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InsectBiochem.Molec.Biol.Vol.23,No.6,pp. 675-680,1993 Printedin GreatBritain.Allrightsreserved 0965-1748/93$6.00+ 0.00 Copyright 1993PergamonPressLtd Sequence of a 17 kDa Vacuolar H+-ATPase Proteolipid Subunit from Insect Midgut and Malpighian Tubules PATRICIA V. PIETRANTONIO,* SARJEET S. GILL*t Received 1 October 1992; revised and accepted 15 January 1993 A 0.4 kb polymerase chain reaction (PCR) product obtained from cDNA made from the midgut and Malpighian tubules of fifth instar larvae of Heliothis virescens was used to screen a larval midgut and Malpighian tubules cDNA library. Four clones were obtained, one of 1.9 kb and others of 1.4 kb. The 1.9 kb clone encodes a 17.2 kDa protein which is highly homologous to other vacuolar ATPases proteolipids. Putative N-glycosylation and DCCD binding sites were observed at amino acid residues 83 and 139, respectively. V-type H+-ATPase 17kDa proteolipid Heliothis virescens Insect Midgut Malpighiantubules INTRODUCTION 1988). Accordingly, the goblet cell apical plasma mem- Lepidopteran larvae encounter high K + concentrations in their diet and their physiology is adapted to higher levels of K ÷ rather than Na ÷ in their hemolymph (Bindokas and Adams, 1988; Harvey et al., 1983). The K ÷ ions, which are absorbed through the midgut, con- tribute to midgut alkalization (Dow, 1984; Dow and Peacock, 1989; Moffett and Koch, 1992), and its influx from the midgut lumen into the columnar cell facilitates amino acid uptake through a voltage dependent symport mechanism (Giordana et al., 1989). The midgut apical membrane actively transports K ÷ ions back to the midgut lumen, creating large membrane potentials (> 100 mV), the gut lumen being electrically positive to both the cell and the hemolymph (Harvey et al., 1983). The K ÷ efflux occurs from the inside of the goblet cell to the goblet cell cavity, and it is a secondary process apparently mediated by an electrogenic H ÷- ATPase pump (Wieczorek et al., 1989). The H+-ATPase pump is localized in the goblet cell apical membrane in the tobacco hornworm Manduca sexta (Klein et al., 1991), and probably facilitates the efflux of K ÷ through a K+/nH ÷ antiport (Wieczorek et al., 1991, Moffett and brane H+-ATPase from M. sexta has been purified and characterized as a vacuolar type (V-type) ATPase (Schweikl et al., 1989). In insects most knowledge of ATPases is primarily physiological and less is known about their primary structures. The deduced amino acid sequence of Drosophila melanogaster 16 kDa proteolipid has been published (Meagher et al., 1990), and the 55 kDa B subunit of Heliothis virescens and M. sexta, and the 67 kDa A subunit of M. sexta V-type ATPases have been recently cloned (Gill and Ross, 1991; Gr~if et al., 1992; Novak et al., 1992). This paper describes the cloning and sequencing of a putative 17 kDa proteolipid or proton translocating V-type ATPase subunit from a cDNA library of the midgut and Malpighian tubules of the tobacco bud- worm, H. virescens, an important agricultural pest. The characterization of the genes that code for ion regulation proteins will provide further understanding of the midgut function, and will enable us to use these proteins as potential targets for disruption to facilitate manipu- lation of insect populations. Koch, 1991). During embryogenesis the lepidopteran goblet cell MATERIALS AND METHODS cavity apparently arises as an intracellular vacuole which later comes in contact with the lumen (Hakim et al., cDNA library construction The construction of the larval H. virescens midgut and *Department of Entomology and Environmental Toxicology Malpighian tubules cDNA library has been described GraduateProgram,UniversityofCalifornia,Riverside,CA9252I, previously (Gill and Ross, 1991). RNA was isolated by U.S.A. homogenization in guanidine isothiocyanate followed by tAuthor for correspondence. Abbreviations used: DIG, Digoxigenin;pfu, plaque formingunits; cesium chloride centrifugation as described (Ausubel DCCD, N, N'-dicyclohexyl-carbodiimide;ORF, open reading et al., 1989). Poly(A) + RNA was purified by passin

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