©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Plasma Membrane V-ATPase Activity by Dissociation of Peripheral Subunits (*)

(Received for publication, August 16, 1994; and in revised form, October 27, 1994)

John-Paul Sumner (1) Julian A. T. Dow (1)(§) Fergus G. P. Earley (2) Ulla Klein (3) Dieter Jäger (3) Helmut Wieczorek (3)(¶)

From the  (1)Department of Cell Biology, University of Glasgow, Glasgow G12 8QQ, United Kingdom, (2)ZENECA Agrochemicals Jeallol's Hill Research Station, Bracknell, BERKS RG12 6EY, United Kingdom, and the (3)Zoological Institute, University of Munich, D-80021 Munich, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(1) sector, whereas the membrane integral V(0) 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(1) domain.


INTRODUCTION

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(1) 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(0) 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. (^1)

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(1) domain.


EXPERIMENTAL PROCEDURES

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)^2 Measurements

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 times1 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 times 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 times 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 Transport

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(2), 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(2), 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, (^3)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 Na(2)HPO(4), 3 mM KCl, 0.02% NaN(3) (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% beta-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).


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.^4 The alkaline phospatase-conjugated secondary antibody probe was either goat anti-rabbit IgG or goat anti-mouse IgG (Sigma).


RESULTS

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.


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.

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 µmolbulletmgbulletmin in stage E moulting larvae compared with 0.39 ± 0.03 µmolbulletmgbulletmin 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.


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(2) and stopped by the addition of 20 mM NH(4)Cl (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(1)-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.


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).



V(1) Subunits Are Missing during Moult

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(1) subunits appeared to be strongly reduced, whereas the bands representing the integral membrane 43- and 17-kDa V(0) 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.

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(1) 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(1) subunits.


DISCUSSION

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(1) subunits from the goblet cell apical membrane, whereas the membrane integral V(0) 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(1) 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 V(1)V(0) 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(1)V(0) holoenzyme.

The disassembly of V(1) subunits from the V(0) domain is well known from in vitro experiments with V-ATPases. The membrane-bound V(1)V(0) holoenzyme is readily dissociated by treatment with chaotropic agents or by cold-inactivation in the presence of ATP (see Nelson, 1992). Dissociation of V(1) and V(0) renders the enzyme unable to hydrolyze ATP or to transport protons. Upon re-association of the V(1) subunits with membranes containing V(0) domains, ATP-driven proton transport is restored (Puopolo and Forgac, 1990). In vivo, V(0) 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(1) subunits (Nelson and Taiz, 1989) but of fully assembled, enzymically inactive, V(1) 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(1) with V(0) domains. The fate of the missing V(1) 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(1) domains remain intact and are re-associated with the apical membrane at ecdysis.

The regulatory signals for disassembly or assembly of the V(1)V(0) 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.


FOOTNOTES

*
This work was supported by a Science Research Council CASE award, Medical Research Council Grant G9120579CB, German Research Foundation Grant Wi 698, European Economic Community Grant SC1*-CT90-0480, and National Institutes of Health Grant AI22444. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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.

§
To whom correspondence may be addressed. Tel.: 44-41-330-4616; Fax: 44-41-330-4501.

To whom correspondence may be addressed: Zoologisches Institut der Universität, Postfach 202136, D-80021 München, Germany. Tel.: 49-89-5902-325; Fax: 49-89-5902-450.

(^1)
A. Lepier and H. Wieczorek, unpublished observations.

(^2)
The abbreviations used are: TEV, transepithelial voltage; GCAM, goblet cell apical membranes; MOPS, 3-(N-morpholino)propanesulfonic acid.

(^3)
U. Klein, M. Timme, F. J. S. Novak, A. Lepier, W. R. Harvey, and H. Wieczorek, unpublished results.


ACKNOWLEDGEMENTS

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.


REFERENCES

  1. Anraku, Y., Hirata, R., Wade, Y., and Ohya, Y. (1992) J. Exp. Biol. 172, 67-81 [Abstract/Free Full Text]
  2. Baldwin, K. M., and Hakim, R. S. (1991) Tissue & Cell 23, 411-422
  3. Bell, R. A., and Joachim, F. G. (1974) Ann. Entomol. Soc. Am. 69, 365-373
  4. Brown, D., Sabolic, I., and Gluck, S. (1991) Kidney Int. 40, Suppl. 33, 79-83
  5. Cioffi, M. (1984) Am. Zool. 24, 139-156
  6. Cioffi, M., and Wolfersberger, M. G. (1983) Tissue & Cell 15, 781-803
  7. Dow, J. A. T. (1984) Am. J. Physiol. 246, R633-R635
  8. Dow, J. A. T. (1992) J. Exp. Biol. 172, 355-375 [Abstract/Free Full Text]
  9. Dow, J. A. T., and O'Donnell, M. J. (1990) J. Exp. Biol. 150, 247-256
  10. Dow, J. A. T., Boyes, B., Harvey, W. R., and Wolfersberger, M. G. (1985). J. Exp. Biol. 116, 685-689
  11. Dow, J. A. T., Goodwin, S. F., and Kaiser, K. (1992) Gene (Amst.) 122, 355-360 [Medline] [Order article via Infotrieve]
  12. Gluck, S. L., Nelson, R. D., Lee, B. S., Wang, Z.-Q., Guo, X.-L., Fu, J.-Y., and Zhang, K. (1992) J. Exp. Biol. 172, 219-229 [Abstract/Free Full Text]
  13. Gräf, R., Novak, F. J. S., Harvey, W. R., and Wieczorek, H. (1992) FEBS Lett. 300, 119-122 [CrossRef][Medline] [Order article via Infotrieve]
  14. Gräf, R., Harvey, W. R., and Wieczorek, H. (1994a) Biochim. Biophys. Acta 1190, 193-196 [Medline] [Order article via Infotrieve]
  15. Gräf, R., Lepier, A., Harvey, W. R., and Wieczorek, H. (1994b) J. Biol. Chem. 269, 3767-3774 [Abstract/Free Full Text]
  16. Graham, L. A., Hill, K. J., and Stevens, T. H. (1994) J. Biol. Chem. 269, 25974-25977 [Abstract/Free Full Text]
  17. Harvey, W. R. (1992) J. Exp. Biol. 172, 1-17
  18. Harvey, W. R., and Nedergaard, S. (1964) Proc. Natl. Acad. Sci. U. S. A. 51, 757-765 [Medline] [Order article via Infotrieve]
  19. Harvey, W. R., and Nelson, N. (1992) J. Exp. Biol. 172
  20. Klein, U., Löffelmann, G., and Wieczorek, H. (1991) J. Exp. Biol. 161, 61-75
  21. Mandel, M., Moriyama, Y., Hulmes, J. D., Pan, Y. E., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5521-5524 [Abstract]
  22. Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981) Science 211, 1437-1438 [Medline] [Order article via Infotrieve]
  23. Myers, M., and Forgac, M. (1993) J. Cell. Physiol. 156, 35-42 [Medline] [Order article via Infotrieve]
  24. Nelson, N. (1992) J. Exp. Biol. 172, 19-27 [Abstract/Free Full Text]
  25. Nelson, N., and Taiz, L. (1989) Trends Biochem. Sci. 14, 113-116 [CrossRef][Medline] [Order article via Infotrieve]
  26. Nelson, H., Mandiyan, S., and Nelson, N. (1994). J. Biol. Chem. 269, 24150-24155 [Abstract/Free Full Text]
  27. Novak, F. J. S., Gräf, R., Waring, R., Wolfersberger, M. G., Wieczorek, H., and Harvey, W. R. (1992) Biochim. Biophys. Acta 1132, 67-71 [Medline] [Order article via Infotrieve]
  28. Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836-14841 [Abstract/Free Full Text]
  29. Schweikl, H., Klein, U., Schindlbeck, M., and Wieczorek, H. (1989) J. Biol. Chem. 264, 11136-11142 [Abstract/Free Full Text]
  30. Truman, J. W. (1992) Prog. Brain Res. 92, 361-374 [Medline] [Order article via Infotrieve]
  31. Wieczorek, H. (1992) J. Exp. Biol. 172, 335-343 [Abstract/Free Full Text]
  32. Wieczorek, H., Weerth, S., Schindlbeck, M., and Klein, U. (1989) J. Biol. Chem. 264, 11143-11148 [Abstract/Free Full Text]
  33. Wieczorek, H., Cioffi, M., Klein, U., Harvey, W. R., Schweikl, H., and Wolfersberger, M. G. (1990) Methods Enzymol. 192, 608-616 [Medline] [Order article via Infotrieve]
  34. Wieczorek, H., Putzenlechner, M., Zeiske, W., and Klein, U. (1991) J. Biol. Chem. 266, 15340-15347 [Abstract/Free Full Text]
  35. Yamamoto, R. T. (1969) J. Econ. Entomol. 62, 1427-1431
  36. Zhang, K., Wang, Z.-Q., and Gluck, S. (1992a) J. Biol. Chem. 267, 9701-9706 [Abstract/Free Full Text]
  37. Zhang, K., Wang, Z.-Q., and Gluck, S. (1992b) J. Biol. Chem. 267, 14539-14542 [Abstract/Free Full Text]
  38. Zhang, J., Myers, M., and Forgac, M. (1992c) J. Biol. Chem. 267, 9773-9778 [Abstract/Free Full Text]
  39. Zheng, X. Y., Spaeth, D. D., Harvey, W. R., and Wolfersberger, M. G. (1992) J. Exp. Biol. 165, 273-278 [Medline] [Order article via Infotrieve]

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