Molecular cloning and characterization of the B subunit of a vacuolar H+-ATPase from the midgut and malpighian tubules of Helicoverpa virescens

ARCHIVES

OF BIOCHEMISTRY

Vol. 291, No. 1, November

AND

BIOPHYSICS

15, pp. 92-99,199l

Molecular Cloning and Characterization of the B Subunit of a Vacuolar H+-ATPase from the Midgut and Malpighian Tubules of Helicoverpa vii-escens Sarjeet

S. Gill1 and Linda

S. Ross

Department of Entomology and the Environmental Riverside, California 92521

Received

May

Toxicology

Graduate

University

of California,

28,199l

Using the polymerase chain reaction (PCR) a 0.8-kb product was amplified from cDNA made from the midgut and Malpighian tubules of fifth instar larvae of Helicoverpu virescens. This 0.8-kb PCR product was then used to isolate a clone of the B subunit of the V-type ATPase from a cDNA library made from the same tissues. The cDNA clone encodes for a protein of 55 kDa which shows very high amino acid homology to other known B subunits of V-type ATPases. The transcript size of the B subunit in the midgut of H. virescens was 2.3 kb, and a transcript of identical size was also detected in the Malpighian tubules. Northern blot analysis revealed the presence of a homologous transcript of 2.6 kb in the midgut of Munducu sextu and PCR analysis also confirmed the presence of such a transcript in the Malpighian tubules and the nervous system of lW. sexta, and in the midgut Malpighian tubules of Culex quinquefasciatus. The presence of the V-type ATPase in the Malpighian tubules of lepidopteran insects suggests that the transport of ions across the cell membrane in this tissue is also probably driven by a similar process as that observed in the midgut of these insects. Q ieei ~cadd~ press, IIIC.

Lepidopteran larvae ingest high concentrations of K+ from their diet, and these high K+ concentrations are also observed in the midgut and the hemolymph of these insects. The Kf level in the hemolymph of these larvae is much higher than the Na+ level, in contrast to that observed in vertebrate blood where Na+ levels are higher (l-3). This hemolymph level of K+ is maintained throughout most of the larval development (4,5). TO regulate this K+ concentration in the hemolymph, lepidopteran larvae have evolved an elaborate midgut 1 To whom

Program,

correspondence

should

be addressed.

pathway. A key feature of this K+ regulation is the efflux of K+ from the inside of the goblet cell to its lumen cavity. This K+ efflux is apparently mediated by an electrogenic H+-ATPase pump (6) which is thought to be localized on the goblet cell apical membrane (1, 7). The H+-ATPase pumps H+ from the goblet cell cytoplasm to its lumen, creating a potential gradient that facilitates the efflux of K+ through a K+/H+ antiport on the goblet cell apical membrane (6,8). The goblet cell apical membrane H+-ATPase of the saturnid Manduca sexta has been purified and shown to be a V-type ATPase as indicated by its biochemical characteristics and its subunit composition, i.e., 70,57,46,29, and 17 kDa (9). This ATPase, which is inhibited by DCCD and N-ethylmaleimide but is vanadate and ouabain insensitive, has substrate specificity for both ATP and GTP, with the ATPase activity being stimulated by Kf (6). Vtype ATPases with similar features have been characterized from a variety of organisms and tissues (9-26). In most of these studies the reported subunit composition is quite similar. Furthermore, there is a high conservation of the structure of individual subunits. Although these Vtype ATPases are conserved both in subunit composition and in structure, it appears that these pumps play a multitude of roles in the various organisms and tissues (27). Other than the subunit composition, little is known about the primary structure of V-type ATPases of insects, the only exception being the structure of the 16-kDa proteolipid of Drosophila melunogaster (28). The subunit proteins of the lepidopteran M. sexta and Helicoverpa uirescens midgut V-type ATPase, however, appear to be very similar to the subunit proteins of V-type ATPase from other sources because there is substantial immunological cross-reactivity of the M. sexta and H. virescens proteins with antibodies raised against noninsect V-type ATPase subunits. Conserved domains in these subunits were therefore used in the design of probes for the iso-

92 Copyright 0 1991 AI1 rights of reproduction

ooo3-9861/91 $3.ocl by Academic Press, Inc. in any form reserved.

THE

VACUOLAR

HC-ATPase

B SUBUNIT

lation of cDNA clones. In this study we report the oligonucleotide and deduced primary amino acid sequence of the B subunit of H. uirescens, an economically important insect pest in agriculture. MATERIALS

AND

METHODS

cDNA library construction. Midguts and Malpighian tubules from 35 (approximately 2.5 g) early fifth instar H. virescens larvae were dissected and stored in liquid nitrogen prior to use. RNA was isolated by homogenization in guanidine iaothiocyanate followed by cesium chloride centrifugation as described (29). Poly(A)+ RNA was purified by passing total RNA through an oligo(dT) column two times. The poly(A)+ RNA was then used for the construct,ion of cDNA using a cDNA synthesis kit from Pharmacia LKB Biotechnology (Piscataway, NJ). Duringfirststrand synthesis, oligo(dT) was used as the primer for the first 30 min of synthesis, whereupon random hexamers were added to prime during the final 30 min of synthesis. After second-strand synthesis, EcoRl adaptors were ligated to the cDNA which was then ligated to EcoRlcut, dephoaphorylated XZAPII arms and packaged by commercially prepared packaging extracts (Strat.agene, La Jolla, CA). The library was plated on Escherichiu coli XL-l Blue and amplified once. The primary library had a complexity of about lo6 clones. PCR amplification of the B subunit of V-type ATPase. Published nucleotide sequences of V-type ATPase B subunits derived from yeast, Neurospora, plants, and bacteria were compared, and three conserved domains were identified (13, 14, 17, 30, 31). Three degenerate oligonucleotide primers (Fig. 4), one sense, one external antisense, and one internal antisense, encompassing these domains were synthesized using an oligonucleotide synthesizer (Applied Biosystems, Emeryville, CA). These primers had sequences of 5’-CAGCTGCAGGTI”MTGAAGG(T/ A)AC(T/A)TC(T/C)GG(T/C/A)AT-3’ (sense primer lS, l/24 degeneracy), 5’-GGGTCGACCTCC(T/G)GTAAT(G/A)TA(T/A)CC(A/G/ C)GT(T/C)AAATC-3’ (antisense external primer 2A, l/48 degeneracy), and 5’-AA(G/A)TC(T/A)GT(A/G)TACAT(G/A)TAACC(T/C/ A)GG(G/A)(T/A)A(T/A)CC-3’ (antisense internal primer 3A, l/384 degeneracy). A polymerase chain reaction (PCR)’ was performed in the presence of the sense 1s primer and antisense external 2A primer (1 jtM) with 500 ng of cDNA as the template. The PCR protocol used was 5 min at 94°C (initial melting), 10 amplification cycles with a slow temperature ramp (1 min at 94’C, 2 min annealing at 37’C, 7”C/min ramp to 72’C, 3 min at 72’C), and 20 cycles of amplification (same as above, without the temperature ramp). The final step at 72°C was extended to 17 min. The resulting PCR product was then used as a template in a second amplification using the sense 1s primer and antisense 3A external primer. The single resultant 0.8-kb PCR product from the first PCR reaction was gel purified by Geneclean (BIOlOl, San Diego, CA) and then labeled by randomly primed incorporation of digoxigenin-labeled deoxyuridine triphosphate following the manufacturer’s instructions (Genius Kit, Boehringer-Mannheim, Indianapolis, IN). Amplification of RNA sequences was performed by first using reverse transcriptase and random hexamers as primers followed by PCR of the cDNA as described by Kawasaki (32). The PCR protocol used was as described above except annealing was performed at 45°C instead of 37’C. RNA used for this PCR amplification was prepared from the midgut, Malpighian tubules, or nervous tissue of last instar larvae using the protocol described above. cDNA library screening and sequencing of clones. The DIG-labeled PCR product was used to screen the cDNA library. Duplicate plaque

2 Abbreviations used: SSC, sodium chloride and sodium citrate; SDS, sodium dodecyl sulfate; DIG, digoxigenin; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; PAGE, polyacrylamide gel electrophoresis; DCCD, dicyclohexylcarbodiimide.

FROM

Helicoverpu

virescens

93

lifts were made on nylon membranes (Micron Separations, Inc., Westboro, MA) which were then screened under stringent hybridization and wash conditions (hybridization at 68°C; two washes in 2X SSC and 0.1% SDS at room temperature for 5 min, followed by two washes in 0.1X SSC and 0.1% SDS at 68°C for 15 min). Positive clones were purified by repeated screenings of isolated plaque plugs. Single-stranded DNA was prepared from pure phage clones following instructions from the supplier of the vector, using the helper phage R408 (Stratagene). Sequencing was performed on five clones using Sequenase 2.0 (U.S. Biochemical, Cleveland, OH). Initial sequences were derived using the T3 primer complementary to the pBluescript SK- (Stratagene) sequence close to the EcoRI cloning site. Subsequent sequences were obtained using primers containing sequences unique to each cfone. Southern and Northern bkting. DNA was separated on 1% agarose, visualized with ethidium bromide, and transferred overnight to nitrocellulose filters. The filters were used for Southern blotting (33) utilizing the DIG-labeled 0.8-kb PCR product from H. uirescens. RNA was separated on a 0.66 M formaldehyde agarose gel. The RNA was transferred to nitrocellulose and baked at 80°C for 2 h under vacuum. The blot was probed according to the manufacturer’s instructions (Genius Kit, Boehringer-Mannheim) using the DIG-labeled 0.8-kb PCR product or a DIG-labeled probe made by labeling the insert from clone 3-6 which contains a B subunit cDNA of 1.5 kb. Rapid amplification of cDNA ends (RACE). The RACE protocol was performed according to the protocol of Frohman (34). Briefly, mRNA from H. virescens midgut was reverse transcribed using TCATCACGTCAATAGCG as the primer. The first-strand cDNA was then poly(A) tailed using terminal transferase, and then double-stranded cDNA was generated by extension of the sense primer GACTCGAGTCGACATCGATT’M’TTTTTTTTTTTTT. This product was then used as the template for PCR amplification using GACTCGAGTCGACATCG and CGTACGCAGGATATCGC as the sense and antisense primers, respectively. As attempts to sequence this PCR product were only partially successful the PCR product was ligated without further modification into the pCRlOO0 vector (Invitrogen, La Jolla, CA). Two positive colonies were analyzed, and the inserts were sequenced by double-stranded sequencing using Sequenase 2.0.

RESULTS Our primary approach to clone the B subunit of the Vtype ATPase from the midgut and Malpighian tubules of H. virescens utilized conserved sequences from homologous genes as sites for the design of primers for gene amplification by PCR. Three degenerate primers, one sense, one external antisense, and one internal antisense, were synthesized. As there was substantial conservation at the nucleotide level, the primers were designed with oligonucleotide degeneracy only at the 3’ end of each primer; overall degeneracies were l/24 and l/48 for the sense and external antisense primers, respectively. Based on the cDNA nucleotide sequence that was subsequently characterized, the sense and external antisense primers had three and four mismatches, respectively. The primers were used to amplify a region of a putative B subunit gene utilizing as the template cDNA that was made from the midgut and the Malpighian tubules of early fifth instar larvae of H. uirescens. The first amplification was performed with the 1s sense and 2A external antisense primers, resulting in the formation of a 0.8-kb fragment (Fig. 1, lane 1). To ascertain that this 0%kb PCR product was correct, a confirmatory amplification was

94

GILL

AND

Kb

1.6 1.0

21

s

FIG. 1. Agarose gel analysis of products obtained in PCR amplification of the B subunit of the V-type ATPase from H. oirescens. Primers constructed as indicated under Materials and Methods were used for PCR amplification of cDNA prepared from H. uirescens midgut and Malpighian tubule tissue. The PCR product obtained by using primers 1s and 2A, as the sense and antisense primers, respectively, is shown in lane 1. The anticipated and observed size of the PCR product using these primers is 0.8 kb. This 0.8-kb PCR product was then used as a template for a second PCR amplification using primer 1s and primer 3A as the sense and antisense primers, respectively. This reaction should yield a PCR product of 0.7 kb because primer 3A is located 100 bp upstream of the location of primer 2s. The observed 0.7-kb PCR product is shown in lane 2.

performed with the 1s sense and 3A internal antisense primers using the 0.8-kb PCR product as the template. This amplification resulted in a product of 0.7 kb (Fig. 1, lane 2). Thus, the PCR products of both the first and the second amplifications were of the expected size based on the sequence of homologous genes. The DIG-labeled O.&J-kb PCR fragment was used for screening the H. uirescens XZAPII cDNA library. Duplicate lifts were made and one of these lifts was probed under high stringency while the other was probed at low stringency. Initially, 3 X lo5 plaques were screened and about 500 of these plaques showed a strong hybridization signal under high stringency hybridization and washes. Six of these positive plaques were selected for further analysis. The cDNAs of these plaques were rescued from XZAPII, and characterized by restriction enzyme digestion. Five clones were sequenced to different extents. Two of the cDNA clones in pBluescript SK-, which were in opposite orientation relative to each other, were then sequenced completely. The largest cDNA of these clones, 2.0 kb, contained the putative ATG start codon, plus three additional upstream nucleotides. To ensure that there were no alternative translation initiation sites, an effort was made to obtain additional sequence information of the 5’ end of the mRNA by means of the RACE protocol using PCR. A 0.4-kb fragment was isolated using this protocol. However, as attempts to obtain sequence information by direct sequencing of this PCR product were not satisfactory, the PCR product was

ROSS

cloned using a commercially available vector, pCR1000. Two colonies were analyzed by restriction mapping and confirmed to be correct by Southern hybridization using an oligonucleotide complimentary to bases 123-139 (Fig. 2). The PCR inserts were then sequenced in both orientations. Only 82 bp of additional sequence information, identical in both of the clones analyzed, was obtained in this manner. The complete cDNA sequence of the B subunit of the V-type ATPase using both approaches is shown in Fig. 2. The putative start codon begins at nucleotide 85, followed by a 1482-bp open reading frame and by at least 624 bp of untranslated sequence. There are no other putative start codons in any other reading frame which result in a protein of the anticipated size. Two polyadenylation sequences were noted in the 3’ untranslated region of the cDNA (Fig. 2). The open reading frame encodes for a protein of 494 amino acids with a predicted molecular weight of 54.9 kDa. The calculated p1 of this protein is 4.8 (35). Three potential glycosylation sites were observed, although it is not known whether the protein is glycosylated in the midgut of H. virescens. The size of the transcript was determined to be 2.3 kb (Fig. 3A, lane 1). The sequence information in Fig. 2 therefore codes for only part of the mRNA; hence about 0.2 kb of the mRNA was not sequenced. Northern hybridization experiments performed with total RNA isolated from only the Malpighian tubules from fifth instar larvae of H. virescens demonstrated that the 2.3-kb Vtype ATPase transcript is also present in the Malpighian tubules of this insect (Fig. 3A, lane 3). Northern hybridization experiments performed under high stringency show that a homologous B subunit transcript of 2.6 kb is present in the midgut of M. se&u larvae (Fig. 3A, lane 2). Under these conditions this transcript gave a weaker signal than that obtained with H. uirescens. Moreover, no transcript was detected from the Malpighian tubule RNA of M. sextu by Northern analysis (Fig. 3A, lane 4). These findings suggest that there may be differences in the nucleotide sequences of these two species as the probe used was specific for the H. virescens transcript. It is also possible that less of the B subunit transcript is present in M. sexta. PCR amplification of RNA from H. uirescem and M. se&z midgut and Malpighian tubule tissues was performed using primers 1s and 2A (Fig. 3B). In order to confirm that the PCR products observed were homologous to the H. virescens midgut 0%kb fragment, we performed Southern blot analysis (33) using the same probe as in the Northern blots (Fig. 3C). The resultant PCR products from all four tissues were the correct size, 0.8 kb, and they appeared to have some degree of homology when probed with the H. virescens V-type ATPase probe, indicating that the gene encoding the B subunit was amplified. This observation demonstrates that the V-type ATPase is also present in the Malpighian tubules of M. sextu larvae.

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FIG. 2. Nucleotide and deduced amino acid sequence of a H. virescens V-type ATPase B subunit cDNA clone. The nucleotide sequence of the cDNA clone and the translated sequence of the B subunit of the V-type ATPase are shown. The putative start codon of the B subunit is at nucleotide 35. No other start codons in any of the reading frames were observed. Two polyadenylation signal sequences are underlined in the 3’ untranslated region of the cDNA. Three possible N-glycoslyation sites are indicated by CHO.

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FIG. 3. (A) Northern blot analysis of RNA isolated from the midgut and Malpighian tubules of H. virescens and M. sezta. The RNA was electrophoresed on a 0.66 M formaldehyde, 1% agarose gel and blotted. The blot was probed with DIG-labeled B subunit V-type ATPase cDNA, from clone 3-6. Lane 1, H. uirescens midgut mRNA (500 ng); lane 2, M. senta midgut mRNA (500 ng); lane 3, H. uirescens Malpighian tubule total RNA (1.0 pg); lane 4, M. se&a Malpighian tubule total RNA (1.0 pg). (B) Ethidium-bromide-stained agarose gel of PCR-amplified RNA. Midgut mRNA (250 ng) or Malpighian tubule total RNA (500 ng) was reverse-transcribed using random hexamers (100 pmol) as primers followed by PCR amplification in the presence of the sense 1s primer (40 pmol) and antisense external 2A primer (40 pmol). A total of 10 ~1 out of a total reaction volume of 100 ~1 was loaded in each lane. Lane 1, PCR product from H. uirescens midgut mRNA; lane 2, PCR product from H. virescens Malpighian tubule total RNA; lane S, size standard l.O-kb DNA ladder (BRL, Gaithersburg, MD); lane 3, PCR product from M. selcta midgut mRNA; lane 4, PCR product from M. serta Malpighian tubule total RNA. (C) Southern blot analysis of the PCR products from B. PCR-amplified RNA was electrophoresed on a 1% agarose gel and blotted (33). The blot was hybridized to the same probe as described in A. Lanes l-4 are as described in B except that only 0.5 ~1 of the total reaction volume was loaded in lanes 1 and 2.

Similar PCR amplification of RNA prepared from the central nervous system of M. senta larvae and the midgut Malpighian tubules of Culex quinquefasciatus larvae using the same two primers indicated above gave rise to 0.8-kb fragments (data not shown). This finding suggests the presence of V-type ATPases, in particular the B subunit, in the midgut Malpighian tubules of C. quinquefasciatus and the nervous system of M. se&a. The deduced amino acid sequence of the B subunit is highly homologous to the amino acid sequencesof known B subunits. The greatest homology is obtained with the B subunit of human kidney, with an amino acid identity and similarity of 85 and 95%, respectively (Fig. 4 and Table I). Similarly, there is significant homology with the B subunits of other eucaryotes. A somewhat lower, but still significant, level of homology is observed with the B subunits of procaryotes. DISCUSSION

A V-type ATPase B subunit cDNA clone was isolated from a cDNA library derived from the midgut/Malpighian tubules of H. uirescens. The predicted molecular mass of the protein encoded by the cDNA is 55 kDa, which is about 2 kDa smaller than the 57-kDa molecular weight of the B subunit observed in SDS-PAGE in another lepidopteran, M. sexta (9). Although the apparent molecular

ROSS

mass in SDS-PAGE for the B subunit for H. virescens is unknown, it is possible that the differences in the size of the transcripts in H. virescens and M. sexta reflect the differences in the size of the proteins that are encoded by these transcripts. Moreover, the predicted and observed molecular weights for the B subunit can vary as noted in the plant Arabidopszk (14). The B subunit of H. virescens is most homologous to the human kidney B subunit, with an amino acid identity of 85% (Table I). In addition, very high homology is observed with other B subunits. Most of the amino acid changes are observed in domains that are also less conserved in the other known B subunits. In particular both the N- and C-terminals show considerable divergence. However, although the various B subunits vary in mass (Fig. 4), they apparently retain essential components required for activity. For example, the putative ATP binding site of the B subunit, NGSIT (amino acids 329-333), is identical to that in the V-type ATPases of Neurospora and yeast and in chromaffin granules. This ATP binding site has previously been shown to be highly conserved among various ATPase subunits, including V-type and FOF1-type ATPases (14). These data support the hypothesis (21,36,37) that these two ATPases underwent gene duplication prior to organismal divergence. Earlier analyses of the B subunit have predicted that this subunit is probably not an integral membrane protein, but rather a peripheral protein (30). The amino acid sequence information obtained herein, which shows little hydrophobicity and a lack of a signal sequence, supports this contention. The integral subunit(s) of the V-type ATPase, i.e., the 16- and 32-kDa proteins from chromaffin granules, in contrast, is substantially more hydrophobic and contains putative membrane spanning domains (38). Both the B subunit and the 16kDa proteolipid are required for the proper function of the V-type ATPase because the disruption of either causesconditional lethality in eukaryotic cells (39). Isoforms of the B subunit have been demonstrated to occur in man, with variants in the amino acid sequence isolated from the brain and the kidney (21,23). Although no isoforms of the B subunit were found in H. virescens, a clone, 3-5, with substantial nucleotide differences was isolated. The deduced amino acid sequence for this clone, however, is identical to that of the clone identified in Fig. 2 (data not shown). The identification of a V-type ATPase in the midgut of H. virescens and M. sexta provides additional evidence that K+ ion regulation is mediated by a H+-ATPase as proposed by Wieczorek et al. (6). Nevertheless, it cannot be ruled out that other ATPases, particularly P-ATPases, may also modulate the K+ ion efflux from the hemocoel to the lumen of the midgut. Further, it is possible that this H+-ATPase, which generates a large potential gradient, could also modulate the efflux or influx of other ions. This ion modulation could be facilitated by the pres-

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AGIVKGAKGS

AGRVEGRGGS AGRVEGRNGS WNVWNWS AGRVWRNGS AGRIEGRKGS AGRVAGRNGS

l

.

.C.

.

I

l

DIGWEVLSIL

DLAWTLLRIF DLAWSLLRIY DIGWKILAHL

DLSWIULRI? DIGWQLLRIF DIGUQLLRIP DQAWSLLRIY

.t

ITQt'lPILTMP

a..

l

.

.

PESELSLIRS

PFU?XUiRIPA RKDKLNRIPK PENQLCRIDN

PKEJlLNRISQ

PWIPA

PKERLKRIPQ PKEHLKRIPQ

t.

NDDNTNPIPD

GDDITHPIPD

NDDITtlPIPD

NDDITNPIPD NDDITHPTPD

ITQIPILTRP 1~IPILTR.F ITQIPILTHP VTQIPILSMP

.

NDDITNPIPD WDIWPIPD

.*

l .

NDDITHPIPD

l

iWV?MIGVR

*

l

.c

YDSALFFRKF

2A

t.....

KYIQKYNPAG EYIKKYRPNY

KTLDQFYSW KIIDEFYSRS

RFLNRFTIBP

AVIDEHSRK STLSEFYPW SXABFYPID

.

LTCYITEGQI

LSGYITBGQX

Ln;lITKGQI

l

.

.

FEAINRV

FIG.

CLFLNLANDP

.

406 494

RKGQ' RGKK=

465

459

STS* 492 AADRKGKGKD

B-I

SM=

SAND*

GRLQDLAPDT

l

VLDRSLFNKG

FVDRQLWNKG YIDRQLNMRQ FVDRGLHNRG WSRBLHRKG

WDRQLKNRQ WWQLWBQ

-P .

l

RKKKLR-

AL*

*.*

513

*.

IYPFINV'LNS

IYPPINVLPS IYPPINVLPS

IYPPINVLPS

IYPPINVLPS

IYPPINVLPS I?P-S

l

.

l

l

504

.

.

WFLNLADDP

TLFLNLBWP TLFLNLAUDP

IYPPINVLPS

FBFXGALBRA

FEWGSl4BRV FBBHGS~RT

YVDRQLRNRQ

NBBAQU'XSD

LETMFFTW

MBTAQFFWD

LTGYITBGQI Ln;rXImI

.

.*

.

LSGSGEPLDG FNPLGBPLDN

FDGSGRFIDN INGSGKPIDN FDGSGRAIDK

FWGSGIDIDK

FNGSGKPIDK FNCSGRPIDR

CLnNLWDP LBTARFFRQDFEBUGSLBRTSLFULANDP

LTGYITEGQI LWlITBGQI

AVWAARGIT

AIVFMWVN SIWCAM'JN

SIVFANG3tR

ITQIPILTllP ITQIPILTNP

XTQIPILTMP

.

LVKR... LEKTV DLLEDHGEDNF LVQR@XTNKGV ..HDGtl.EENF . . . . . . . . . . . . ..VPGS.ESAF . . . . . . . . . . . . ..VRGE.ESN?

LVR....FTKOV

.

KISBBHLCRI

PASIDLLGRI

PVSBDRLGRI PVSLDHLGNI GVSBDKLGRI

PVSIDWLQIO

PVSSDlCCCRV

PVSBDML.GRV

NETARFFKSD FKQNGTUGN'J MTARFFKSDFEBltGSNDlWCLFLNLAMDP -

.**

WFTGETLKL VRFURGLEV

VBFTGBSLQ.

VQFTGCVLKT

VBFTGBSLRI

LTIX'ITBGQI

rtOfSPTOSI(

TToLD.KlECG

TSGIDVKKTT TSGIDTSGIDVBKTK

ZSGI-CBRODILRZ

TSGIDARKTT CLFTCDILRT DAlUtTS CZFTGDILRT

..liDGN.LENF

*

GVTFVQWFU

DIWIQI~

NRAWQWBfJ

BKAWQVFBG

DRAIVQLFBG

--

TKAIVQVFBG

IAAQICRQAG

l

RRCLWDSQU

RRGKVLDSSS

B RSGQVLEARG

RQGQVLEIRG

-w-w

RSGQVLEVAG

..LDYH.DDNF MVFAANGVN ..VDYS.LENF AIWMWVN ..LoD1I.KDHFAIVFMHGVN~D

“**

VTLl’LPDGTQ VTINLPDGTT VEIEMPDGSIC

VBLTLPDGTV VNIRIGDGST

VWSLWGTL

VliIRPDGTQ

IAAQICRQAG LVK....KSKAV IAAQfCRQAG LVK....WKDV IAAQICXQAGLVK...IPGRSV

GVSDMXNBL

KTEPVGYKEI

tlIKGPLIAVQ

NVKFPRYNBI

GVNGPLVILD

KVWPKYQEI

QIAGPLVFVB

TDNSSYAEAL TmlsSYABAL TDNSSYADAL TDNSSYADAL

TDNSSYAEAL

l

DVKTPRTSBI

oVWPLVILD GVl‘GFLVILE GVAGPLVILD KVKFPRYNBI

RVKFAQYASI

SVNGPLWLD

4. Amino acid sequence homology of the V-type ATPase B subunit from H. uirescens (Hv) to the B subunits from human chroma5n granules (HuK, 21), human brain (HUB, 23); yeast (SC, 30); Aradopsis (AH, 14), Neurosporu (NC, 12), Methanosarcino (MB, 17), and Sulfolobw (SA, 13). Amino acid identity is indicated by an asterisk. The three domains used for primer construction are underlined. Only the complete sequences of yeast, Aradopsis, Neurosporo, and Sulfo.!&us, and the partial sequence of Methanococcus (31) were available at the time the primers were designed. The conserved amino acid sequences used for the design of primers lS, 2A, and 3A are underlined.

KPTTKDTROT

.. . ..

LSRLKWGIG

..

EGNTRFZHSD EGMTRKDHGD AGKTREDHKA EGKTWDWIJ

LSRLWKSAIG LSRLNKSAIG

LSRLUNSGlG

VSNQLIAKYA VSNQLYANYA VSNQLYAKYA

VSIIQLYACVA

.*

SGRTRKDIIGD

VSNQLYWYA

VSNQLYACYA

l

EHGMIVLVIL EKDRGVLAIL

QCKRHVLVIL QcswvLVIL QTERNVLTIL KffiMVLVIL QLBKNVLVIL

QCEKHVLVIL

LSRLRKSAIG

EGNTRKDNGD EGI4TRKDHAD

**

TRSYPEEFIQ .

TGISTXDGTN TGISAIDGLN

ARLPPMFIQ .

TCISTXDVMN TGISAIDTMN

l

TGISAIDGNM WISAIov# TGVSAIDTW

TGISPIDVXN

SRIYPBKMQ

.

LNVREYSKIS

TPRIRYNTVG WVMYKTIT

EIGl4EYRTVS

KPRLNYRTVS

QPNLIxKTvs

HPRVTYRT'JC

CRIYPBKJIIQ SRIXPaglIQ ARIYPEWIIS CRTYPCWIQ ARFXPQENIS

LSRIJtKSAIQB-ESD

LSRLNKSAIG

LSRUDLSAIG

l

ALTLAEYLAF

l .

PSLKILTPKT

222

.

ALTAMYLAY

216 .

ALTTAEFLAX ~s!AwFIAY ALTTAKrLAI ALTTABXLAY ALTTMYYAY

TIBRIITPRI

ALTTAEFLAY

.*

TIERIITPRL TIBRIITPNL TIKRIITPRL

l

TIERIITPRL

GPKWAEDYL GPPILPMYL GPKVLASLYL GPRIWDQLL GPPVIGGEFR t.

DINGQPINPH DIMGQPINPQ DIQGQPINPW DINGSPINPY DISGSSINPS DINGSPINPY DINGAAMNPY NINGDPINPA

VIJwSw?Is KXAVBQGFNV DLDIEBGOTL. NA DPRVPSSXNV

NUT LWSQMBBN BIV LSDKELFAIN n%SHUGTN

RSL

MQAVTRNYIT

SCNLGMREN

DSRPGGLPGS

GPWMAEDFL GPWLASDFL

HAltBI

TIZRIITPRL AMRIVTPRII

241 249 250 243

262 163

mu sus BT SC w NC IQ M

EUB BUN NT W w NC w sn

136 31 114 123 120 113 9s 104

NUB ml8 BT SC AI NC w BL

1 1 1

1

1

1 1 1

Nlu Nus NT SC AN NC WI sa

.

98

GILL TABLE

AND

I

Amino Acid Homology of the B Subunit of H. virescens to that of Other V-Type Subunits Percentage identity Percentage similarity HV HuK SC AT NC MB SA

HV

HuK

SC

AT

NC

MB

SA

93 85 87 83 74 71

85

76 74

79 74 73 80 74 69

74 74 82 71 74 70

57 55 58 58 58

53 53 54 54 53 57 -

84 85 83 73 70

83 89 74 71

73

ence of a number of antiporters in the cell membrane. The capability of a specific antiport in transporting ions across the cell membrane would depend upon the selectivity of that specific antiporter protein. A number of such antiporters have been alluded to and also proposed by Wieczorek et al. (6). The H+-ATPase also could play a major role in the alkalinization of the insect midgut. Rapid and extensive excretion of H+ ions from the goblet cell cavity into its lumen, followed by an exchange of H+/K+ through an antiport, could also facilitate the simultaneous release of carbonate or bicarbonate ions. The release of these ions would lead to the midgut alkaline pH. Indeed, the goblet cells of the anterior and middle midgut have high levels of carbonic anhydrase (40). In the posterior lepidopteran midgut, the goblet cell cavities, in contrast, are more acidic (6, 41). The transcript size of the B subunit in the midgut of H. virescens was 2.3 kb, and a similar-size transcript was also detected in the Malpighian tubules. Using PCR analyses and Northern blotting the presence of homologous transcripts were also detected in the midgut and Malpighian tubules of M. sextu. The presence of the V-type ATPase in the Malpighian tubules of lepidopteran insects suggeststhat the transport of ions across the cell membrane in this tissue is also probably driven by a similar process to that observed in the midgut of these insects. In addition to the midgut, similar V-type H+-ATPases have also been postulated to occur in the salivary glands and the Malpighian tubules of insects. For example, in the salivary glands of the dipteran Calliphora erythrocephula, K+ is actively transported across the apical cell membranes (42,43). In addition to K+, Na+ is also transported in these tissues when the K+ concentration is low or absent. Similarly, ATPase activity has been demonstrated in the Malpighian tubules of Rhodnius, with the pump having a higher affinity for Na+ than for K+ (44). The localization of V-type ATPases in the Malpighian tubules of both H. virescens and M. sexta suggests that the midgut ion transport model may be also applicable

ROSS

for these tissues, whereby the V-type H+-ATPase drives the monovalent cation (M+) transport. This Mf transport occurs through an antiport which exchanges Mf for H+. However, unlike the midgut, ouabain sensitive P-ATPases,such as the Na+/K+-ATPase, are also present in the Malpighian tubules of Drosophila and Rhodnius (45,46). In Rhodnius this Na+/K+-ATPase is located on the basal membrane (46). The presence of a V-type ATPase in the goblet cell apical membrane of lepidopteran insects is not surprising becausethe cavity of this cell may arise from an embryonic cell vacuole (47). However, the finding that a V-type ATPase is also present in the Malpighian tubules of these insects suggeststhat V-type ATPases may be more common in insect epithelia than previously thought. The location of this V-type ATPase in the Malpighian tubules is not known. If the current model of ion transport in the Malpighian tubule is correct (44,46), then it is likely that the V-type ATPase in the Malpighian tubules is also present on the apical membrane of Malpighian cells as previously observed with the M. sexta midgut V-type ATPase (6, 7). The V-type ATPases appear to be present in a wide variety of tissues. For example, V-ATPases have been shown to occur in synaptic vesicles (48), the insect nervous system as demonstrated in this study, the brush border membrane vesicles of mammalian kidney (21), and the mosquito midgut Malpighian tubules, among others. Although the primary function of the ATPase in these tissuesis probably to transport protons across membranes, it is possible that the potential gradient that is generated by this transport of protons could drive the transport of other ions across these cell membranes. If this is to occur it is likely that a second cell membrane component is probably required to modulate or regulate the flow of these ions across the cell membrane. Potentially, antiports and/ or symports may serve as these regulatory components, whereby their presence or absence in a particular tissue and their ion selectivity could determine the process(es) that is ultimately driven by these pumps. In some manner, these two processes,that is, the development of a potential gradient and the selectivity of ion transport, may be coupled. ACKNOWLEDGMENTS The authorsthank E. Cowles, P. V. Pietrantonio, for the critical review of this manuscript. This work by grant 2 SO7 RR07010-23 from NIH.

and R. T. Leonard was support in part

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CeU 108,