From the 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
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ABSTRACT |
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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).
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.
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 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).
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).
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 ( 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.
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).
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 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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/Å2). 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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).
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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 ( ) 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).
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.
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Fig. 3.
Electron micrograph of the negatively stained
A3B3EG complex. Scale bar
represents 50 nm.
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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.
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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/ 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
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).
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.
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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, 13, 14). 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 (13, 21) 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 and
.
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.
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FOOTNOTES |
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* 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
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ABBREVIATIONS |
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The abbreviation used is: LDAO, N,N-dimethyldodecylamine N-oxide.
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REFERENCES |
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