(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/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.
H 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. (
)
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/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.
Maintaining the very high active K 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.
In this investigation, we report on events
during a larval/larval moult. We measured a reversible switching off
and on of K 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.
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. The alkaline phospatase-conjugated secondary antibody probe was
either goat anti-rabbit IgG or goat anti-mouse IgG (Sigma).
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 transport.
These
results provide evidence that the K 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.
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 and stopped by the addition of 20 mM NH
Cl (final concentrations). Equal concentrations (25
µg/ml) of membrane protein were used in each
assay.
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 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.
Here we report on functional and structural changes in the
plasma membrane V-ATPase of larval M. sexta midgut during
moulting. K 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.
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 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.
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
VV
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 V
V
holoenzyme.
The
disassembly of V subunits from the V
domain is
well known from in vitro experiments with V-ATPases. The
membrane-bound V
V
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).
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 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.
The regulatory signals for disassembly or assembly of the
VV
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.
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.