The Membrane Domain of the Na+-motive V-ATPase from Enterococcus hirae Contains a Heptameric Rotor*

Takeshi Murata {ddagger} §, Ignacio Arechaga {ddagger} §, Ian M. Fearnley {ddagger}, Yoshimi Kakinuma ¶, Ichiro Yamato || and John E. Walker {ddagger} **

From the {ddagger}Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, United Kingdom, the Department of Applied Chemistry, Muroran Institute of Technology, 27-1 Mizumoto-cho, Muroran-shi, Hokkaido 050-8585, Japan, and the ||Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan

Received for publication, February 14, 2003 , and in revised form, March 14, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In F-ATPases, ATP hydrolysis is coupled to translocation of ions through membranes by rotation of a ring of c subunits in the membrane. The ring is attached to a central shaft that penetrates the catalytic domain, which has pseudo-3-fold symmetry. The ion translocation pathway lies between the external circumference of the ring and another hydrophobic protein. The H+ or Na+:ATP ratio depends upon the number of ring protomers, each of which has an essential carboxylate involved directly in ion translocation. This number and the ratio differ according to the source, and 10, 11, and 14 protomers have been found in various enzymes, with corresponding calculated H+ or Na+:ATP ratios of 3.3, 3.7, and 4.7. V-ATPases are related in structure and function to F-ATPases. Oligomers of subunit K from the Na+-motive V-ATPase of Enterococcus hirae also form membrane rings but, as reported here, with 7-fold symmetry. Each protomer has one essential carboxylate. Thus, hydrolysis of one ATP provides energy to extrude 2.3 sodium ions. Symmetry mismatch between the catalytic and membrane domains appears to be an intrinsic feature of both V- and F-ATPases.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In eukaryotic cells, vital processes such as protein trafficking, endocytosis, neurotransmitter release, and intracellular pH regulation depend on the acidification and energization of organelles (1, 2). The energy is provided by ATP hydrolysis and is coupled to ion movement through the membranes. The reaction is catalyzed by V-ATPases, multisubunit assemblies that are related in structure and mechanism to F-ATPases found in eubacteria, mitochondria, and chloroplasts. Similar to the F-ATPases, V-ATPases have a globular catalytic domain, V1, with 3-fold symmetry connected by central and peripheral stalks to an intrinsic membrane domain, Vo. It is likely that, as demonstrated in F-ATPases, the central stalk in the V-ATPases also couples energy released by ATP hydrolysis in the globular catalytic sector (V1) to ion translocation across the membrane domain (Vo). Eukaryotic V-ATPases contain 13 different polypeptides (3). Eight of them (subunits A–H) form the V1 domain, and the rest, subunits a, c, c', c'' and, d, are in Vo. The c and c' subunits are 16-kDa proteolipids that are probably folded into four membrane-spanning {alpha}-helices (five in the 23-kDa subunit c''). Their sequences consist of two tandem repeats, each corresponding to one c subunit in F-ATPases, except that V-type c subunits have a single essential carboxylate in the fourth (C-terminal) {alpha}-helix, whereas each F-type c subunit has an essential carboxylate in its C-terminal {alpha}-helix (4, 5). In the V-ATPase from Saccharomyces cerevisiae, all three kinds of c subunits are essential (6). They assemble into Vo, where by analogy with Fo they may form a ring that rotates as an ensemble together with the central stalk (1). However, their stoichiometry is uncertain, and the estimates range from c3-c1'-c2'' (7) to c4–5-c1'-c1'' (5, 8).

V-ATPases are also found in prokaryotes (9). The enzyme in the nonrespiring bacterium Enterococcus hirae acts as a primary sodium extrusion system (10). Its subunit composition is simpler than that of its eukaryotic counterparts, and its nine subunits are encoded in the ntp operon (11, 12). NtpK is the homologue of eukaryotic V-type subunit c (13). No isoforms of this subunit have been encountered in E. hirae. As described below, oligomeric assemblies of NtpK have been purified from the V-ATPase from E. hirae. By single particle analysis, the oligomers have been shown to be rings with 7-fold symmetry.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of NtpK Oligomers—The V-ATPase was purified from cells of E. hirae as described previously (14). The purified complex (5 mg/ml protein) was redissolved in a buffer (pH 7.5) containing 20 mM Tris-HCl, 5 mM MgCl2, 1 mM dithiothreitol, 20% glycerol, and 0.05% dodecylmaltoside. Isopropanol (10%) was added, and the solution was kept at 18 °C for at least 24 h. Aggregates that formed during this period were removed by centrifugation (10,000 x g, 10 min) at room temperature. The supernatant was loaded onto an anion-exchange column (Bio-Scale DEAE2; Bio-Rad) equilibrated at room temperature with a buffer (pH 7.5) consisting of 20 mM Tris-HCl, 10% glycerol, 3% isopropanol, 400 mM NaCl, and 0.05% dodecylmaltoside. The proteins were eluted with a linear gradient of NaCl (0.4–1.0 M) with a flow rate of 0.5 ml/min. NtpK eluted at 0.53–0.57 M NaCl. Fractions containing NtpK were pooled and concentrated by ultrafiltration (Centricon YM-10; Amicon, Beverly, MA). The concentrate was injected into a gel filtration column (Superose 6 HR 10/30; Amersham Biosciences) equilibrated in the same buffer containing 0.1 M NaCl. The fractions were eluted with a flow rate of 0.25 ml/min. NtpK was recovered in fractions appearing at approximately 0.66 column volumes.

Analytical Methods—Protein concentrations were determined by the BCA method (Pierce) using bovine serum albumin as standard. SDS-PAGE was carried out using the Schägger and Von Jagow (15) and Laemmli (16) systems. The gels (16.5% and 12–22% acrylamide, respectively) were stained with silver. NtpK bands were detected with a rabbit antiserum against NtpK and with goat anti-rabbit IgG (alkaline-phosphatase conjugate) (17). Protein bands observed in SDS-PAGE gels were analyzed by N-terminal sequence analysis in an Applied Biosystems Procise model 494 protein sequencer and peptide mass finger-printing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The peptides were sequenced by tandem mass spectrometry using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) as described elsewhere (18). {alpha}-Helical regions in protein sequences were predicted with SOSUI. (sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html).

Electron Microscopy and Image Analysis—Purified NtpK rings (5 µl, 2.6 µg/ml) were adsorbed onto freshly glow-discharged carbon-coated grids and stained with 1% (w/v) uranyl acetate. The images were recorded at 42,000x nominal magnification on Kodak SO 163 film using a Philips Tecnai-12 microscope operating at 100 kV under "low dose" conditions (10 e-2). Negatives were scanned at a resolution of 7 µm/pixel using a Zeiss-SCAI scanner and demagnified three times by linear interpolation, giving a final pixel size of 5 Å. A total of 4,534 particles were selected using XIMDISP, which is part of the MRC image processing package software (19), and extracted in 54 x 54-pixel square boxes. The images were normalized, band pass-filtered, and analyzed with IMAGIC5 software (20). The particles were aligned translationally (i.e. centered) using a rotational average of the total sum of the particles as a reference. The centered particles were placed by multivariate statistical analysis into 25 classes. These 25 classes, containing average images of all 4,534 centered particles, were used as references for multi-reference alignment. This operation was performed to prevent bias that could appear if only selected views were used as references. The multi-reference aligned particles were also classified in 25 classes (again, containing all 4,534 particles) that, in turn, were used for a new cycle of alignment. This process was iterated until stable results were obtained. Finally, the aligned particles were classified into 100 classes. Classes corresponding to views of the ring perpendicular to the planar membrane were calculated. The resulting classes were subjected to Fourier transformation, and their rotational power spectra were calculated (21).

Representative views were selected for three-dimensional reconstruction. Euler angles were assigned and refined by applying a C7 point group symmetry. This point group symmetry was suggested by the rotational power spectra of the rings. As control, other symmetries were tested, but they did not give reliable models. A three-dimensional model was calculated by standard angular reconstitution methods using sinogram-based angle assignments with IMAGIC (20). Projections in the asymmetrical triangle of this initial model were used as references for a further multi-reference alignment of the original filtered images. The class averages were calculated, and representative views were selected for calculation of a new three-dimensional model. This process was iterated until stable results were obtained.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Characterization of Oligomers of NtpK—The purified bacterial V-ATPase complex was treated with isopropanol, and subunit K was isolated by ion-exchange chromatography in the presence of dodecylmaltoside (Fig. 1). It eluted in fractions 55–59. These pooled fractions were passed through a gel filtration column to remove minor amounts of NtpG. The purified protein was examined by two different SDS-PAGE gel systems. One or three bands (apparent molecular masses, 11, 22, and 33 kDa, corresponding to monomer, dimer, and trimer) were detected by silver staining (Fig. 2, B and D). In the latter system, an antibody against subunit K reacted with all three bands, and a fourth faint band was also detected at a higher molecular mass than the other bands (Fig. 2C).



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FIG. 1.
Purification of NtpK from the V-ATPase from E. hirae. The enzyme was disrupted with isopropanol, and insoluble material was removed by centrifugation. The supernatant was applied to an anion-exchange column (Bioscale DEAE2; Bio-Rad) equilibrated in a buffer (pH 7.5) consisting of 20 mM Tris-HCl, 10% glycerol, 3% isopropanol, 400 mM NaCl, and 0.05% dodecylmaltoside. The proteins were eluted with a linear gradient (dotted line) of NaCl (0.4–1.0 M) with a flow rate of 0.5 ml/min. The absorbance of the eluate was monitored at 280 nm (closed squares). NtpK eluted in fractions 55–59 at 0.53–0.57 M NaCl. A minor impurity was removed by gel filtration. For further details, see "Experimental Procedures."

 


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FIG. 2.
Analysis of purified NtpK by SDS-PAGE and Western blotting. SDS-PAGE was carried out using the Schägger (lanes A–C) and Laemmli (lanes D and E) systems. Lanes A and E, V1Vo-ATPase (3 µg) purified from E. hirae; lanes B–D, purified NtpK (0.5 µg;). Lanes A, B, D, and E were silver-stained. The bands in lane C were detected with NtpK antiserum.

 

To verify that the silver staining bands contained subunit K and no other subunit of the V-ATPase, they were examined by N-terminal sequence analysis. Following treatment with methanolic HCl to remove the presumed N-{alpha}-formyl group of subunit K (and other components of Vo), no sequences arising from impurities were observed. The purity of subunit K was also assessed by mass spectrometric analysis of tryptic peptides. Two tryptic peptides with monoisotopic masses of 1054.6 and 1818.8 were detected in digests of monomer and dimer and one in the trimer digest. These masses correspond to tryptic peptides T2 and T4 (residues 37–46 and 113–116) of subunit K. Their identities were confirmed by sequencing by tandem mass spectrometry in conjunction with collision-induced fragmentation. Peptide T4 from the trimer band was also detected by this means (Table I). No peptide was detected that arose from any other protein.


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TABLE I
Mass spectrometric analysis of tryptic peptides from preparations of NtpK

 

From these analyses it was concluded that purified protein contains subunit K only and that the three bands in Fig. 2B are different oligomeric states of NtpK, probably corresponding to monomers, dimers, and trimers derived from the same native oligomeric state of NtpK that persist under some experimental conditions in SDS. Other oligomeric membrane proteins such as subunit c from some F-ATPases (22) and phospholamban (23) show similar behavior. Oligomers are also present in gel analyses of the V-type proteolipid isolated from gap junctions of Nephrops norvegicus (24). Another characteristic feature of hydrophobic proteins, also displayed by the oligomers of NtpK, is that their apparent molecular masses determined by SDS-PAGE are significantly lower than the calculated values.

Single Particle Analysis of Oligomers of NtpK—Purified NtpK was stained with uranyl acetate and examined by electron microscopy (Fig. 3A). From the micrographs, 4,534 individual particles were selected, aligned, and classified. Representative views of some of these class averages are shown in Fig. 3B. Analysis of class averages showed views with a strong 7-fold symmetry signal in its rotational power spectrum (Fig. 4). This class is likely to represent views from above or below the ring. Chloroplast c rings have a wide and a narrow end, with external diameters of 74 and 59 Å, probably corresponding to the luminal and stromal sides, respectively (25). The NtpK rings also have different dimensions when viewed from "up" and "down," but it is not possible to deduce which is in contact with V1 and its central stalk. Weak signals for other symmetries were also present (Fig. 4), but their contributions were insignificant (less than 30% of the 7-fold power) and similar to those found in chloroplast c rings (25). The rotational power spectra of other class averages were analyzed, but no significant symmetry other than 7-fold symmetry was observed. Analysis of rotational power spectra is recognized as a powerful method of determination of symmetries and has been used widely (21, 25, 26, 27). An alternative approach used, for example, to distinguish between 12- and 13-fold symmetries in phage portal assemblies is to analyze eigenimages (28). Twenty-four eigenimages were generated in the analysis of K rings. They were compatible with 7-fold symmetry, and no other symmetries were suggested.



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FIG. 3.
Analysis by electron microscopy of purified NtpK. A, original images stained with uranyl acetate (scale bar, 100 nm). A total of 4,534 particles were selected and classified. B, representative views of the 100 class averages obtained. Selected projections of this analysis were used for the calculation of a three-dimensional model. Projections of this initial three-dimensional model were used as reference for multi-reference alignment of the original filtered images. C, representative views of class averages after the refined alignment. Views from this analysis were used for the calculation of the final three-dimensional model. D, projections in the asymmetrical triangle of the final three-dimensional model with similar Euler angles to those in B and C.

 


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FIG. 4.
Rotational power spectra of classes corresponding to a view of the NtpK ring perpendicular to the planar membrane. A, panel a, class average after initial analysis of images. Panel b, similar view obtained after multi-reference alignment using projections of the initial three-dimensional model. Panel c, projection of the final three-dimensional model with same Euler angles as b. B, all of these images were subjected to Fourier transformation, and their rotational power spectra were calculated. The symbols {blacksquare}, {blacktriangleup}, and {circ} correspond to the power spectra of panels a–c, respectively.

 

A three-dimensional reconstruction of the K ring was calculated. Because of the 7-fold symmetry observed in the rotational power spectra of projections perpendicular to the planar membrane, a C7 point group symmetry was used in the assignment of Euler angles. As control, other symmetries were tested, but they did not give reliable models. Projections in the asymmetrical triangle of an initial model were used as references for multi-reference alignment of the original filtered images. Representative views of class averages obtained after iteration of this process are shown in Fig. 3C. A final three-dimensional model was calculated using representative views from this analysis. By calculation of the Fourier shell correlation of two three-dimensional models from two half data sets, the resolution of this model is estimated to be not better than 35 Å. Projections of the final model with similar Euler angles to the refined class averages are shown in Fig. 3D, and a surface representation of the final three-dimensional model is shown in Fig. 5. Because of the difficulty in obtaining a reliable estimate of the protein-detergent density of the NtpK ring and its excluded volume in negative stain, no attempt was made to adjust the threshold of the surface representation to the estimated value of 112 kDa (assuming seven copies of a 16-kDa monomer). Variations in the threshold of the surface representation affect mainly the dimensions of the inner diameter of the ring. Nonetheless, dimensions of the external and inner diameters of the ring in the original raw class averages (Fig. 4A) have been estimated to be 108 and 23 Å, respectively. Because of the contributions from bound detergent and the flattening effect of negative stain, these dimensions are likely to be overestimates. Although it is difficult to evaluate the contributions of detergent to the dimensions of the ring, it is worth noting that the size of monomeric dodecylmaltoside is ~18 Å, which would mean that the diameter of the NtpK ring would be only 72 Å. The dimensions of the NtpK ring are comparable with those of chloroplast c ring (25) and of a three-dimensional reconstruction of the Vo domain from the V-ATPase of clathrin-coated vesicles isolated from brain (29). These latter particles are asymmetric with a cross-section approximating an ellipse (110 x 140 Å), and they contain not only c protomers but also subunits a and d plus the glycoprotein Ac45.



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FIG. 5.
Surface representation of Ntp K ring three-dimensional model. A, top (or bottom) view of the K ring. B, side view. C, bottom (or top) view. The threshold used for the surface representation is arbitrary because no reliable estimations of the protein-detergent density and the volume excluded by the negative stain were available.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is accepted widely that V-ATPases are related in structure and mechanism to the F-ATPases, which have been studied in much greater detail. For example, atomic structures have been established for the catalytic domain at different points in the catalytic cycle (30, 31, 32), and the rotary mechanism of coupling of the proton-motive force to catalytic events in the F1 domain (33, 34) has been demonstrated and analyzed by direct observation (35, 36). By analogy, the V-ATPase is thought also to operate by a rotary mechanism, and this notion is supported by recent experiments (37). In the F-ATPases the rotary element consists of an ensemble made from the central stalk (containing subunits {gamma}, {delta}, and {epsilon} in the mitochondrial enzyme) and a ring of hydrophobic c subunits in the Fo membrane domain (34, 38). The equivalent subunits in the V-ATPases are possibly D, F, and d, respectively (39), which form the central stalk, and the hydrophobic subunits c, c', and c'' (or K in the E. hirae enzyme) (1). Although mutational and cross-linking experiments conducted on the bacterial F-ATPase from Escherichia coli were interpreted as supporting the presence of a ring of c subunits in Fo with estimated stoichiometries ranging from 9–12 (40, 41, 42), direct unequivocal proof of the presence of the c ring in F-ATPases came from x-ray crystallography of an F1-c ring complex from S. cerevisiae (43). Subsequently, rings were observed by electron microscopy and single particle analysis of isolated E. coli c oligomers (22) and by atomic force microscopic examination of c oligomers from the plant chloroplast F-ATPase (25) and from the Na+-motive F-ATPase from Ilyobacter tartaricum (44). The latter ring has been analyzed further by electron crystallography (45). A surprising aspect of these analyses was that the number of c subunits in the rings is not constant in all F-ATPases. In S. cerevisiae, plant chloroplasts, and I. tartaricum there are 10, 14, and 11 subunits, respectively, in the ring, and so far no c ring has been found with clearly established 3-fold symmetry. Therefore, given that according to extant models, each 360° rotation of the central stalk and the attached c ring generates three ATP molecules and that each c subunit contains a carboxyl residue that enters the proton translocating pathway in turn, the proton (or sodium ion):ATP ratios associated with 10-, 14-, and 11-fold ring symmetries are 3.3, 4.6, and 3.7, respectively (46, 47, 48). It has been proposed that this mismatch of symmetries between the F1 and Fo domains is an important intrinsic feature of the enzyme that might help to reduce potential energy barriers to rotation that might exist in an enzyme with matching symmetries in F1 and Fo (43).

The Vo membrane domains of V-ATPases contain a homologue of the F-type subunit c (also known as subunit c in many species or subunit K in E. hirae). It consists of two fused tandem repeats of sequences corresponding to the F-type subunit (4). In the enzymes from vacuoles of S. cerevisiae, the isoforms c' and c'' are also present (6). By amino acid analysis, their stoichiometry in coated vesicles was estimated to be (c,c')5–6·c''1 (8), but dimers of subunit c'' were detected in cross-linking studies of the enzyme from the yeast vacuole (7). These uncertainties notwithstanding, it is believed widely that the number of proteolipids in Vo is six and that the c subunits form a ring (1, 5). However, the evidence that the ring is formed from c subunits is not definitive. By electron microscopy in negative stain of naturally occurring ordered sheets of a 16-kDa proteolipid isolated from gap junctions of the lobster N. norvegicus (a homologue of the subunit c of V-ATPases) rings with 6-fold symmetry were observed (49, 50), and the protein functions as a Vo proteolipid when incorporated into the yeast V-ATPase (51). To date, no observation has been made of the rings or of their symmetry either in the intact yeast or lobster V-ATPase or in c subunits isolated directly from the enzymes.

In contrast to eukaryotic V-ATPases, the enzyme from E. hirae has only one type of proteolipid, called NtpK. As described above, oligomers of the protein have been isolated by disrupting the enzyme with isopropanol. In the isolation of c rings from chloroplast F-ATPase (25), the enzyme was disrupted with SDS and isolated by SDS-PAGE. The c rings from the I. tartaricus enzyme resist boiling in SDS for 5 min (52). Therefore, the c rings appear to be stable assemblies. In the analysis of the I. tartaricus c oligomers, both intact and partially disrupted rings were observed. The partially disrupted rings had the same radius as intact rings (53), indicating that only rings with a unique radius (and therefore with unique symmetry) are present. There was no indication that isolation and disruption of rings led to the formation of rings with other radii and symmetries. The E. hirae K rings, prepared under milder conditions than the F-ATPase rings, contained only intact oligomers with 7-fold symmetry. Therefore, on balance, it is likely that these 7-fold rings represent the oligomeric state of subunit K in the intact enzyme. The sizes of the rings and of the seven individual features are consistent with the one K subunit being the repeating element. As in other species, the protein contains the two tandem repeats. Each repeat corresponds to the F-type c subunit and probably contains two antiparallel transmembrane {alpha}-helices linked by a loop (Fig. 6). It should be remembered that so far the c rings have been observed either in association with the F1-domain but in the absence of subunit a (in the S. cerevisiae F-type enzyme) or in other cases the absence of all other subunits. Therefore, the ring stoichiometries have yet to be determined in an intact F- or V-ATPase.



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FIG. 6.
Sequences of proteolipids from various V- and F-ATPases. The N- and C-terminal residues are numbered on the left and right, respectively. The following sequences are shown: subunit NtpK from the V-ATPase of E. hirae (Eh-K); yeast V-ATPase subunits c (Yv-c), and c' (Yv-c'); N. norvegicus 16-kDa proteolipid (Nn-16k); c subunits from F-ATPases from E. coli (Ec-c), yeast mitochondria (Ym-c), and Propionigenium modestum (Pm-c). Conserved proline residues in loops between helices 1 and 2 and helices 3 and 4 are shown in black, as are conserved functionally essential side chain carboxyls. Other conserved amino acids are highlighted in gray. The dashes show insertions. Potential {alpha}-helices are boxed. The black bars below the sequences show the transmembrane {alpha}-helices predicted with SOSUI.

 

In the F-type c oligomers in S. cerevisiae, the N- and C-terminal {alpha}-helices pack in internal and external rings, respectively, and so the essential carboxylates are accessible from the external surface (43). Based on evolutionary considerations, it might be anticipated that each of the tandem repeats in subunit K (and in V-type c subunits) would be arranged similar to an F-type c subunit, thus placing {alpha}-helices 1 and 3 in the internal ring and {alpha}-helices 2 and 4 in the external ring, with the essential carboxylate in helix 4 accessible from the outside of the ring. Because the K rings have 7-fold symmetry, each tandem repeat is clearly not exactly equivalent to an F-type c subunit in the 14-fold symmetrical ring of the chloroplast F-type ATPase (Fig. 7B). Therefore, an arrangement similar to that in Fig. 7A would be required to explain the 7-fold symmetry. This type of arrangement has been proposed for the V-type ring in the S. cerevisiae vacuole (5) but not in the same enzyme where the natural c subunit has been replaced by the N. norvegicus 16-kDa proteolipid. In the latter case, a model was proposed based on cross-linking experiments in which helix 1 makes an inner ring with helices 2 and 3 at the same radius outside it and with helix 4 sitting alone outside helices 2 and 3 (Fig. 7C) (54). In the absence of more detailed structural data, it is not possible to know which of these and other possible variants is the correct interpretation. However, it is worth noting that like the N-terminal helices of F-type c subunits, helices 1 and 3 of subunit K are relatively rich in glycine and alanine residues in comparison with helices 2 and 4, which might favor the model in Fig. 7A.



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FIG. 7.
Schematic models of the heptameric NtpK ring from the V-ATPase from E. hirae and of rings made from related proteins. A–C show possible cross-sectional packing arrangements of {alpha}-helices in NtpK rings in E. hirae, in c rings in the F-ATPase from chloroplasts, and of the 16-kDa subunit from N. norvegicus, respectively. Helices 1–4 are the four helices from N to C terminus in V-type proteolipids, and helices 1 and 2 are the N- and C-terminal helices of F-type subunit c. The arrangement in A is based on the electron microscopy described in this paper and on evolutionary considerations (see text). In B, the arrangement of c rings isolated from the F-ATPases in chloroplasts is derived from atomic force microscopy (25), which demonstrated their 14-fold symmetry, and on known general features of F-type c subunits. In part C, the model is based on cross-linking data (54).

 

This interpretation of the heptameric rings of subunit K in E. hirae makes the seven essential carboxylates accessible from the outside of the ring, and by analogy with models proposed for the mechanism of Fo and the F-ATPase, it implies that the coupling ratio Na+:ATP is 2.3. The ratio has been measured in other V-ATPases. Values ranging from 1.75 to 3.28 depending on cytoplasmic and luminal pH were estimated in patch clamp experiments on vacuoles from beetroot (55), from 1.8 to 2.3 in tonoplast-enriched vesicles from lemons (56), from 1.52 to 1.75 in chromaffin granules (57), and from 3.2 to 3.8 in the enzyme from S. cerevisiae vacuoles (58). It remains to be discovered what c ring symmetries are associated with these enzymes. However, if symmetry mismatch between catalytic and membrane sectors, now demonstrated in the E. hirae enzyme, is a general intrinsic feature of V-ATPases as it appears to be in F-ATPases, 3-fold and related symmetries are excluded. It is formally possible that the extra transmembrane helix in c'' subunit of eukaryotic V-ATPases could contribute to the creation of symmetry mismatch in a ring with six c subunits in a stoichiometry (ch, c'k, and c''l; h + k + l = 6). The N-terminal helix of subunit c'' in the S. cerevisiae V-ATPase has been deleted without apparent effect on the activity of the enzyme (59), but there is no information on the stoichiometry of c, c', and the modified c'' when this deletion is introduced. In any case, in the enzyme with intact c'', the arrangement would contain 25 transmembrane helices and not the 28 transmembrane spans associated with the 7-fold symmetric K rings in E. hirae.


    FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

** To whom correspondence should be addressed. Tel.: 44-1223-252701; Fax: 44-1223-252705; E-mail: walker{at}mrc-dunn.cam.ac.uk.


    ACKNOWLEDGMENTS
 
We are very grateful to Drs. J. Rubinstein, J. Fernández, and P. Rosenthal for helpful advice on the electron microscopic analysis of the rings.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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