the nucleotide-induced interaction of the A subunit with the B subunit. There are at least two possible models for how nucleotide binding to the A subunit induces interaction between the A and B subunits. One possible model is that binding of the nucleotide changes the conformation of the A subunit into one that interacts with the B subunit. This is based on several lines of evidence that indicate that the catalytic beta subunit of F1-ATPase, which has some similarity to the A subunit of V-ATPase, undergoes a large conformational change upon binding of ATP (38-40). Another possible model is that bound nucleotide acts as "glue" that pastes the A and B subunits together. This idea is based on the fact that ATP is bound to the catalytic site that is formed at the interface between the A and B subunits in A1B1 heterodimer. In the reconstitution of V1 complex, we also detected A1B1, A3B3, and A2B2DF subcomplexes. It is very likely that these subcomplexes are intermediates in the assembly process. Other possible subcomplexes, such as A2B2 or A1B1DF, might exist, but could not be detected by our methods. Based on the results of this study, a model for the assembly process for T. thermophilus V1 from its components is shown in Fig. 8. At the first step of assembly, the A subunit binds ATP, followed by binding of the B subunit, to form the A1B1 subcomplex. There might be at least two pathways from the A1B1 to the V1 complex. One is that two A1B1 and one DF assemble into A2B2DF, followed by assemble into the V1 complex by binding another A1B1 complex. The other is that A1B1 first trimerizes into the A3B3 subcomplex, which then binds the DF dimer to form the V1 complex. Although we showed that nucleotide is required for assembly of A and B to A1B1 but not for assembly of A3B3 and DF to V1, it is not clear whether or not other assembly steps require nucleotide. Of course, it should be taken into account that the assembly in vivo is more complex. For example, it has been shown for yeast V-ATPase that some populations of V1 subunits are incorporated into the membrane-embedded Vo fraction before whole V1 complex is assembled (33). This means V1 is sometimes assembled on the Vo complex, and that Vo complex might affect assembly of V1 subunits. However, assembly properties derived from in vitro reconstitution experiments would also be important for assembly in vivo.


Figure 8
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  		FIGURE 8.
Model of assembly pathway of V1 components. The A subunit interacts first with ATP, followed by binding to the B subunit, forming the A1B1 subcomplex. Then, the A1B1 and DF subcomplexes assemble into V1 complex via A2B2DF or A3B3 subcomplexes. The A3B3 subcomplex reversibly dissociates into the A1B1 subcomplex by ATP (see text for detail).

 
V-ATPase belongs to a group of Walker-type ATPases, which shares a conserved motif, GXXXXGK(T/S), called the Walker motif. It is known that some of Walker-type ATPases have a ring-shaped oligomeric structure like the V1 complex, and interestingly, require nucleotide for subunit oligomerization. These include E. coli F1-ATPase (41), circadian clock protein KaiC (42, 43), and AAA+ chaperone ClpB (27). Nucleotide-dependent oligomerization in these complexes might proceed through the mechanism similar to that of V-ATPase.

Interestingly, the A3B3 subcomplex disassembles in the presence of nucleotide. How does nucleotide induce disassembly of A3B3? It seems unlikely that ATP binding to the catalytic site causes disassembly because the A3B3 with the TSSA mutation shows continuous ATPase activity. It might be possible that the A3B3 subcomplex is "programmed" to disassemble after it hydrolyzes a certain number of ATP molecules. If so, the A3B3(TSSA) would hydrolyze a certain number of ATP molecules, on average, independent of ATP concentration. However, this mechanism is ruled out because the average number of ATPs hydrolyzed per single A3B3(TSSA) molecule before inactivation, which is given by an initial ATPase activity times a lifetime of A3B3(TSSA) (tA3B3 = 1/ka-i), varied largely with the concentration of ATP (Fig. 7F) and because wild-type A3B3, which does not show ATPase activity probably due to ADP inhibition, also disassembles by ATP. It has been suggested that a low affinity non-catalytic ATP binding site is formed on the B subunit in the V1 complex (44-46) and also on the isolated B subunit (32). Thus, another possibility is that ATP binding to this low affinity site weakens the interaction between A and B subunits. This model well explains why the number of ATP hydrolyzed by the A3B3 subcomplex before inactivation is large and varies with [ATP] (Fig. 7F). Why T. thermophilus V-ATPase has a system of ATP-dependent disassembly that seems quite wasteful is not clear. One possible explanation is that this system prevents the accumulation of a non-productive dead-end complex, A3B3-Vo, which is formed when A3B3 is mixed with the Vo complex in vitro.5 Accumulation of A3B3-Vo will waste Vo complex, resulting in the reduction of the amount of functional V-ATPase complex. Because the A3B3 moiety in A3B3-Vo also is disassembled by nucleotide in vitro,5 the amount of A3B3 and A3B3-Vo must be kept at a very low level in vivo.


   FOOTNOTES
 
* This work was supported by Grants-in-Aid 1837005 and 18657041 from the Ministry of Education, Science, Sports and Culture of Japan (to K. Y.). 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. Back

1 Present address: Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047, Japan. Back

2 To whom correspondence should be addressed. Tel.: 81-45-922-5238; Fax: 81-45-922-5239; E-mail: kyokoyama-ra{at}res.titech.ac.jp.

3 The abbreviations used are: V-ATPase, vacuolar-type H+-ATPase; mant-ADP, N-methylanthraniloyl ADP; AMP-PNP, 5'-adenylyl-beta,{gamma}-imidodiphosphate; FRET, fluorescence resonance energy transfer; KTAA, K234A and T235A mutations in the A subunit; TSSA, S232A and T235S mutations in the A subunit; MOPS, 4-morpholinepropanesulfonic acid. Back

4 All nucleotides used in this study were a 1:1 mixture with MgCl2. For example, 1 mM ATP means 1 mM ATP and 1 mM MgCl2. Back

5 K. Yokoyama, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Daniela Stock for kindly providing structural coordinates for the A3B3 subcomplex model, Masahiro Nakano and Masafumi Toei for discussions and experimental suggestions, and Ryota Iino and William S. Allison for critical assessment of the manuscript.



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