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(Received for publication, August 16,
1994; and in revised form, October 27, 1994) From the
The plasma membrane V-ATPase of Manduca sexta larval
midgut is an electrogenic proton pump located in goblet cell apical
membranes (GCAM); it energizes, by the voltage component of its proton
motive force, an electrophoretic K
H V-ATPases usually energize the transport of acid into
organelles or out of cells (see Harvey and Nelson(1992)). The plasma
membrane V-ATPase in the larval midgut of M. sexta is an
exception to this rule since it does not energize acid transport to the
cell exterior (Wieczorek, 1992). By contrast, it produces, due to the
absence of functional anion channels, a high transmembrane voltage in
excess of 250 mV across the goblet cell apical membrane (Dow, 1992)
that drives an electrophoretic K Maintaining the very high active K In this investigation, we report on events
during a larval/larval moult. We measured a reversible switching off
and on of K
Figure 4:
SDS-polyacrylamide gel electrophoresis of
highly purified goblet cell apical membranes. Silver stains of GCAM
isolated from fifth instar feeding larvae (lanea) or
from stage E moulting larvae (laneb). N,N`-dicyclohexylcarbodiimide labeling of GCAM
isolated from fifth instar feeding larvae (lanec) or
from stage E moulting larvae (laned). 0.5 µg of
protein was applied on each lane. Arrows indicate defined
V-ATPase subunits.
Figure 5:
Western blots of partially purified goblet
cell apical membranes. 10 µg of GCAM isolated from fifth instar
feeding larvae (lanesb and d) and from
stage E moulting larvae (lanesc and e).
Immunostaining was performed with a monoclonal antibody against 67-kDa
subunit A (lanesb and c) or with
monospecific polyclonal antibodies against the 14-kDa subunit (lanesd and e). Lanea,
purified V-ATPase (2 µg) immunostained with anti-holenzyme serum
(Wieczorek et al., 1991) to indicate the position of
the subunits.
Figure 6:
Dot blots of partially purified goblet
cell apical membranes after SDS treatment. Serial dilutions of GCAM
isolated from fifth instar feeding larvae (rows labeled a) or from stage E moulting larvae (rows labeled b), spotted onto the nitrocellulose membrane (1 µl/spot),
air dried, and probed with anti-V-ATPase antibodies. i,
polyclonal antiserum against V-ATPase holoenzyme; ii,
monoclonal antibody against 67-kDa subunit A; iii, monoclonal
antibody against 28-kDa subunit E (cf.
Gräf et al., 1994a); iv,
monospecific polyclonal antibodies against the 14-kDa
subunit.
As primary antibodies for
immunostaining, a polyclonal rabbit immune serum against the native
V-ATPase (Wieczorek et al., 1991), monospecific rabbit
antibodies to the 14-kDa subunit (Gräf et
al., 1994b) or protein G-purified monoclonal mouse antibodies to
the 67-kDa subunit A) or to the 28-kDa subunit E ) were used.
Figure 1:
Time course of TEV during moult.
Timings relative to formation of the head capsule at time 0,
corresponding to stage C. Each time point is the mean of at least four
independent measurements. Errorbars indicate
standard error of the mean; when not visible, they are smaller than the
plot symbols. The time course of changes in the TEV agreed with those
in the short circuit current (not shown), indicating that TEV was a
good measure of active K
These
results provide evidence that the K
Figure 2:
ATP-dependent proton transport as
determined by the fluorescence quenching of acridine orange. Original
data from representative experiments on goblet cell apical membrane
vesicles. The reactions were started by the addition of 1 mM MgCl
Figure 3:
Immunocytochemical labeling of V-ATPase.
Cryosections of M. sexta posterior midgut, labeled with the
monoclonal antibody directed to the peripheral 56-kDa subunit B and an
undefined 20-kDa polypeptide are shown. a, fourth instar
feeding larva with specific labeling of the goblet cell apical
membrane; b, stage E moulting larva without immunoreactivity; c, fifth instar feeding larva with regained immunoreactivity; d, fifth instar feeding larva, control incubation without
primary antibody. Scale, 10 µm. The same results were obtained when
the monoclonal antibody directed to the peripheral 67-kDa subunit A was
used (not shown).
To verify that the difference
in band patterns on SDS gels accurately reflected differences in the
V-ATPase subunit composition, Western blots after SDS-polyacrylamide
gel electrophoresis of partially purified GCAM from fifth instar
feeding larvae and from stage E moulting larvae were stained with a
monoclonal antibody to the 67-kDa subunit and with monospecific
antibodies to the 14-kDa subunit (Fig. 5). GCAM from stage E
moulting larvae exhibited no (67-kDa subunit) or nearly no (14-kDa
subunit) immunoreactivity as compared with GCAM from fifth instar
feeding larvae. To quantify the loss of V Here we report on functional and structural changes in the
plasma membrane V-ATPase of larval M. sexta midgut during
moulting. K During a moult,
the number of midgut cells increases about 4-fold by intercalation of
new goblet and columnar cells between the mature differentiated cells
already present in the epithelium (Baldwin and Hakim, 1991). The mature
cells remain intact and mostly unchanged. In stage E moulting larvae,
the newly developing cells have already grown and differentiated to a
columnar shape and extend regularly among the mature cells. Therefore,
one could argue that lowered specific enzyme activity may be due to the
increased tissue mass of the newly emerged cells, especially that of
goblet cells, in which the plasma membrane V-ATPase has not yet been
assembled. This explanation may be true in part. However, the
immunocytochemical results clearly demonstrated that at moulting stage
E, antibodies against peripheral subunits do not label the mature
goblet membrane. Furthermore, analysis of the protein pattern of
inactivated GCAM revealed that only the peripheral subunits were
missing from the V-ATPase subunit profile. Both findings strongly
suggest that V Control of pump activity in vivo by regulation of the
number of V-ATPase molecules has been described in kidney epithelial
cells (Brown et al., 1991). In these cells, pump concentration
is increased by integration of vesicles heavily loaded with complete
V The
disassembly of V The
present study on the plasma membrane V-ATPase in M. sexta midgut is the first clear demonstration of in vivo control of V-ATPase activity by regulation of the association of
V The regulatory signals for disassembly or assembly of the
V
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
Z26918[GenBank]. ZENECA Agrochemicals in the
U.K. is part of ZENECA Ltd., registered in England No. 2710846.
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5649-5653
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/nH antiport and thus K secretion (Wieczorek, H.,
Putzenlechner, M., Zeiske, W., and Klein, U.(1991) J. Biol Chem. 266, 15340-15347). Midgut transepithelial voltage,
indicating net active K transport, was found to be
more than 100 mV during intermoult stages but was abolished during
moulting. Simultaneously, ATP hydrolysis and ATP-dependent proton
transport in GCAM vesicles were found to be reduced to 10-15% of
the intermoult level. Immunocytochemistry of midgut cryosections as
well as SDS-polyacrylamide gel electrophoresis and immunoblots of GCAM
demonstrated that loss of ATPase activity paralleled the disappearance
of specific subunits. The subunits missing were those considered to
compose the peripheral V
sector, whereas the membrane
integral V
subunits remained in the GCAM of moulting
larvae. The results provide, for the first time, evidence that a
V-ATPase activity can be controlled in vivo by the loss of the
peripheral V domain.
translocating V-ATPases are ubiquitous in
endomembranes (Anraku et al., 1992) but also occur in many
plasma membranes (Gluck et al., 1992). The plasma membrane
V-ATPase in the larval midgut epithelium of Manduca sexta (Lepidoptera, Sphingidae) is a typical representative of these
heteromultimeric proteins (Wieczorek, 1992): amino acid sequences
deduced from cDNAs encoding four M. sexta V-ATPase subunits
show substantial similarities to subunits of V-ATPases from other
sources (67-kDa subunit A (Gräf et al.,
1992); 56-kDa subunit B (Novak et al., 1992); 28-kDa subunit E
(Gräf et al., 1994a); 17-kDa subunit c
(Dow et al., 1992)). The peripheral V part of the M. sexta V-ATPase appears to consist of at least the subunits
A, B and E, which are common to all V-ATPases, along with a 14-kDa
subunit (Gräf et al., 1994b), which has
since been identified and sequenced in Drosophila melanogaster (EMBL/GenBank accession number Z26918) and yeast (Graham et al.,
1994; Nelson et al., 1994) although no homologous subunits have yet
been identified in vertebrates. The membrane integral V part appears to consist of at least the 43-kDa subunit and the
17-kDa subunit c (Gräf et al., 1994b); the
latter subunit evidently forms the proton-conducting pore, and, like
its counterparts in other V-ATPases (Mandel et al., 1988), it
is labeled by N,N`-dicyclohexylcarbodiimide. ()/nH antiport (Wieczorek et al., 1991). The combined activity
of V-ATPase and K/nH antiport in the
same membrane results in net K secretion. Thus both
elements constitute the electrogenic active transport mechanism, which
had already been detected in the lepidopteran midgut 30 years ago
(Harvey and Nedergaard, 1964). The K active transport
mechanism not only energizes the absorption of amino acids but also, at
least in part, the alkalinization of the midgut lumen; the luminal
fluid produced is the most alkaline in a biological system and can
exceed pH 12 (Dow, 1984; Harvey, 1992; Dow, 1992). The unique potency
of the V-ATPase in lepidopteran midgut, and the ease with which it can
be measured by the electrical signature of the transport, render it an
attractive model system for studies of regulation of the V-ATPase. transport rates
measured must require a huge amount of energy; the minimum cost has
been estimated to be 10% of a larva's total ATP production (Dow,
1984). Therefore one would expect, for reasons of economy, that
K transport should be strongly regulated. However,
larvae taken at different times of day or at different nutritional
states show uniformly high rates of transport, as assessed by
electrical and pH measurements. The only indication for regulation of
ion transport was given by Cioffi(1984), who cited her own unpublished
evidence that K fluxes fell when larvae moulted from
one instar to the next. transport that was correlated with active
and inactive states of the V-ATPase. These results provide evidence,
for the first time, that a V-ATPase activity can be controlled in
vivo by the loss of the peripheral V domain.
Rearing and Staging of Larvae
M. sexta larvae were reared on a standard artificial diet (Yamamoto, 1969;
Bell and Joachim, 1974). Larvae were maintained in a 16-h light/8-h
dark photo period at 25-27 °C. Eggs were either kindly
supplied by Dr. Stuart Reynolds at the University of Bath or were from
the Munich laboratory M. sexta culture. Larvae were studied
during the final (fourth to fifth instar) larval moult. Staging of
larvae was based upon development of external morphological features
(Baldwin and Hakim, 1991) as summarized below. Fourth instar intermoult
larvae feed and gain mass until they attain the weight of approximately
1.5 g (stage A). They stop feeding upon entry into the moult (stage B).
Development of an extensively green head capsule and dropping of the
head are the first external indications of entry into the moult (stage
C). The following processes up to ecdysis take approximately 24 h in
total. The base of the head capsule becomes opaque (stage D) and then
the head capsule becomes entirely opaque, and the mandibles are present
but unpigmented (stage E). The mandibles become pigmented (stage F),
and finally ecdysis occurs. The onset of ecdysis is defined as the
point when integument begins to be shed, and the larvae lose their head
capsule (stage G). About 2 h after the moult (stage H), the larvae
resume feeding and are then considered to be fifth instar intermoult
larvae. Where not stated otherwise, moulting stage E larvae weighing
about 1.5 g, approximately 12-18 h after development of a green
head capsule (``stage E moulting larvae''), were used and
compared with feeding fifth instar intermoult larvae of about 5-7
g (``fifth instar feeding larvae'').Transepithelial Voltage (TEV)
Voltage measurements were taken at hourly
time points during the time course of the fourth to fifth instar moult.
TEV across the midgut was measured using an Ussing-type chamber (Dow et al., 1985). Measurements were made on a flat sample of
mid-midgut stretched across an aperture 6 mm in diameter. This was
inserted into the chamber using a holder. ``Manduca saline'' (Dow and O'Donnell, 1990) was oxygenated and
circulated on either side of the epithelium. Open circuit voltage was
measured using silver chloride electrodes and displayed on a chart
recorder via a 
Measurements
1 high impedance preamplifier. Stable TEV
readings were obtained 15 min after mounting the midgut in the chamber. Purification of Goblet Cell Apical Membranes
(GCAM)
Preparation of GCAM from fifth instar feeding larvae was
carried out according to Cioffi & Wolfersberger(1983) and Wieczorek et al.(1990). The preparation of membranes from the much
smaller stage E moulting larvae required some subtle modifications. After removal of the longitudinal muscles, the tissue pieces
from 10 stage E moulting larvae were pooled and sonicated. Sonication
time was reduced by approximately 30%. The sample was washed,
aspirated, and filtered as described for fifth instar feeding larvae.
The filtrate was spun at 250 g for 2 min, and the
resulting supernatant was discarded. Pellets from 30 larvae were
accumulated on ice. Following the published protocols, the combined
pellets were layered onto a 45:41:37% (w/w) discontinuous sucrose
density gradient and spun overnight at 77,000 g. Band
2 contained partially purified GCAM. Thirty stage E moulting larvae
yielded approximately 5 µg of partially purified GCAM protein/100
µg (wet weight) of tissue. This figure was in the same order of
magnitude as the protein yield from fifth instar feeding larvae.
Preparation of highly purified membranes from partially purified GCAM
was as described previously (Wieczorek et al., 1990).V-ATPase Activity and ATP-dependent H
Assays of V-ATPase activity in partially
purified GCAM were performed at an ambient temperature of 24-27
°C and consisted of approximately 25-30 µg of membrane
protein/ml, 1 mM Tris-ATP, 1 mM MgCl Transport, 10
mM MOPS-Tris (pH 7.0), 0.5 mM sodium azide, and 0.1
mM sodium orthovanadate. The reaction was started by the
addition of ATP/MgCl, incubated for 5 min, and stopped by
immersion of the samples in liquid nitrogen. All further conditions,
including the determination of inorganic phosphate, were as described
previously (Wieczorek et al., 1990). ATP-dependent vesicle
acidification was measured by the quench in fluorescence of acridine
orange (Wieczorek et al., 1989). Assays had the same
composition as for the determination of V-ATPase activity except the
inclusion of 0.9 µM acridine orange.Immunocytochemistry
Larval midguts were prepared
under ice-cold fixative (2.5% glutaraldehyde, 2% formaldehyde in 0.1 M Sørensen phosphate buffer (pH 7.4)) and fixed in the
same solution for 1-2 h on ice. Procedures for embedding,
cryosectioning, and immunolabeling were modified slightly from those
described by Klein et al.(1991). Two protein G-purified
monoclonal antibodies to the native V-ATPase, (
)one to the
67-kDa subunit of the V-ATPase) and one to the 56-kDa subunit B, and an
undefined polypeptide of about 20 kDa) were applied for labeling.
Incubations with the primary antibody solutions were carried out
overnight at room temperature. The antibodies were visualized by 5-nm
gold-conjugated secondary antibodies (goat anti-mouse IgG, whole
molecule, Sigma) diluted as recommended by the producer. For light
microscopical inspection, the gold particle labeling was intensified by
silver staining (silver enhancement kit, Boehringer Mannheim). The
sections were rinsed for 5 min in phosphate-buffered saline (140 mM NaCl, 10 mM NaHPO
, 3 mM KCl, 0.02% NaN (pH 7.4)) and desalted by rinsing 6
times for 2 min in distilled water. Silver labeling was completed after
about 15 min in the dark. The reaction was stopped by rinsing 5 times
for 2 min in distilled water. Finally the sections were covered by
Mowiol 4-88 (Hoechst, Frankfurt, Germany) and investigated with a
Zeiss Axioplan light microscope by normal or difference interference
illumination. To test for unspecific binding of secondary antibody or
unspecific silver depositions, control incubations were performed with
blocking solution instead of primary antibody or without any antibody
incubation but treatment for silver enhancement only.Other Methods
For dot blots and for
SDS-polyacrylamide gel electrophoresis, samples were resuspended in 125
mM Tris-HCl (pH 6.8), 2% SDS, and 2% 
-mercaptoethanol and
with additional 5% sucrose and 0.05% bromphenol blue in the case of
SDS-polyacrylamide gel electrophoresis. Samples were heated at 95
°C for 30 s (see Fig. 4) or 5 min (see Fig. 5and Fig. 6). SDS-polyacrylamide gel electrophoresis, Western
blotting on nitrocellulose membranes (BA85), immunostaining, and
protein determination with Amido Black were performed as described
previously (Schweikl et al., 1989; Wieczorek et al.,
1990, 1991). Silver-stained protein was visualized according to Merril et al.((1) . Labeling with N,N`-dicyclohexylcarbodiimide was performed according
to Zheng et al.(1992).
The alkaline phospatase-conjugated secondary antibody probe was
either goat anti-rabbit IgG or goat anti-mouse IgG (Sigma).
Net Epithelial Ion Transport Is Abolished during the
Moult
Fourth instar feeding larvae generated a transepithelial
voltage of +91 ± 10 mV (n = 4, mean
± S.D.), lumen side positive (Fig. 1). The high TEV
indicated that active electrogenic K transport across
the GCAM to the midgut lumen was as intense as it is in fifth instar
feeding larvae. Approximately 2 h before development of the head
capsule, the larvae stopped feeding and purged the gut lumen of food
(stage B). This behavior, the first sign of entry into the moult, did
not affect the TEV, which remained stable until development of the head
capsule (stage C), whereupon it fell abruptly to -5 ± 4 mV (n = 4, mean ± S.D.), lumen side negative. The
slightly negative residual voltage, presumably due to unknown ion
transport processes in the epithelium, which take longer to shut down,
was reproducible and could be inhibited with 1 mM azide
(results not shown). Approximately 1 h after head capsule development,
the TEV had stabilized at 0 mV and remained at zero until ecdysis
(stages D-F). Upon ecdysis, the TEV rose to +32 ± 13
mV (n = 5, mean ± S.D.), lumen side positive.
The TEV continued rising steadily until it leveled off at approximately
+100 mV some 4 h after ecdysis. The re-establishment of the active
K transport mechanism after ecdysis was not triggered
by the resumption of feeding, as it could be observed in larvae
deprived of access to food since the start of the moult.
transport.
transport
mechanism is active during feeding stages and is inactive during moult.
To analyze further the differences between active and inactive states,
we used fifth instar feeding larvae to produce membranes with active K transport mechanisms and stage E
moulting larvae to produce membranes with inactive K transport mechanisms. Stage E larvae were
chosen because these larvae were midway between head capsule
development and ecdysis.The V-ATPase Is Inactivated during
Moult
Membrane-bound V-ATPase activity and ATP-dependent proton
transport were assayed on partially purified GCAM preparations.
Specific V-ATPase activity was 0.06 ± 0.02
µmol
mg
min in
stage E moulting larvae compared with 0.39 ± 0.03
µmolmgmin in
fifth instar feeding larvae (n = 3, means ±
S.D.). Thus there was a significant 84% reduction in V-ATPase activity
in moulting larvae. An equally strong decrease was observed for
ATP-dependent proton transport (Fig. 2); the maximal specific
fluorescence quench obtained by using vesicles from stage E moulting
larvae was 8 ± 5% (mean ± S.D., n = 4)
that of fifth instar feeding larvae-derived vesicles.
and stopped by the addition of 20 mM NHCl (final concentrations). Equal concentrations (25
µg/ml) of membrane protein were used in each
assay.
The Immunoreactivity of the GCAM
Disappears
Inactivation of V-ATPase activity and proton
transport during the moult could be accomplished either by inactivation
or by down-regulation of the proton pump. Therefore, cryosections of
larval midgut were probed for immunolocalization of the V-ATPase
molecule with two monoclonal antibodies to peripheral
V-part subunits. In fourth instar feeding larvae (Fig. 3a) and in fifth instar feeding larvae (Fig. 3c), there was intense labeling in the area of
the goblet cell apical membrane, whereas stage E moulting larvae
exhibited only faint or undetectable labeling (Fig. 3b, cf. the control, Fig. 3d). This result means
that in stage E moulting larvae, the mature goblet cells had lost their
immunoreactivity and that the newly developing goblet cells had not yet
gained it.
V
To
determine whether the V-ATPase holoenzyme or just specific subunits
were down-regulated during moult, the polypeptide composition of highly
purified GCAM, isolated from either stage E moulting larvae or fifth
instar feeding larvae, was analyzed by SDS-polyacrylamide gel
electrophoresis (Fig. 4). Although the overall band patterns of
the membrane samples were similar, there were significant differences
in the subunit profiles of the V-ATPase. GCAM from fifth instar feeding
larvae (Fig. 4, lanea) showed the full
complement of defined insect V-ATPase subunits: 67, 56, 43, 28, 17, and
14 kDa. By contrast, in GCAM from stage E moulting larvae (Fig. 4, laneb), the peripheral 67-, 56-,
28-, and 14-kDa V Subunits Are Missing during Moult subunits appeared to be strongly reduced,
whereas the bands representing the integral membrane 43- and 17-kDa
V subunits were stained as strongly or even more strongly
as compared with GCAM from fifth instar feeding larvae. Specific
labeling of the 17-kDa subunit by N,N`-dicyclohexylcarbodiimide demonstrated that this
subunit was present in both membrane preparations (Fig. 4, lanesc and d). Compared with active
membranes from fifth instar feeding larvae, labeling on inactive
membranes from stage E moulting larvae was more intense. The stronger
appearance of the membrane integral subunits in silver staining and
DCCD labeling suggested that these subunits were present as a higher
proportion of total membrane protein. subunits, serial
dilutions of partially purified GCAM from fifth instar feeding larvae
and from stage E moulting larvae were dot blotted and probed with
various antibodies against the V-ATPase. As deduced from staining
intensity, binding of polyclonal antibodies against the holoenzyme
indicated that GCAM from stage E moulting larvae contained
approximately 50% less V-ATPase protein than identical amounts of GCAM
from fifth instar feeding larvae (Fig. 6, i.). GCAM
blots were also probed with the antibodies used for the Western blots
in Fig. 5and with a monoclonal antibody against the 28-kDa
subunit (Fig. 6, ii-iv). All three subunits were
at least 5-10-fold reduced in inactive stage E membranes. Taken
together, results from silver-stained SDS gels, from Western blots and
from dot blots, were consistent with the immunocytochemical results in
which monoclonal antibodies had failed to label the peripheral V subunits.
secretion, which is energized by the
electrogenic proton pumping V-ATPase via an electrophoretic
K/nH antiporter, is switched off
temporarily during the moult. The decrease in K transporting capability is paralleled by an almost complete loss
of plasma membrane V-ATPase activity. Inactivity of ATP hydrolysis and
proton pumping corresponds with the transient absence of peripheral
V subunits from the goblet cell apical membrane, whereas
the membrane integral V subunits remain. subunits disappear from the membrane in the
early moulting stages and that the immunoreactivity, complete subunit
pattern, and full pump activity are re-established after ecdysis. V holoenzymes. However, there is no
ultrastructural evidence that such an endo-exocytotic regulation of
V-ATPase occurs in M. sexta midgut. Bovine kidney cells have
also been reported to contain a cytosolic inhibitor protein of 6.3 kDa,
as well as a cytosolic activator protein of approximately 35 kDa, both
affecting V-ATPases specifically (Zhang et al., 1992a, 1992b).
So far, we have found no direct evidence that such proteins are
associated with the M. sexta plasma membrane V-ATPase.
However, we cannot exclude the presence of factors that may stabilize
or destabilize the VV holoenzyme. subunits from the V domain is
well known from in vitro experiments with V-ATPases. The
membrane-bound VV holoenzyme is readily
dissociated by treatment with chaotropic agents or by cold-inactivation
in the presence of ATP (see Nelson, 1992). Dissociation of V and V renders the enzyme unable to hydrolyze ATP or
to transport protons. Upon re-association of the V subunits
with membranes containing V domains, ATP-driven proton
transport is restored (Puopolo and Forgac, 1990). In vivo,
V domains are thought to be present in membranes in excess
(Zhang et al., 1992c) and correspondingly, it is thought,
there is a cytoplasmic pool not only of V subunits (Nelson
and Taiz, 1989) but of fully assembled, enzymically inactive, V domains of unknown function (Myers and Forgac, 1993). with V domains. The fate of the missing
V subunits is not known since any unattached subunits will
be lost during membrane purification. Preliminary studies using
Northern blots have indicated no significant change in mRNA
concentration for peripheral subunits over the time course of the moult
(results not shown), so we may speculate that the V domains
remain intact and are re-associated with the apical membrane at
ecdysis.V holoenzyme now need to be elucidated. The
switching of V-ATPase activity during the moult demonstrated here
occurs in synchrony with hormonal signals (Truman, 1992). V-ATPase
activity is turned off during an ecdysone peak in the presence of
juvenile hormone and returns when ecdysis is stimulated by eclosion
hormone. It seems not unlikely that V-ATPase activity is modulated,
either directly or indirectly, by these hormones. The unique transport
capabilities, their strict regulation during moult and the ease of
biochemical accessibility make M. sexta midgut an exciting
system for further studies of regulation of V-ATPase activity.
)))
We thank Alexandra Lepier for much help with the N,N`-dicyclohexylcarbodiimide labeling, Dr. Ralph
Gräf for preparing the anti 14-kDa monospecific
antibodies, and Dr. William R. Harvey for critically reading the
manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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N. Kitagawa, H. Mazon, A. J. R. Heck, and S. Wilkens Stoichiometry of the Peripheral Stalk Subunits E and G of Yeast V1-ATPase Determined by Mass Spectrometry J. Biol. Chem., February 8, 2008; 283(6): 3329 - 3337. [Abstract] [Full Text] [PDF]
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 J. Rein, M. Voss, W. Blenau, B. Walz, and O. Baumann Hormone-induced assembly and activation of V-ATPase in blowfly salivary glands is mediated by protein kinase A Am J Physiol Cell Physiol, January 1, 2008; 294(1): C56 - C65. [Abstract] [Full Text] [PDF]
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M. Voss, O. Vitavska, B. Walz, H. Wieczorek, and O. Baumann Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A J. Biol. Chem., November 16, 2007; 282(46): 33735 - 33742. [Abstract] [Full Text] [PDF]
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A. M. Smardon and P. M. Kane RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase J. Biol. Chem., September 7, 2007; 282(36): 26185 - 26194. [Abstract] [Full Text] [PDF]
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J. Qi and M. Forgac Cellular Environment Is Important in Controlling V-ATPase Dissociation and Its Dependence on Activity J. Biol. Chem., August 24, 2007; 282(34): 24743 - 24751. [Abstract] [Full Text] [PDF]
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N. Kroning, M. Willenborg, N. Tholema, I. Hanelt, R. Schmid, and E. P. Bakker ATP Binding to the KTN/RCK Subunit KtrA from the K+-uptake System KtrAB of Vibrio alginolyticus: ITS ROLE IN THE FORMATION OF THE KtrAB COMPLEX AND ITS REQUIREMENT IN VIVO J. Biol. Chem., May 11, 2007; 282(19): 14018 - 14027. [Abstract] [Full Text] [PDF]
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 S. Liegeois, A. Benedetto, G. Michaux, G. Belliard, and M. Labouesse Genes Required for Osmoregulation and Apical Secretion in Caenorhabditis elegans Genetics, February 1, 2007; 175(2): 709 - 724. [Abstract] [Full Text] [PDF]
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M. L. Patrick, K. Aimanova, H. R. Sanders, and S. S. Gill P-type Na+/K+-ATPase and V-type H+-ATPase expression patterns in the osmoregulatory organs of larval and adult mosquito Aedes aegypti J. Exp. Biol., December 1, 2006; 209(23): 4638 - 4651. [Abstract] [Full Text] [PDF]
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M. A. Owegi, D. L. Pappas, M. W. Finch Jr.,, S. A. Bilbo, C. A. Resendiz, L. J. Jacquemin, A. Warrier, J. D. Trombley, K. M. McCulloch, K. L. M. Margalef, et al. Identification of a Domain in the Vo Subunit d That Is Critical for Coupling of the Yeast Vacuolar Proton-translocating ATPase J. Biol. Chem., October 6, 2006; 281(40): 30001 - 30014. [Abstract] [Full Text] [PDF]
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M. Ohira, A. M. Smardon, C. M. H. Charsky, J. Liu, M. Tarsio, and P. M. Kane The E and G Subunits of the Yeast V-ATPase Interact Tightly and Are Both Present at More Than One Copy per V1 Complex J. Biol. Chem., August 11, 2006; 281(32): 22752 - 22760. [Abstract] [Full Text] [PDF]
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S. Terhzaz, T. D. Southall, K. S. Lilley, L. Kean, A. K. Allan, S. A. Davies, and J. A. T. Dow Differential Gel Electrophoresis and Transgenic Mitochondrial Calcium Reporters Demonstrate Spatiotemporal Filtering in Calcium Control of Mitochondria J. Biol. Chem., July 7, 2006; 281(27): 18849 - 18858. [Abstract] [Full Text] [PDF]
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 Q. Yang, G. Li, S. K. Singh, E. A. Alexander, and J. H. Schwartz Vacuolar H+-ATPase B1 Subunit Mutations that Cause Inherited Distal Renal Tubular Acidosis Affect Proton Pump Assembly and Trafficking in Inner Medullary Collecting Duct Cells J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1858 - 1866. [Abstract] [Full Text] [PDF]
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J. Rein, B. Zimmermann, C. Hille, I. Lang, B. Walz, and O. Baumann Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina J. Exp. Biol., May 1, 2006; 209(9): 1716 - 1724. [Abstract] [Full Text] [PDF]
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 P. Dames, B. Zimmermann, R. Schmidt, J. Rein, M. Voss, B. Schewe, B. Walz, and O. Baumann cAMP regulates plasma membrane vacuolar-type H+-ATPase assembly and activity in blowfly salivary glands. PNAS, March 7, 2006; 103(10): 3926 - 3931. [Abstract] [Full Text] [PDF]
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 P. M. Kane The Where, When, and How of Organelle Acidification by the Yeast Vacuolar H+-ATPase Microbiol. Mol. Biol. Rev., March 1, 2006; 70(1): 177 - 191. [Abstract] [Full Text] [PDF]
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K. W. Beyenbach and H. Wieczorek The V-type H+ ATPase: molecular structure and function, physiological roles and regulation J. Exp. Biol., February 15, 2006; 209(4): 577 - 589. [Abstract] [Full Text] [PDF]
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T. Inoue and M. Forgac Cysteine-mediated Cross-linking Indicates That Subunit C of the V-ATPase Is in Close Proximity to Subunits E and G of the V1 Domain and Subunit a of the V0 Domain J. Biol. Chem., July 29, 2005; 280(30): 27896 - 27903. [Abstract] [Full Text] [PDF]
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M. A. Owegi, A. L. Carenbauer, N. M. Wick, J. F. Brown, K. L. Terhune, S. A. Bilbo, R. S. Weaver, R. Shircliff, N. Newcomb, and K. J. Parra-Belky Mutational Analysis of the Stator Subunit E of the Yeast V-ATPase J. Biol. Chem., May 6, 2005; 280(18): 18393 - 18402. [Abstract] [Full Text] [PDF]
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 Y. Y. Sautin, M. Lu, A. Gaugler, L. Zhang, and S. L. Gluck Phosphatidylinositol 3-Kinase-Mediated Effects of Glucose on Vacuolar H+-ATPase Assembly, Translocation, and Acidification of Intracellular Compartments in Renal Epithelial Cells Mol. Cell. Biol., January 15, 2005; 25(2): 575 - 589. [Abstract] [Full Text] [PDF]
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O. Vitavska, H. Merzendorfer, and H. Wieczorek The V-ATPase Subunit C Binds to Polymeric F-actin as Well as to Monomeric G-actin and Induces Cross-linking of Actin Filaments J. Biol. Chem., January 14, 2005; 280(2): 1070 - 1076. [Abstract] [Full Text] [PDF]
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E. Shao and M. Forgac Involvement of the Nonhomologous Region of Subunit A of the Yeast V-ATPase in Coupling and in Vivo Dissociation J. Biol. Chem., November 19, 2004; 279(47): 48663 - 48670. [Abstract] [Full Text] [PDF]
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 C. A. Wagner, K. E. Finberg, S. Breton, V. Marshansky, D. Brown, and J. P. Geibel Renal Vacuolar H+-ATPase Physiol Rev, October 1, 2004; 84(4): 1263 - 1314. [Abstract] [Full Text] [PDF]
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S. Wilkens, T. Inoue, and M. Forgac Three-Dimensional Structure of the Vacuolar ATPase: LOCALIZATION OF SUBUNIT H BY DIFFERENCE IMAGING AND CHEMICAL CROSS-LINKING J. Biol. Chem., October 1, 2004; 279(40): 41942 - 41949. [Abstract] [Full Text] [PDF]
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M. Iwata, H. Imamura, E. Stambouli, C. Ikeda, M. Tamakoshi, K. Nagata, H. Makyio, B. Hankamer, J. Barber, M. Yoshida, et al. Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase PNAS, January 6, 2004; 101(1): 59 - 64. [Abstract] [Full Text] [PDF]
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 N. Morel, J.-C. Dedieu, and J.-M. Philippe Specific sorting of the a1 isoform of the V-H+ATPase a subunit to nerve terminals where it associates with both synaptic vesicles and the presynaptic plasma membrane J. Cell Sci., December 1, 2003; 116(23): 4751 - 4762. [Abstract] [Full Text] [PDF]
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Z. Zhang, C. Charsky, P. M. Kane, and S. Wilkens Yeast V1-ATPase: AFFINITY PURIFICATION AND STRUCTURAL FEATURES BY ELECTRON MICROSCOPY J. Biol. Chem., November 21, 2003; 278(47): 47299 - 47306. [Abstract] [Full Text] [PDF]
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A. K. Pullikuth, V. Filippov, and S. S. Gill Phylogeny and cloning of ion transporters in mosquitoes J. Exp. Biol., November 1, 2003; 206(21): 3857 - 3868. [Abstract] [Full Text] [PDF]
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K. Yokoyama, K. Nagata, H. Imamura, S. Ohkuma, M. Yoshida, and M. Tamakoshi Subunit Arrangement in V-ATPase from Thermus thermophilus J. Biol. Chem., October 24, 2003; 278(43): 42686 - 42691. [Abstract] [Full Text] [PDF]
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B. Zimmermann, P. Dames, B. Walz, and O. Baumann Distribution and serotonin-induced activation of vacuolar-type H+-ATPase in the salivary glands of the blowfly Calliphora vicina J. Exp. Biol., June 1, 2003; 206(11): 1867 - 1876. [Abstract] [Full Text] [PDF]
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O. Vitavska, H. Wieczorek, and H. Merzendorfer A Novel Role for Subunit C in Mediating Binding of the H+-V-ATPase to the Actin Cytoskeleton J. Biol. Chem., May 9, 2003; 278(20): 18499 - 18505. [Abstract] [Full Text] [PDF]
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E. Shao, T. Nishi, S. Kawasaki-Nishi, and M. Forgac Mutational Analysis of the Non-homologous Region of Subunit A of the Yeast V-ATPase J. Biol. Chem., April 4, 2003; 278(15): 12985 - 12991. [Abstract] [Full Text] [PDF]
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 E. S. Trombetta, M. Ebersold, W. Garrett, M. Pypaert, and I. Mellman Activation of Lysosomal Function During Dendritic Cell Maturation Science, February 28, 2003; 299(5611): 1400 - 1403. [Abstract] [Full Text] [PDF]
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V. F. Rizzo, U. Coskun, M. Radermacher, T. Ruiz, A. Armbruster, and G. Gruber Resolution of the V1 ATPase from Manduca sexta into Subcomplexes and Visualization of an ATPase-active A3B3EG Complex by Electron Microscopy J. Biol. Chem., January 3, 2003; 278(1): 270 - 275. [Abstract] [Full Text] [PDF]
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M. Huss, G. Ingenhorst, S. Konig, M. Gassel, S. Drose, A. Zeeck, K. Altendorf, and H. Wieczorek Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c J. Biol. Chem., October 18, 2002; 277(43): 40544 - 40548. [Abstract] [Full Text] [PDF]
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M. Clarke, J. Kohler, Q. Arana, T. Liu, J. Heuser, and G. Gerisch Dynamics of the vacuolar H+-ATPase in the contractile vacuole complex and the endosomal pathway of Dictyostelium cells J. Cell Sci., July 15, 2002; 115(14): 2893 - 2905. [Abstract] [Full Text] [PDF]
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S. Reineke, H. Wieczorek, and H. Merzendorfer Expression of Manduca sexta V-ATPase genes mvB, mvG and mvd is regulated by ecdysteroids J. Exp. Biol., April 15, 2002; 205(8): 1059 - 1067. [Abstract] [Full Text] [PDF]
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A. M. Smardon, M. Tarsio, and P. M. Kane The RAVE Complex Is Essential for Stable Assembly of the Yeast V-ATPase J. Biol. Chem., April 12, 2002; 277(16): 13831 - 13839. [Abstract] [Full Text] [PDF]
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W. Zeiske, H. Meyer, and H. Wieczorek Insect midgut K+ secretion: concerted run-down of apical/basolateral transporters with extra-/intracellular acidity J. Exp. Biol., February 15, 2002; 205(4): 463 - 474. [Abstract] [Full Text] [PDF]
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S. Kawasaki-Nishi, K. Bowers, T. Nishi, M. Forgac, and T. H. Stevens The Amino-terminal Domain of the Vacuolar Proton-translocating ATPase a Subunit Controls Targeting and in Vivo Dissociation, and the Carboxyl-terminal Domain Affects Coupling of Proton Transport and ATP Hydrolysis J. Biol. Chem., December 7, 2001; 276(50): 47411 - 47420. [Abstract] [Full Text] [PDF]
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G. Gruber, H. Wieczorek, W. R. Harvey, and V. Muller Structure-function relationships of A-, F- and V-ATPases J. Exp. Biol., January 8, 2001; 204(15): 2597 - 2605. [Abstract] [Full Text] [PDF]
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D Weihrauch, A Ziegler, D Siebers, and D. Towle Molecular characterization of V-type H(+)-ATPase (B-subunit) in gills of euryhaline crabs and its physiological role in osmoregulatory ion uptake J. Exp. Biol., January 1, 2001; 204(1): 25 - 37. [Abstract] [PDF]
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T. Nishi and M. Forgac Molecular Cloning and Expression of Three Isoforms of the 100-kDa a Subunit of the Mouse Vacuolar Proton-translocating ATPase J. Biol. Chem., March 15, 2000; 275(10): 6824 - 6830. [Abstract] [Full Text] [PDF]
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O Peyronnet, V Vachon, J. Schwartz, and R Laprade Ion channel activity from the midgut brush-border membrane of gypsy moth (Lymantria dispar) larvae J. Exp. Biol., January 6, 2000; 203(12): 1835 - 1844. [Abstract] [PDF]
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M Forgac Structure, mechanism and regulation of the clathrin-coated vesicle and yeast vacuolar H(+)-ATPases J. Exp. Biol., January 1, 2000; 203(1): 71 - 80. [Abstract]
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P. Kane and K. Parra Assembly and regulation of the yeast vacuolar H(+)-ATPase J. Exp. Biol., January 1, 2000; 203(1): 81 - 87. [Abstract]
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E. Bowman and B. Bowman Cellular role of the V-ATPase in Neurospora crassa: analysis of mutants resistant to concanamycin or lacking the catalytic subunit A J. Exp. Biol., January 1, 2000; 203(1): 97 - 106. [Abstract]
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K. J. MacLeod, E. Vasilyeva, K. Merdek, P. D. Vogel, and M. Forgac Photoaffinity Labeling of Wild-type and Mutant Forms of the Yeast V-ATPase A Subunit by 2-Azido-[32P]ADP J. Biol. Chem., November 12, 1999; 274(46): 32869 - 32874. [Abstract] [Full Text] [PDF]
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C. Landolt-Marticorena, W. H. Kahr, P. Zawarinski, J. Correa, and M. F. Manolson Substrate- and Inhibitor-induced Conformational Changes in the Yeast V-ATPase Provide Evidence for Communication between the Catalytic and Proton-translocating Sectors J. Biol. Chem., September 10, 1999; 274(37): 26057 - 26064. [Abstract] [Full Text] [PDF]
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P. M. Kane, M. Tarsio, and J. Liu Early Steps in Assembly of the Yeast Vacuolar H+-ATPase J. Biol. Chem., June 11, 1999; 274(24): 17275 - 17283. [Abstract] [Full Text] [PDF]
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H. Merzendorfer, M. Huss, R. Schmid, W. R. Harvey, and H. Wieczorek A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively J. Biol. Chem., June 11, 1999; 274(24): 17372 - 17378. [Abstract] [Full Text] [PDF]
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M. Forgac Structure and Properties of the Vacuolar (H+)-ATPases J. Biol. Chem., May 7, 1999; 274(19): 12951 - 12954. [Full Text] [PDF]
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N. Nelson and W. R. Harvey Vacuolar and Plasma Membrane Proton-Adenosinetriphosphatases Physiol Rev, April 1, 1999; 79(2): 361 - 385. [Abstract] [Full Text] [PDF]
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K. J. Parra and P. M. Kane Reversible Association between the V1 and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect Mol. Cell. Biol., December 1, 1998; 18(12): 7064 - 7074. [Abstract] [Full Text]
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 C. Bauerle, C. Magembe, and D. P. Briskin Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase Plant Physiology, July 1, 1998; 117(3): 859 - 867. [Abstract] [Full Text] [PDF]
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J. Ludwig, S. Kerscher, U. Brandt, K. Pfeiffer, F. Getlawi, D. K. Apps, and H. Schagger Identification and Characterization of a Novel 9.2-kDa Membrane Sector-associated Protein of Vacuolar Proton-ATPase from Chromaffin Granules J. Biol. Chem., May 1, 1998; 273(18): 10939 - 10947. [Abstract] [Full Text] [PDF]
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 H. Niessen, G. W. Meisenholder, H.-L. Li, S. L. Gluck, B. S. Lee, B. Bowman, R. L. Engler, B. M. Babior, and R. A. Gottlieb Granulocyte Colony-Stimulating Factor Upregulates the Vacuolar Proton ATPase in Human Neutrophils Blood, December 1, 1997; 90(11): 4598 - 4601. [Abstract] [Full Text] [PDF]
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R. Hirata, L. A. Graham, A. Takatsuki, T. H. Stevens, and Y. Anraku VMA11 and VMA16 Encode Second and Third Proteolipid Subunits of the Saccharomyces cerevisiae Vacuolar Membrane H+-ATPase J. Biol. Chem., February 21, 1997; 272(8): 4795 - 4803. [Abstract] [Full Text] [PDF]
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R. Graf, W. R. Harvey, and H. Wieczorek Purification and Properties of a Cytosolic V1-ATPase J. Biol. Chem., August 23, 1996; 271(34): 20908 - 20913. [Abstract] [Full Text] [PDF]
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K. J. Parra and P. M. Kane Wild-type and Mutant Vacuolar Membranes Support pH-dependent Reassembly of the Yeast Vacuolar H+-ATPase in Vitro J. Biol. Chem., August 9, 1996; 271(32): 19592 - 19598. [Abstract] [Full Text] [PDF]
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A. Nanda, J. H. Brumell, T. Nordstrom, L. Kjeldsen, H. Sengelov, N. Borregaard, O. D. Rotstein, and S. Grinstein Activation of Proton Pumping in Human Neutrophils Occurs by Exocytosis of Vesicles Bearing Vacuolar-type H+-ATPases J. Biol. Chem., July 5, 1996; 271(27): 15963 - 15970. [Abstract] [Full Text] [PDF]
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P. M. Kane and P. M. Kane Disassembly and Reassembly of the Yeast Vacuolar H[IMAGE]-ATPase in Vivo J. Biol. Chem., July 14, 1995; 270(28): 17025 - 17032. [Abstract] [Full Text] [PDF]
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C. Landolt-Marticorena, K. M. Williams, J. Correa, W. Chen, and M. F. Manolson Evidence That the NH2 Terminus of Vph1p, an Integral Subunit of the V0 Sector of the Yeast V-ATPase, Interacts Directly with the Vma1p and Vma13p Subunits of the V1 Sector J. Biol. Chem., May 12, 2000; 275(20): 15449 - 15457. [Abstract] [Full Text] [PDF]
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K. J. Parra, K. L. Keenan, and P. M. Kane The H Subunit (Vma13p) of the Yeast V-ATPase Inhibits the ATPase Activity of Cytosolic V1 Complexes J. Biol. Chem., July 7, 2000; 275(28): 21761 - 21767. [Abstract] [Full Text] [PDF]
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G. Gruber, D. I. Svergun, J. Godovac-Zimmermann, W. R. Harvey, H. Wieczorek, and M. H. J. Koch Evidence for Major Structural Changes in the Manduca sexta Midgut V1 ATPase Due to Redox Modulation. A SMALL ANGLE X-RAY SCATTERING STUDY J. Biol. Chem., September 22, 2000; 275(39): 30082 - 30087. [Abstract] [Full Text] [PDF]
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T. Xu and M. Forgac Subunit D (Vma8p) of the Yeast Vacuolar H+-ATPase Plays a Role in Coupling of Proton Transport and ATP Hydrolysis J. Biol. Chem., July 14, 2000; 275(29): 22075 - 22081. [Abstract] [Full Text] [PDF]
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S. Kawasaki-Nishi, T. Nishi, and M. Forgac Yeast V-ATPase Complexes Containing Different Isoforms of the 100-kDa a-subunit Differ in Coupling Efficiency and in Vivo Dissociation J. Biol. Chem., May 18, 2001; 276(21): 17941 - 17948. [Abstract] [Full Text] [PDF]
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T. Xu and M. Forgac Microtubules Are Involved in Glucose-dependent Dissociation of the Yeast Vacuolar [H+]-ATPase in Vivo J. Biol. Chem., June 29, 2001; 276(27): 24855 - 24861. [Abstract] [Full Text] [PDF]
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K. Keenan Curtis and P. M. Kane Novel Vacuolar H+-ATPase Complexes Resulting from Overproduction of Vma5p and Vma13p J. Biol. Chem., January 18, 2002; 277(4): 2716 - 2724. [Abstract] [Full Text] [PDF]
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K. K. Curtis, S. A. Francis, Y. Oluwatosin, and P. M. Kane Mutational Analysis of the Subunit C (Vma5p) of the Yeast Vacuolar H+-ATPase J. Biol. Chem., March 8, 2002; 277(11): 8979 - 8988. [Abstract] [Full Text] [PDF]
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