Gene, 122 (1992) 355-360 @ 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

355

0378-l 119/92/$05.00

06797

Analysis of the gene encoding a 16kDa proteolipid subunit of the vacuolar H’-ATPase from Manduca sexta midgut and tubules (Proton

pump;

V-ATPase;

insects;

transepithelial

potential;

larvae; transport;

Lepidoptera;

tobacco

hornworm)

Julian A.T. Dow a, Stephen F. Goodwin b and Kim Kaiserb Departments of a Cell Biology and ofb Genetics, University of Glasgow, Glasgow G12 8QQ. UK. Tel. (44-41) 339 8855 x6228 (S.F.G.);(44-41)

339 8855 x51 12

(K.K.) Received

by R.W. Davies:

22 May 1992; Revised/Accepted:

28 July/30 July 1992; Received

at publishers:

20 August

1992

SUMMARY

Vacuolar ATPases (V-ATPases), originally characterised as components of endomembranes, have also been implicated in epithelial ion transport, both in vertebrates and in insects. The ATPase is particularly noteworthy in lepidopteran larvae, where it generates large transepithelial potential differences and short-circuit currents across the midgut epithelium. A cDNA library from Manduca sexta larval midguts and Malpighian tubules was screened with a Drosophila melanogaster cDNA encoding the 16-kDa proteolipid subunit of the V-ATPase, and a 1.4-kb cDNA sequenced in its entirety. The sequence contains a long open reading frame, encoding a putative peptide of 156 amino acids (aa) and with an M, of 1.5 967, in close agreement with values previously suggested by sodium dodecyl sulfate-polyacrylamide gels of M.sexta midgut proteins. Correspondence of the deduced aa sequence with those of other species, particularly D. melanogaster, was extremely close. Northern blots ofM.sexta midgut mRNA at high stringency revealed two transcripts of 1.4 and 1.9 kb, whereas genomic Southern blots suggest that there is only a single copy of the gene in M. sexta. The possibility that members of the 16-kDa gene family might serve multiple roles in transport and membrane communication is discussed.

INTRODUCTION

Electrogenic active transport of alkali metal cations in insects has widely been accredited to a ‘common cation pump’ (Harvey et al., 1983). This was thought to be an electrogenic ATPase, capable of transporting either sodium or potassium, according to availability, and was considered

Correspondence to: Dr. J.A.T. Dow, Department

of Cell Biology, Univer-

sity of Glasgow, Glasgow G12 8QQ, UK. Tel. (44-41) 330 4616; Fax (44-41) 330 4501; e-mail [email protected] Abbreviations: aa, amino acid(s); ATPase, adenosine triphosphatase; bp, base pair(s): DCCD, dicyclohexylcarbodiimide; GCG, Genetics Computer Group, Madison, WI; kb, kilobase or 1000 bp; oligo, oligodeoxyribonucleotide; ORF, open reading frame(s): SDS, sodium dodecyl sulfate; UTR, untranslated region(s); V-ATPase, vacuolar ATPase.

to be virtually ubiquitous in insects. Recently, persuasive evidence has suggested that the pump is composed of a plasma-membrane vacuolar H’-ATPase, together with a (possibly electrogenic) alkali metal/H + exchanger, analogous with, or in some cases identical to, the amiloridesensitive Na+ /H+ exchanger (Schweikl et al., 1989; Wieczorek et al., 1989; Klein et al., 1991). Thus protons flow in a futile cycle, resulting in the macroscopic signature of an alkali metal (Na’ or K+ ) electrogenic ion pump. The ‘pump’ is widely distributed in invertebrates: it is believed to be located in salivary glands, Malpighian tubules, and cuticular sensillae of probably all insects (Harvey et al., 1983). In particular, it is found a fortiori in the midguts of larval Lepidoptera, within a unique ‘goblet’ cell, where it produces an unparalleled electrical signature. Under perfusion in vitro, open-circuit potentials can be in excess of 150 mV (lumen positive), and the short circuit

356 current can comfortably exceed 1 mA cm-’ tissue (Harvey et al., 1983; Dow, 1986). The midgut is also characterised by a uniquely high luminal pH, in excess of 12 (Dow, 1984; Dow and Peacock, 1989; Dow and O’Donnell, 1990). A vacuolar ATPase has been purified from midgut by membrane fractionation (Wieczorek et al., 1989), and the goblet cell apical membrane can be stained by antibodies raised against V-ATPases in other species. Similarly, immune sera raised against the Munducn ATPase cross-react with V-ATPases isolated from other species (Klein et al., 1991). The V-ATPase is a multi-subunit complex, related to the F,/F, ATPase of bacteria, nitochandria and chloroplasts (Nelson and Nelson, 1989; Nelson and Taiz, 1989; Stone et al., 1990). The transmembrane proton pore is believed to be formed from six identical 16-kDa proteolipid subunits, the gene encoding which has now been cloned from several species (Mandel et al., 1988; Meagher et al., 1990; Hanada 0

et al., 1991; Toyama et al., 1991); the deduced aa sequences are highly conserved. Characteristically, DCCD inhibits V-ATPases: this has been shown to be due to binding to a conserved glutamic acid residue in the 16-kDa subunit (Jehmlich et al., 1991). This subunit also binds the bovine papillomavirus E5 oncoprotein (Goldstein et al., 1991; 1992). In addition, the aa sequence of the 16-kDa subunit resembles a tandem repeat of the 8-kDa F, ATPase subunit, suggesting a common ancestry by gene duplication (Nelson and Nelson, 1989; Nelson et al., 1990). Interestingly, it has recently been reported that a related, but nonidentical, yeast gene is essential for correct insertion of the 16-kDa proteolipid into the membrane (Umemoto et al., 1991). Evidence is thus accumulating that there is a 16-kDa proteolipid gene family. In this paper, we report the cloning and sequencing of a 1.4-kb cDNA encoding a 16-kDa protein virtually identi-

GG TGC AGA TAG TCA TCA CAG TTT TTG GGA CCT GTA AAT ACT CCC ACA CTA AAC GAT AAA

60 ATG GCC GAA AAT CCA ATC TAC GGA CCC TTC TTT GGA GTT ATG GGG GCG GCG TCT GCT ATT m et ala glu asn pro ile tyr gly pro phe phe gly val met gly ala ala ser ala ile 120 ATC TTC AGC GCC CTG GGA GCC GCC TAT GGC ACA GCC AAG TCG GGC ACC GGT ATC GCC GCC ile phe ser ala leu gly ala ala tyr gly thr ala lys ser gly thr gly ile ala ala 180 ATG TCG GTG ATG CGG CCC GAG CTG ATC ATG AAG TCC ATC ATC CCC GTC GTC ATG GCG GGT met ser val met arg pro glu leu ile met lys ser ile ile pro val val m et ala gly 240 ATC ATT GCC ATT TAC GGG CTG GTC GTG GCT GTA CTT ATT GCG GGT TCC CTG GAC TCG CCC ile ile ala ile tyr gly leu val val ala val leu ile ala gly ser leu asp ser pro 300 TCC AAT AAC TAC ACC CTG TAC AGA GGG TTC ATC CAC CTT GGC GCC GGC CTT GCC GTC GGA ser asn asn tyr thr leu tyr arg gly phe ile his leu gly ala gly leu ala val gly 360 TTC TCC GGT CTC GCG GCC GGT TTC GCC ATA GGC ATC GTT GGT GAT GCG GGC GTC CGC GGC phe ser gly leu ala ala gly phe ala ile gly ile val gly asp ala gly val arg gly 420 ACA GCT CAG CAG CCT AGG TTA TTC GTC GGA ATG ATC CTT ATC CTC ATT TTC GCC GAA GTA thr ala gln gin pro arg leu phe val gly met ile leu ile leu ile phe ala glu val 480 TTG GGT CTG TAC GGT CTC ATC GTC GCC ATC TAC CTG TAC ACG MA CAG TAA GCT GAA CAC leu gly leu tyr gly leu ile val ala ile tyr leu tyr thr lys gln OCH 540 ACC ACT CCC GTC GCC GTG CTC CGA GTT TCT ATG CCA GCC TGC TCA GTC CGC GCG ATG ACG 600 GCG TAC TAA TTT ATT TAP. TTT ATT ACC GCA GTG TTA TTT TTC GGT GAC CGG RGA ACT ATT 660 AAT TTC ACC TAT CGA CAT ATC AGT TTG TTT GCG CGT ACC ATC CGA CGA TAT CAC TTA TAG 720 ATT ATG TTA GCG TTA AGG CGA AAC TAA TGT ACA GTT AGA GTA GAA ATT TAA GTC CAA GCG 780 AGT TAT CTT AAA TAA TTT ATG MA

CCA TTG TTC TCG GGT ACT GTG TAC CAG AGT ATC ACT

840 GAG ATG AGG TTA AAC CTG GCT TGA GTA TTA CTT GCT ATT AGC TTT CAG TGT TAT AGT GGA 900 TGT GTT GTA TCC CCA TTA TAC TGT TAC GCC TTT AAA ATG TTA TCT TAT TCT CTC TTG GAG 960 TGA CTG TTG ACA MT

TAA GAG CGA GTC ACA AAC ATT GTA TTG CCA GGA ATC AGT AAA T.&A

1020 ACA TGG TTT GTA CAT TCC ATA TTA TTT TGT TTC TAC TTG ATT ATA CTG CGT TTT ATT TTC 1080 CTT TTC GAT CTT ATT ATG GGT GCG ATA TAT TTT MA 1140 ATT AGG AAG GCA MT

AAA MT

ATA ATG ATA TAA TAA TAA

GCT TTC ATG ACA TTA TAT AAA CTG TCG TAA TAA AAT TAC CTT TAA

1200 TAA TAA TAA TAA TAC ATT TCT GAC GAC TTA TTA TAA TAA AAG TAT TTC AAA TAT ATA TTT 1260 ATT CTG TAT ACC TCA TAG AAA TGA TGT TTG MT

GAT TAT AAA TTG GTA AGT AAA GTA CAT

1340 CTA GGT AAA ATT GTA TTT TAT TTT AGG TTT CAT GTT AGA GAA TGT TAT AAT CTT TGG TGT 1400 TAT TGA ATG AAT TGT GTT AAT MA Fig. 1. &NA

ad

signal is underlined.

putative

aa sequences

The cDNA

sequence

representing

ATA TTT TGA AAA CAA CTA CTG TAA

the 16-kDa proteolipid

has been published

subunit of the vacuolar

in the EMBL database

(Heidelberg)

ATPase

of M. senta. The presumed

under accession

No. X65051.

polyadenylation

357 cal to sequences previously reported for the 16-kDa proteolipid subunit of the vacuolar ATPase in other species. The known sequence which most closely resembles it is that for Drosophila (Meagher et al., 1990). Genomic Southern blots suggest a single gene, and Northern blot analysis demonstrates the presence of two transcripts, of 1.4 and 1.9 kb, in midgut.

EXPERIMENTAL

AND DISCUSSION

(a) separation of library M. sextu were purchased as larvae from the Department of Zoology, University of Cambridge, or obtained as eggs as a kind gift of Dr. Stuart Reynolds (University of Bath), and reared as described previously (Dow and Peacock, 1989; Dow and O’Donnell, 1990). Larvae were immobilised by chilling in ice for 20 min, and midguts were rapidly dissected and thoroughly rinsed in sterile ~~~~~c~ saline (Dow and O’Donnell, 1990) before freezing in liquid nitrogen. Total RNA was extracted from the frozen tissue as described (Chomczynski and Sacchi, 1987). Poly(A)‘RNA was purified by the polyATtractTM (Promega, UK) method; successful enrichment was demonstrated by Northern blotting, using a near fulllength Drosophila 16-kDa V-ATPase subunit cDNA (Meagher et al., 1990) as a probe. No information on the developmental or spatial expression patterns of the putative ATPase genes within the midgut is available, and so RNA was pooled from all midgut regions, and from Malpighian tubules (which also contain the pump), from a range of sizes of larvae of both 4th and 5th instars. In this way, although V-ATPase cDNAs might not be optimally represented in the library, they were almost certain to occur. The cDNA library was constructed in the jlZap II (Stratagene, La Jolla, CA) phagemid, according to the manufacturer’s instructions. The library was estimated to contain lo6 members, with an average insert size of around 1.5 kb. (b) Isolation and sequencing of clones The library was screened by plaque hyb~disation. Plaque lifts were probed with the Drosophila 16-kDa subunit cDNA (Meagher et al., 1990). Positives were obtained at approx. 1:lOOO and were purified by a further round of plating. Inserts of four recomb~ant phage were excised as pBluescript plasmids. Double-stranded sequencing was performed according to the SequenaseTM II protocol (US Biochemical, Cleveland, OH), with the aid of synthetic oligo primers. The 5 ’ and 3 ’ ends of all four cDNAs were sequenced, and found to be identical, except for small differences ( < 90 bp) in length at the 5’ end. The 1ongestcDNA was sequenced over its entire length on both strands.

50 MAENPIYGPF SSDNPIYGPF z&NGPEYASF SKSGPEYASF ....... ..i%A D IK?@?PPYSSF ........ ..MS TPGAPEYSAF .......... MS TDLCPVYAPF ....... ..MSS VFSGDETAPF .... ..MSYDLA TAERAAYAPF

............ ..... ..MSSEV ....... ..MSE ....... ..MSE

Drvhatp .. Eovatpplc .. Hspchsuca . .

Mmvp . . .. .. oat I. Lumbric . . Torpedo Schizo

Ysctfp? .. Ysvmall . . Yscppal ~KES~DDDM ............................

...........................

.

..MSTQLA

S NIYAPLYAPF

..* wnduca AVGFSGLAAG FAIGIVGDAG VRGTAQQPRL Dmvhatr,AVGFSGLAAG FAIGIVGDAG VRGTAQQPRL Bovatpplc SVGLSGLAAG FAIGIVGDAG VRGTAQQPRL Hspchsuca SVGLSGLAAG FAIGIVGDAG VRGTAQQPRL Mmmvp SVGLSGLAAG FAIGIVGDAG VRCTAQQPRL Torpedo SVGLSGLAAG FAIGIVGDAG VRGTAQQPRL Schizo SVGLAGLAAG FAIGIVGDAG VRGTAQQPRL Oat ACGLAGLAAG MAAIGIYGDAGVRANAQQBKL Lumbric TCGLCGLGAG YAIGIVGDAG VRGTAQQPRL Ysctfp3 LCGICLFE . ,......... .. .... .... Ysvmall CVGFACLSSG YAIGMVGDVG VRKYMHQFSL Yscppal TVGASNLICG IAVGITGATA AISDAADSAL

Manduca Dmvhatp Bovatpplc Hspchsuca Mmmvp Torpedo Schizo Oat Lumbric Ysctfg3 Ysvmall Yscppai

> VAIYLYTKQ. VAIYLYTK.. VALILSTK.. VALILSTK.. VALILSTK.. VALILSTK.. VALLLNTRAT VGIILSSRAG VALILGTSZT ........ .. VALILNTRGS VGLLMAGKAS