Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy*

Vincenzo F. RizzoDagger §, Ünal CoskunDagger §, Michael Radermacher, Teresa Ruiz, Andrea ArmbrüsterDagger , and Gerhard GrüberDagger ||

From the Dagger  Universität des Saarlandes, Fachrichtung 2.5-Biophysik, D-66421 Homburg, Germany and the  Department of Molecular Physiology and Biophysics, College of Medicine, University of Vermont, Burlington, Vermont 05405

Received for publication, August 22, 2002, and in revised form, October 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The effect of the ATPase activity of Manduca sexta V1 ATPase by the amphipathic detergent lauryldimethylamine oxide (LDAO) and the relationship of these activities to the subunit composition of V1 were studied. The V1 was highly activated in the presence of 0.04-0.06% LDAO combined with release of the subunits H, C, and F from the enzyme. Increase of LDAO concentration to 0.1-0.2% caused the characterized subcomplexes A3B3HEGF and A3B3EG with a remaining ATPase activity of 52 and 65%, respectively. The hydrolytic-active A3B3EG subcomplex has been visualized by electron microscopy showing six major masses of density in a pseudo-hexagonal arrangement surrounding a seventh mass. The compositions of the various subcomplexes and fragments of V1 provide an organization of the subunits in the enzyme in the framework of the known three-dimensional reconstruction of the V1 ATPase from M. sexta (Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37).

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vacuolar ATPases (V1VO ATPases) define an ubiquitous class of proton pumps, which utilize ATP hydrolysis to maintain an acidic pH inside the vacuole (1). The electrochemical ion gradient created across the vacuolar membrane is used for the accumulation of positively charged substrates such as calcium and basic amino acids (2). In addition to this storage function, the vacuolar compartment has secretory and proteolytic functions (3, 4). The V-ATPases, consisting of at least thirteen distinct subunits (A3:B3:C:D:E:F:Gy:Hz:a:d:c:c':c") are morphologically subdivided in two components: a membrane-bound domain, VO, that contains the ion channel, and an extrinsic domain, V1, in which ATP hydrolysis takes place (4, 5). The two major subunits A and B, in a stoichiometry of A3:B3, contain the nucleotide-binding sites and are connected to the VO part by the so-called stalk subunits C-H (6). Seen from the side the structures of the V1 ATPase, recently identified from Caloramator fervidus (7) and the tobacco hornworm, M. sexta (8, 9), revealed a molecule with a single, compact stalk.

The V1 ATPase from M. sexta, which reversibly dissociates from the VO part as an in vivo regulatory mechanism (10), is the object of our studies and comprises the eight subunits A, B, H, C, D, E, G, and F with apparent molecular masses of 67, 56, 54, 40, 32, 28, 14, and 16 kDa, respectively (11). Low resolution structural studies of this V1 complex using small-angle x-ray scattering have shown that the hydrated enzyme is an elongated molecule. The x-ray data define a mushroom-shaped V1 ATPase, which consists of an ~145 Å headpiece, joined by an elongated stalk (8). Image processing of electron micrographs of negatively stained V1 (9, 12, 13) has revealed that the headpiece consists of a pseudo-hexagonal arrangement of six masses surrounding a seventh mass. These barrel-shaped masses of approximately 30 Å in diameter and 80 Å in length, which consist of the major subunits A and B, are arranged in an alternating manner (9). The hexagonal barrel of subunits A and B encloses a cavity of ~32 Å in which part of the central stalk is asymmetrically located. The stalk protrudes from the bottom side of the headpiece forming an angle of ~7° with the vertical axis of the molecule. At the upper end of the hexagonal barrel extensions can be observed, assumed to belong to the N termini of subunit A (9, 13). Further insights into the topology of the M. sexta V1 ATPase were obtained by differential protease sensitivity, release by chaotropic agents (13), and cross-linking studies (13, 14). These studies resulted in a model in which the subunits H, C, D, G, and F are exposed in the enzyme, whereas subunit E is shielded in the complex (6, 13, 14).

Here we report an investigation of the structure-function relationship of the V1 stalk subunits in M. sexta using the detergent LDAO.1 We show that the detergent liberates a highly active A3B3DEG complex and various hydrolytic-active subcomplexes, A3B3HEGF, A3B3HDEG, and A3B3EG. Electron microscopy has been used to visualize directly the two-dimensional structure of the A3B3EG subcomplex demonstrating a hexagonal modulation surrounding a central cavity with an interior mass.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- All chemicals were at least of analytical grade and were obtained from BIOMOL (Hamburg, Germany), Merck (Darmstadt, Germany), Promega (Madison, WI), ROTH (Karlsruhe, Germany), Sigma, or Serva (Heidelberg, Germany).

Purification of V1 ATPase-- Tobacco hornworms were reared as described at the web site manduca.entomology.wisc.edu/manual/cover.html. The Manduca eggs were a generous gift of Dr. J. Dolzer and PD Dr. M. Stengl, Philipps-University of Marburg, and Prof. Trenczek, University of Giessen. The V1 ATPase from M. sexta was isolated according to Gräf et al. (15) with the following differences. (i) V1 ATPase purification occurred in the presence of the protease inhibitors Rotistapi (ROTH) and Pefabloc SC (final concentration of 8 mM; BIOMOL). (ii) Only the fractions that eluted from the Mono-Q HR 10/10 column (Pharmacia) at 250-280 mM NaCl and contained the complete V1 complex, as judged from SDS-PAGE, were pooled, concentrated on a Centricon 100K concentrator (Schleicher & Schuell), and applied on a SephacrylTM S-300 HR column (10/30, Pharmacia) instead of a Superdex HR200 (10/30) column as described previously (15). The V1 complex eluted in a single symmetrical peak containing the eight subunits A-H. The purity and homogeneity of the protein sample was analyzed by Native-PAGE (16) and SDS-PAGE (17). SDS gels were stained with Coomassie Brilliant Blue G250, Blue R250 (18), or with silver (19). Protein concentrations were determined by the bicinchonic acid assay (Pierce). ATPase activity, which is stimulated by Ca2+, was performed in the presence of an ATP-regenerating system as described by Lötscher et al. (20).

Isolation of V1 Subcomplexes-- Dissociation of the V1 complex was induced by incubation of the enzyme in buffer A (20 mM Tris/HCl (pH 8.1) and 150 mM NaCl) with or without the addition of 9.6 mM 2-mercaptoethanol and different concentrations (0.05 to 0.5% (w/v)) of LDAO. The sample was shaken for 1 h on ice and then applied at a flow rate of 0.5 ml/min to a SephacrylTM S-300 HR column (10/30, Pharmacia) equilibrated with buffer A with or without the addition of 2-mercaptoethanol and detergent. Fractions containing pure subcomplexes were pooled and reapplied to the same Sephacryl column equilibrated with buffer A and 9.6 mM 2-mercaptoethanol but without LDAO, to remove the detergent.

Electron Microscopy and Two-dimensional Image Analysis-- For electron microscopy the protein was diluted in 20 mM Tris/HCl (pH 8.1) and 150 mM NaCl to 20-40 µg/ml. The sample was applied to 400 mesh carbon-coated copper grids and deep stained with uranyl acetate. Micrographs were recorded at a calibrated magnification of 58 300× on a Philips CM 120 electron microscope under low electron dose conditions (10 e-2). The negatives were scanned on a flat-bed SCAI (Zeiss) microdensitometer with 7 µm pixel size. The images were reduced by binning to a final pixel size of 21 µm, corresponding to 3.6 Å on the scale of the specimen. Particles were selected from the micrographs using the sole selection criterion that they were far enough apart from their neighbors such that no overlap occurred in the 0° image. For image processing the SPIDER software (21), version 5.0 with extension was used. Images of 2132 particles were windowed from 11 micrographs and normalized in contrast with the outer area of each image, excluding a round masked area around the center (9). The particles were translationally/rotationally aligned using a combination of correlation methods, starting with a first reference created by reference-free alignment (22), followed by reference-based alignments as previously described for the V1 ATPase (9). After each alignment correspondence analysis was applied (23) and the images were classified using Diday's method of moving centers (24) followed by hierarchical ascendant classification. In each step 6 class-averages were created and used as reference for a multi-reference alignment. For each correspondence analysis classification, 100 iterations were performed. All alignments were based on cross-correlations of two-dimensional Radon transforms (25). The resolutions of the final classes were determined using the Fourier ring correlation (26, 27) with a cut-off criterion of five times the noise correlation (FRC5) (28).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of LDAO on the V1 ATPase Activity-- The Ca2+-ATPase activity of the isolated V1 was about 1.7 ± 0.2 µmol of ATP hydrolyzed/min/mg when performed in the presence of an ATP-regenerating system. The effect of various concentrations of LDAO on the ATPase activity of V1 is shown in Fig. 1. The presence of 0.04-0.08% (w/v) of this detergent causes a stimulation of Ca2+-ATPase activity, with a maximal activity of 3.2 ± 0.3 µmol ATP hydrolyzed/min/mg. The activity of V1 in 0.1 and 0.2% LDAO was 52 and 65%, respectively, compared with the activity of the untreated enzyme. Higher concentrations of LDAO (0.4 and 0.5% (w/v)) inhibited the enzyme to less than 10% of total ATPase activity. When the enzyme was applied onto a Native-PAGE in the presence of 0.4% or 0.5% of detergent two new bands (Fig. 1B, I and II) with higher molecular mass were obtained. Both bands were cut out from the gel, destained, and subjected to SDS-PAGE (Fig. 1C), indicating that both contain a V1 subcomplex, consisting of the subunits A, B, D, E, and G and forming oligomers. The ratio of the protein band of V1 relative to the bands I and II was 52:24:25, respectively, based on the quantitation of the staining intensity of the three bands. Therefore, the data imply that the inhibition of ATPase activity is mainly caused by the presence of higher LDAO concentration (see below).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of LDAO on the ATPase activity of V1 from M. sexta. A, the Ca2+-dependent ATPase activity of V1 was assayed at 37 °C using an ATP-regenerating system in the presence of various concentrations of LDAO. B, V1 ATPase (lane 1) and V1 in the presence of 0.4% (lane 2) and 0.5% (lane 3) of LDAO were applied to a native polyacrylamide gel (4-15%; BIO-RAD) and stained with Coomassie Blue G-250. Relative intensities of the protein bands I and II and of V1 (for details see "Results") were determined by scanning the gel of panel B with an Epson (2450 Photo) flat-bed scanner. The intensities of each protein band were digitized and calculated by the program AIDA 3.11.2002 (advanced image data analyzer; RAYTEST). C, the bands I and II in the gel from panel B were cut out, destained, soaked in buffer consisting of 20 mM Tris-HCl (pH 8.1), 50 mM dithiothreitol, and 0.5% SDS and placed onto a 17.5% total acrylamide and 0.4% cross-linked acrylamide gel. The gel was stained with silver (19).

Resolution of V1 into Subcomplexes-- To establish whether the changes of ATPase activity might be caused by the resolution of the enzyme into subcomplexes, size-exclusion chromatography has been performed in the presence of LDAO. V1, incubated with 0.05% LDAO and 2-mercaptoethanol, was applied to a Sephacryl S-300 HR column and a major peak eluted at about 18 min (Fig. 2, panel A) and several smaller ones after 44 min. The fractions of the major peak were pooled and subsequently reapplied onto the same column in absence of detergent. The protein eluted as an active V1 complex (3.0 µmol ATP hydrolyzed/min/mg) without the subunits H, C, and F as shown by SDS-PAGE (Fig. 2, panel A).


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Size-exclusion chromatography of the subcomplexes of V1. A, V1 was treated with 0.05% (panel A), 0.1% (panel A and B) or 0.5% (panel D) of LDAO and in the absence (panel C) or presence of 2-mercaptoethanol (panel A, B, and D). Afterward, the samples were applied onto a SephacrylTM S-300 HR column in the presence of detergent. B, the indicated (black-square) fractions were pooled and reapplied to a SephacrylTM S-300 HR column in the absence of LDAO but presence of 2-mercaptoethanol (panels A-C). Panel D shows the elution diagram after the second column step in the absence of LDAO. The detergent-free samples were subjected to SDS-PAGE and stained either with Coomassie Blue R250 (panel A, blot B), G250 (panel A and C), or with silver (Ref. 19, panel D).

Application of V1 in the presence of 0.1% LDAO and 2-mercaptoethanol to a Sephacryl S-300 HR column results in a major peak accompanied by a shoulder (24 min) (Fig. 2, panel B). Detergent of the pooled fractions of the major peak was removed as described above. The eluted protein with an ATPase activity of 0.9 ± 0.1 µmol ATP hydrolyzed/min/mg contains a six-subunit enzyme composed of the polypeptides A, B, H, E, G, and F (Fig. 2, panel B).

Several lines of evidence indicate that redox-modulation of the V-ATPase, proposed as a mechanism of regulation of this complex (29-31), leads to structural changes in the enzyme (3, 29, 32). In this connection we incubated V1 ATPase with 0.1% LDAO in the absence of the reducing agent, 2-mercaptoethanol. Size-exclusion chromatography of this mixture, which was done as described above, resulted in an exclusion diagram with a main peak at 21 min and several minor peaks at 53 min (Fig. 2, panel C). SDS-PAGE of the main peak, which was freed of detergent by chromatography in the presence of 2-mercaptoethanol, yielded an A3B3EG subcomplex (Fig. 2, panel C) with an ATPase activity of 1.0 ± 0.1 µmol ATP hydrolyzed/min/mg.

To study the inhibitory effect of high LDAO concentrations (>=  0.5%) as described above in more detail, V1 was incubated with 0.5% detergent plus 2-mercaptoethanol and applied to the column. The protein eluted as a broad peak. Detergent was removed by a subsequent column step (Sephacryl S-300 HR), and three peaks were eluted (Fig. 2, panel D). The first peak contained a V1 complex without the subunits C and F, followed by a subcomplex consisting of the subunits A, B, and E in a stoichiometry of 1.8:1:1, based on the quantitation of the staining intensity of the three bands of theses subunits, respectively (Fig. 2, panel D). The ATPase activity of the V1(-C,F) complex was 0.8 µmol ATP hydrolyzed/min/mg, whereby the A2BE subcomplex had lost its enzyme activity. Peak three contained only detergent. Thus, the V1 ATPase is mainly inhibited by the presence of 0.5% LDAO (Fig. 1B) and dissociates in subcomplexes after removing of the detergent.

Electron Microscopic Characterization of the A3B3EG Subcomplex-- Electron micrographs of the negatively stained A3B3EG subcomplex show a homogenous distribution of this complex (Fig. 3). A visual representation of map factor 1 versus 2 of the first correspondence analysis of the A3B3EG subcomplex (Fig. 4) reveals the variation of the subcomplex. Particles with a well pronounced pseudo-hexagonal arrangement and a centrically seventh mass can be observed (indicated by arrows in Fig. 4), as well as particles, which are most probably slightly tilted, with an elongated seventh mass toward the bottom and left side of the map.


View larger version (88K):
[in this window]
[in a new window]
 
Fig. 3.   Electron micrograph of the negatively stained A3B3EG complex. Scale bar represents 50 nm.


View larger version (163K):
[in this window]
[in a new window]
 
Fig. 4.   Viusal representation of the factor map (factor 1 versus 2) obtained by correspondence analysis of an image set of 2080 particles. The map gives an overview over the variations in the set of images (images close to each other are similar, images far from each other most dissimilar). The hexagonal view is indicated by arrows. Toward the bottom and left more asymmetrical views can be observed.

Three averages of the A3B3EG subcomplex calculated from 501, 490, and 456 particles (Fig. 5) show a pseudo-hexagonal arrangement of six protein densities with an overall diameter of ~115 Å. This is in agreement with structural data of the V1 ATPase determined recently by electron microscopy (13), which likewise showed six major masses of density that were grouped in a pseudo-hexagonal arrangement with a diameter of ~130 Å. The center of the particle shows a seventh mass, which would be consistent with the presence of a central stalk. Fig. 5 (panel II) shows an average in which an additional density can be observed near one of the outer densities. This may be caused by small tilts of the A3B3EG subcomplex perpendicular to its hexagonal axis, sufficient to displace the central mass without apparent distortion of the outer hexagon. In the previous three-dimensional study of the V1 ATPase (9) it was observed that many particles exhibited an inclination of about 20° relative to the on-axis view. The resolution of the three averaged images was 29 Å, as determined by Fourier ring correlation FRC5 (28) (Fig. 5B).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 5.   Average images of the A3B3EG complex. A, the averages of I, II, and III were calculated from 501, 490, and 456 particles, respectively. Bar represents 10 nm. B, Fourier ring correlation curve (solid line) and a noise correlation reference curve (5/radical N for FRC5) for average I, which is representative for all averages. The resolution of the averages were determined to be 29 Å using the FRC5 criterion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The results presented here show that addition of various amounts of the zwitterionoic detergent LDAO to the V1 ATPase from M. sexta induces the dissociation of the enzyme into the subcomplexes A3B3DEG, A3B3HEGF, and A3B3EG with altered rates of ATPase activity. Below the critical micelle concentration of 0.05% in a buffer >=  pH 7.0 (33) the monomeric detergent leads to release of the subunits H, C, and F combined with an enhancement of ATP-hydrolytic activity. By assaying the A3B3DEG subcomplex after chromatography in the absence of LDAO, where activation of ATPase activity was essentially the same, it was possible to conclude that the enhancement is either due to the release of an ensemble of the subunits H, C, and F or to the removal of one of these three polypeptides (see below).

Increasing LDAO concentrations to 0.1% shows that the reduced V1 can be converted into an A3B3HEGF complex, whereby under oxidizing conditions the complex dissociates further into a smaller subcomplex composed of the subunits A, B, E, and G. Most recent studies on the oxidized and reduced V1 ATPase from M. sexta have also shown that the subunits H and F are less accessible to proteolytic digestion in the reduced form (32). This is in line with the data presented in which the presence of 0.1% LDAO leads to the dissociation of these two subunits only under oxidizing conditions. The ATPase activity of the A3B3HEGF and A3B3EG subcomplex after detergent removal is about 52 and 58% of that of V1 ATPase, respectively (see above). This observation that both complexes lacking subunit D still retain a high ATPase activity is important, because this subunit has been proposed as a structural and functional homologue of the gamma  subunit of F-ATPases (34, 35). Recently, prolonged digestion of the M. sexta V1 with trypsin resulted in an active enzyme lacking subunit D (13). This is consistent with a previous report of Xie (36), which suggested that subunit D is not essential for ATP hydrolysis in the bovine enzyme. However, the resulting hydrolytic-active A3B3EG-domain of M. sexta forms what can be called a "core" complex (see Fig. 6). For the H+-ATPase of clathrin-coated vesicles it has been demonstrated that at least the four subunits A, B, C, and E are necessary for ATP hydrolysis (37, 38). In these studies recombinant subunits E and C were reconstituted with a biochemically prepared A-B complex, resulting in an A3B3E subcomplex with reproducible reconstitution of ATPase activity (37) and a 6-fold stimulation of hydrolytic activity when recombinant C was added to the A3B3E complex. Addition of subunit C to the A-B subcomplex resulted in slight activity (38). Most recent studies reveal that subunit C (39-41) together with subunit H (42, 43) might play a role in bridging the V1 and VO domains rather than acting as a core subunit (40) (Fig. 6). Together with our data this reinforces the hypothesis that at least the subunits A, B, and E might constitute part of the minimal ATP-hydrolytic core of V1. The presence of both stalk subunits G and F in the A3B3E complex is not surprising because of their close proximity in the enzyme (Fig. 6), demonstrated by cross-linking experiments in the V1 ATPase from yeast (43), clathrin-coated vesicles (44), and M. sexta (13). Furthermore, an arrangement of the subunits A, B, E, and G close to each other is in accordance with the formation of an A2BEG subcomplex of M. sexta upon treatment with chaotopic iodide, but without hydrolytic activity (13, 45). Notably, from recent studies using a bifunctional cross-link reagent it has been proposed that subunit E (Vma4p), with an apparent molecular mass of 26 kDa, is located at the outer surface of the A3B3 hexamer in the V1VO ATPase from yeast (46). Close inspection of the recently determined three-dimensional structure of the V1 ATPase from M. sexta (9) clearly indicates that no additional mass is located at the outer surface of the A3B3 hexamer of V1 alone.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 6.   Model of the subunit arrangement in the V1 ATPase from M. sexta. The V1 subunits are placed into a surface view of the structure of the V1 ATPase without subunit C (light gray) determined at 18 Å resolution (9). The model is based on the combination of presented biochemical and structural data and topology studies described recently (13, 14). Because a nucleotide-dependent arrangement of subunit E has been observed in V1 (13) the model presented describes the subunit topology in the presence CaATP, in which E-G and E-F cross-linked products can be formed. The position of subunit C (dark gray) bases on most recent electron micrographs of a V1 subcomplex of the C. fervidus V-ATPase (41) and the V1(-C) complex from M. sexta (9), indicating that subunit C is located at the bottom of the central stalk (9, 41) and connected to the center of VO (41).

However, a direct correlation between the dissociation of the individual subunits H, C, and F forming different subcomplexes and the change of ATPase activity is not clear at the moment. Nevertheless, the increase of hydrolytic activity after removal of the three subunits H, C, and F implies that it needs such an ensemble of subunits, whose simultaneous dissociation might lead to structural and thereby functional alterations in the remaining subcomplex.

To date the best structural model for M. sexta V1 ATPase is an 18 Å three-dimensional reconstruction obtained from single-particle images of the molecules embedded in deep stain (9). The V1 model consists of a headpiece of 130 Å in diameter, with the six major subunits A and B alternating around a cavity and a compact central stalk. Inside the cavity the stalk can be seen connected to only two of the major subunits (9). Zero-length cross-linking studies (13), photo-affinity labeling (14) as well as the formation of an A2BE complex (see Fig. 2D) indicated that the catalytic A subunit of V1 is close to subunit E (Fig. 6), which is proposed to be located in the cavity of the A3B3 hexamer (9, 1314). The presented two-dimensional averages of the A3B3EG subcomplex from M. sexta determined from negatively stained specimens reveals a hexagonal modulation as seen previously in projection images of V1 molecules in stain (1321) and are interpreted as three copies each of the nucleotide-binding subunits A and B (9). The two-dimensional analysis of the A3B3EG domain shows also that the A3B3 hexamer is occluded by a seventh mass centrally to the hexamer. This hexagonal modulation of the A3B3 hexamer with an internal seventh mass is remarkably similar to the subunit arrangement of the closely related F1 complex. Cryo-electron micrographs of F1 from Escherichia coli that were labeled with Fab fragments of monoclonal antibodies (47) or with monomaleimido gold (48-50) have shown that the additional seventh mass includes the central stalk subunits gamma  and epsilon .

In summary, the amphipathic detergent LDAO is a useful tool for examining the composition of the ATP-hydrolytic core of the M. sexta V1 ATPase. The data presented show that at least the subunits A, B, E, and G are essential for ATP-hydrolysis in this enzyme. Previous experiments (13, 14) on the interactions between the different subunits in the M. sexta V1 using differential protease sensitivity and cross-linking studies and the results presented here suggested that subunit E, from which no isoform exists in Manduca V-ATPase (51, 52), contributes to the coupling element in the V1 ATPase of M. sexta.

    FOOTNOTES

* This research was supported by Grant GR 1475/9-1 from the Deutsche Forschungsgemeinschaft and by the NSF Grant DBI 95 155 18.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ These authors contributed equally to this work.

|| To whom correspondence should be addressed: Universität des Saarlandes, Fachrichtung 2.5-Biophysik, Universitätsbau 76, D-66421 Homburg, Germany. Tel.: 49-6841-162-6085; Fax: 49-6841-162-6086; E-mail: ggrueber@med-rz.uni-saarland.de.

Published, JBC Papers in Press, October 31, 2002, DOI 10.1074/jbc.M208623200

    ABBREVIATIONS

The abbreviation used is: LDAO, N,N-dimethyldodecylamine N-oxide.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Perzov, N., Padler-Karavani, V., Nelson, H., and Nelson, N. (2001) FEBS Lett. 504, 223-228[CrossRef][Medline] [Order article via Infotrieve]
2. Wieczorek, H., Brown, D., Grinstein, S., Ehrenfeld, J., and Harvey, W. R. (1999) Bioessays 21, 637-648[CrossRef][Medline] [Order article via Infotrieve]
3. Nishi, T., and Forgac, M. (2002) Nat. Rev. Mol. Cell. Biol. 3, 94-103[CrossRef][Medline] [Order article via Infotrieve]
4. Graham, L. A., Powell, B., and Stevens, T. H. (2000) J. Exp. Biol. 203, 61-70[Abstract]
5. Bowman, E. J., and Bowman, B. J. (2000) J. Exp. Biol. 203, 97-106[Abstract]
6. Grüber, G., Wieczorek, H., Harvey, W. R., and Müller, V. (2001) J. Exp. Biol. 204, 2597-2605[Abstract/Free Full Text]
7. Boekema, E. J., Ubbink-Kok, T., Lolkema, J. S., Brisson, A., and Konings, W. N. (1998) Photosyn. Research 57, 267-273[CrossRef]
8. Svergun, D. I., Konrad, S., Huss, M., Koch, M. H., Wieczorek, H., Altendorf, K., Volkov, V. V., and Grüber, G. (1998) Biochemistry 37, 17659-17663[CrossRef][Medline] [Order article via Infotrieve]
9. Radermacher, M., Ruiz, T., Wieczorek, H., and Grüber, G. (2001) J. Struct. Biol. 135, 26-37[CrossRef][Medline] [Order article via Infotrieve]
10. Sumner, J. P., Dow, J. A. T., Earley, F. G., Klein, U., Jäger, D., and Wieczorek, H. (1995) J. Biol. Chem. 270, 5649-5653[Abstract/Free Full Text]
11. Wieczorek, H., Grüber, G., Harvey, W. R., Huss, M., Merzendorfer, H., and Zeiske, W. (2000) J. Exp. Biol. 203, 127-135[Abstract]
12. Radermacher, M., Ruiz, T., Harvey, W. R., Wieczorek, H., and Grüber, G. (1999) FEBS Lett. 453, 383-386[CrossRef][Medline] [Order article via Infotrieve]
13. 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[CrossRef][Medline] [Order article via Infotrieve]
14. Schäfer, H. J., Coskun, Ü., Eger, O., Godovac-Zimmermann, J., Wieczorek, H., Kagawa, Y., and Grüber, G. (2001) Biochem. Biophys. Res. Commun. 286, 1218-1227[CrossRef][Medline] [Order article via Infotrieve]
15. Gräf, R., Harvey, W. R., and Wieczorek, H. (1996) J. Biol. Chem. 271, 20908-20913[Abstract/Free Full Text]
16. Schägger, H., Cramer, W. A., and v. Jagow, G. (1994) Anal. Biochem. 217, 220-230[CrossRef][Medline] [Order article via Infotrieve]
17. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
18. Weber, K., and Osborne, M. (1969) J. Biol. Chem. 244, 4406-4412[Abstract/Free Full Text]
19. Damerval, C., le Guillioux, M., Blaisomeau, J., and de Vienne, D. (1987) Electrophoresis 8, 158-159
20. Lötscher, H.-R., de Jong, C., and Capaldi, R. A. (1984) Biochemistry 23, 4140-4143[Medline] [Order article via Infotrieve]
21. Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996) J. Struct. Biol. 11, 190-199[CrossRef]
22. Marabini, R., Masegosa, I. M., San Martin, M. C., Marco, S., Fernández, J. J., de la Fraga, L. G., Vaquerizo, C., and Carazo, J. M. (1996) J. Struct. Biol. 116, 237-240[CrossRef][Medline] [Order article via Infotrieve]
23. van Heel, M., and Frank, J. (1981) Ultramicroscopy 6, 187-194[Medline] [Order article via Infotrieve]
24. Diday, E. (1971) Rev. Stat. Appl. 19, 19-34
25. Radermacher, M. (2002) Scanning Microsc. Int. Suppl., in press
26. Saxton, W. O., and Baumeister, W. (1982) J. Microscopy 127, 127-138[Medline] [Order article via Infotrieve]
27. van Hell, M., Keegstra, W., Schutter, W., and van Bruggen, E. J. F. (1982) Life Chemistry Reports (Suppl. 1) , pp. 69-73, EMBO Workshop, Leeds
28. Radermacher, M. (1988) J. Electron Microsc. Tech. 9, 359-394[Medline] [Order article via Infotrieve]
29. Forgac, M. (2000) J. Exp. Biol. 203, 71-80[Abstract]
30. Dschida, W. J. A., and Bowman, B. J. (1995) J. Biol. Chem. 270, 1557-1563[Abstract/Free Full Text]
31. Oluwatosin, Y. E., and Kane, P. M. (1995) J. Biol. Chem. 272, 28149-28157[Abstract/Free Full Text]
32. Grüber, G., Svergun, D. I., Godovac-Zimmermann, J., Harvey, W. R., Wieczorek, H., and Koch, M. H. J. (2000) J. Biol. Chem. 275, 30082-30087[Abstract/Free Full Text]
33. Helenius, A., McCaslin, D. R., Fries, E., and Tanford, C. (1979) Methods Enzymol. 56, 734-749[Medline] [Order article via Infotrieve]
34. Nelson, H., Mandiyan, S., and Nelson, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 497-501[Abstract]
35. Xu, T., and Forgac, M. (2000) J. Biol. Chem. 275, 22075-22081[Abstract/Free Full Text]
36. Xie, X. S. (1996) J. Biol. Chem. 271, 30980-30985[Abstract/Free Full Text]
37. Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867[Abstract/Free Full Text]
38. Peng, S. B., Zhang, Y., Tsai, S. J., Xie, X. S., and Stone, D. K. (1994) J. Biol. Chem. 269, 11356-11360[Abstract/Free Full Text]
39. Kane, P. M., Tarsio, M., and Liu, J. (1999) J. Biol. Chem. 274, 17275-17283[Abstract/Free Full Text]
40. Curtis, K. K., Francis, S. A., Oluwatosin, Y., and Kane, P. M. (2002) J. Biol. Chem. 277, 8979-8988[Abstract/Free Full Text]
41. Chuban, Y., Ubbink-Kok, T., Keegstra, W., Lolkema, J. S., and Boekema, E. J. (2002) EMBO reports 10, 982-987[CrossRef]
42. Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286-18292[Abstract/Free Full Text]
43. Tomashek, J. J., Graham, L. A., Hutchins, M. U., Stevens, T. H., and Klionsky, D. J. (1997) J. Biol. Chem. 272, 26787-26793[Abstract/Free Full Text]
44. Xu, T., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 28909-28915[Abstract/Free Full Text]
45. Huss, M. (2001) Structure, Function and Regulation of the Plasmamembrane V-ATPase from Manduca sextaPh. D. thesis , University of Osnabrück, Germany
46. Arata, Y., Baleja, J. D., and Forgac, M. (2002) J. Biol. Chem. 277, 3357-3363[Abstract/Free Full Text]
47. Gogol, E. P., Aggeler, R., Sagermann, M., and Capaldi, R. A. (1989) Biochemistry 28, 4717-4724[Medline] [Order article via Infotrieve]
48. Wilkens, S., and Capaldi, R. A. (1992) Arch. Biochem. Biophys. 299, 105-110[Medline] [Order article via Infotrieve]
49. Gogol, E. P. (1994) Microsc. Res. Tech. 27, 294-306[Medline] [Order article via Infotrieve]
50. Wilkens, S., and Capaldi, R. A. (1994) Biol. Chem. Hoppe-Seyler 375, 43-51[Medline] [Order article via Infotrieve]
51. Gräf, R., Harvey, W. R., and Wieczorek, H. (1994) Biochim. Biophys. Acta 1190, 193-196[Medline] [Order article via Infotrieve]
52. Merzendorfer, H., Reineke, S., Zhao, X.-F., Jacobmeier, B., Harvey, W. R., and Wieczorek, H. (2000) Biochim. Biophys. Acta 1467, 369-379[Medline] [Order article via Infotrieve]


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