Title: A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively
Abstract: Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment withN-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel Vosubunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta. Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal anti- body to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment withN-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody. Thus, extensive N-glycosylation of the novel Vosubunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the Vo complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta. H+ V-ATPases are a class of ion transport proteins that couple ATP hydrolysis to the movement of protons across membranes. In endomembranes they function, in concert with chloride channels, as acidifiers of intracellular compartments, whereas in plasma membranes their roles are dependent on the cell type. V-ATPases consist of a peripheral V1 complex, which is responsible for the hydrolysis of ATP, and a membrane-bound Vo complex which is responsible for the translocation of protons. Although the subunit composition may depend upon the source of the enzyme, at least seven subunits of the V1 complex, subunits A to G, appear to be universal V-ATPase components (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (522) Google Scholar). By contrast, the subunit composition of the Vo complex is less clear. There is no doubt that a 16–17-kDa proteolipid, the proton "channel," is a major constituent of the Vo complex. A membrane-associated subunit in the 40-kDa range and an ∼100-kDa transmembrane subunit may be two additional essential Vo components (1Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (522) Google Scholar). Recently, a novel 9.2-kDa membrane sector-associated polypeptide was reported from bovine chromaffin granules (2Ludwig J. Kerscher S. Brandt U. Pfeiffer K. Getlawi F. Apps D.K. Schägger H. J. Biol. Chem. 1998; 273: 10939-10947Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Its sequence and structure show some similarity to Vma21p, a yeast protein involved in the assembly of the V-ATPase; whether or not it is a constitutive V-ATPase subunit remains an open question. In the larval midgut epithelium of the model insect, Manduca sexta (Lepidoptera, Sphingidae), a plasma membrane V-ATPase is present in the apical membranes of goblet cells where it energizes the alkalinization of the gut lumen to a pH of more than 11 (3Azuma M. Harvey W.R. Wieczorek H. FEBS Lett. 1995; 361: 153-156Crossref PubMed Scopus (90) Google Scholar). For the V1 complex, amino acid sequences of five insect subunits A, B, E, F, and G have been deduced from cloned cDNAs (4Merzendorfer H. Gräf R. Huss M. Harvey W.R. Wieczorek H. J. Exp. Biol. 1997; 200: 225-235Crossref PubMed Google Scholar), and evidence for the existence of subunit D has been derived from partial amino acid sequencing. 1M. Huss, R. Schmid, W. R. Harvey, and H. Wieczorek, unpublished data. 1M. Huss, R. Schmid, W. R. Harvey, and H. Wieczorek, unpublished data. For the Vo complex, only sequences of the 17-kDa proteolipid and of the subunit M40 have been derived from cDNAs to date (5Dow J.A.T. Goodwin S.F. Kaiser K. Gene (Amst.). 1992; 122: 355-360Crossref PubMed Scopus (38) Google Scholar, 6Merzendorfer H. Harvey W.R. Wieczorek H. FEBS Lett. 1997; 411: 239-244Crossref PubMed Scopus (21) Google Scholar), although evidence for a 100-kDa subunit is appearing on the horizon. Based on a partial amino acid sequence obtained from a 20-kDa polypeptide band that is present in gels after SDS-PAGE 2The abbreviations used are: ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MBP, maltose-binding protein; bp, base pair; DCCD, dicyclohexylcarbodiimide 2The abbreviations used are: ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MBP, maltose-binding protein; bp, base pair; DCCD, dicyclohexylcarbodiimideof the insect holoenzyme, we have cloned and sequenced the cDNA encoding a 9.7-kDa protein that is remarkably similar to the bovine 9.2-kDa Vo-associated protein. The insect protein is glycosylated extensively, with sugar residues contributing to half of its apparent molecular mass. We provide evidence here that the 9.7-kDa protein is a constitutive subunit of the Vo complex of the mature V-ATPase holoenzyme. Larvae of M. sexta (Lepidoptera, Sphingidae) were reared under long day conditions (16 h of light) at 27 °C using a synthetic diet modified according to Bell et al. (7Bell R.A. Joachim F.G. Ann. Entomol. Soc. Am. 1974; 69: 365-373Crossref Google Scholar). V-ATPase was isolated from the goblet cell apical membranes of larval M. sexta midgut as described previously (8Schweikl H. Klein U. Schindlbeck M. Wieczorek H. J. Biol. Chem. 1989; 264: 11136-11142Abstract Full Text PDF PubMed Google Scholar, 9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar). Three hundred μg of purified V-ATPase were subjected to preparative SDS-PAGE and stained with Coomassie Blue. The 20-kDa band was excised from the gel and concentrated as described by Rider et al. (10Rider M.H. Puype M. Van Damme J. Gevaert K. De Boeck S. D′Alayer J. Rasmussen H.H. Celis J.H. Vanderkerckhofe J. Eur. J. Biochem. 1995; 230: 258-266Crossref PubMed Scopus (48) Google Scholar) but using 5% polyacrylamide as spacer and a 15% polyacrylamide gel underneath. The resulting protein spot was blotted onto a polyvinylidene difluoride membrane (Immobilon P) using a buffer system consisting of 10 mmNaHCO3 and 3 mm Na2CO3. After staining with Amido Black, the spot was excised and installed into the blot cartridge of a model 473A protein sequencer (Applied Biosystems), and its amino acid sequence was determined as described previously (11Schmid R. Bernhardt J. Antelmann H. Völker A. Mach H. Völker U. Hecker M. Microbiology. 1997; 143: 991-998Crossref PubMed Scopus (62) Google Scholar). The N-terminal amino acid sequence of the M. sexta 20-kDa protein was used to design the degenerate primer pM20-deg (5′-ATGGC(T/C)TTCTTCGTICC(TCA)AT(TC)AC(TC)GTITTC-3′) which was optimized according to the codon usage of M. sextaproteins (12Frohlich D.R. Wells M.A. J. Mol. Evol. 1994; 38: 476-481Crossref PubMed Scopus (9) Google Scholar). Direct PCR (13Gussow D. Clackson T. Nucleic Acids Res. 1989; 17: 4000Crossref PubMed Scopus (281) Google Scholar) was performed in the presence of the primers pM20-deg (100 pmol) and pT7 (20 pmol, 5′-AATACGACTCACTATAGGGC-3′), the latter corresponding to the T7 promoter of the λ-Zap II DNA and 2 × 105plaque-forming units from an M. sexta larval midgut λ-Zap II cDNA library (14Gräf R. Novak F.J.S. Harvey W.R. Wieczorek H. FEBS Lett. 1992; 300: 119-122Crossref PubMed Scopus (28) Google Scholar). The reaction was carried out with AmpliTaq DNA polymerase (Perkin-Elmer) in a buffer consisting of 50 mm KCl, 1.5 mm MgCl2, 0.01% gelatin, 200 μm of each dNTP, and 10 mmTris-HCl (pH 8.3). After initial denaturation at 94 °C for 2 min, temperature cycles were set as follows: 94 °C for 30 s, 53 °C for 60 s, 73 °C for 40 s, 40 repeats. The resulting PCR product was cloned into the pUC18 vector using the SureClone Ligation Kit from Amersham Pharmacia Biotech and sequenced to verify the origin of the PCR product. To obtain full-length clones, 4 × 105 plaque-forming units of the λ-Zap II cDNA library (14Gräf R. Novak F.J.S. Harvey W.R. Wieczorek H. FEBS Lett. 1992; 300: 119-122Crossref PubMed Scopus (28) Google Scholar) were screened using a digoxigenin-11-dUTP labeled probe that had been generated by PCR (15Lion T. Haas O.A. Anal. Biochem. 1990; 188: 335-337Crossref PubMed Scopus (122) Google Scholar) in the presence of the primers pM20–1FWD (5′-GCATTGTGTGCCCTATCTTT-3′) and pM20–3REV (5′-CAGTAGTGGAATGACATCGG-3′). This approach led to the isolation of five independent phage clones. The phagemid sections of two clones were rescued by in vivo excision and subsequent infection ofEscherichia coli XL1-Blue (16Short J.M. Fernandez J.M. Sorge J.A. Huse W.D. Nucleic Acids Res. 1988; 16: 7583-7600Crossref PubMed Scopus (1080) Google Scholar). The derived pBluescript SK(−) plasmid clones pcM20BSK-A and pcM20BSK-B were purified using the QIAprep Spin Miniprep Kit from Qiagen and sequenced in both directions. Sequencing of all plasmids was performed by the dideoxynucleotide chain termination method (17Sanger F. Niklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52610) Google Scholar) using Sequenase 2.0 (18Tabor S. Richardson C.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4767-4771Crossref PubMed Scopus (1687) Google Scholar) from Amersham Pharmacia Biotech and appropriate sets of custom-synthesized 20-mer oligonucleotides prepared by MWG Biotec, Germany. Poly(A) RNA was prepared from either the midgut or Malpighian tubules of fifth instar larvae using the Quickprep Micro mRNA purification Kit from Amersham Pharmacia Biotech. Aliquots of 3.5 μg were loaded onto a 1% agarose, 2% formaldehyde gel. Sample preparation, gel electrophoresis, and Northern transfer on Hybond N membranes (Amersham Pharmacia Biotech) were performed as described previously (6Merzendorfer H. Harvey W.R. Wieczorek H. FEBS Lett. 1997; 411: 239-244Crossref PubMed Scopus (21) Google Scholar). After UV irradiation, the poly(A) RNA was hybridized with a digoxigenin-11-dUTP-labeled single-stranded RNA probe. This probe had been generated by in vitro transcription using the DIG RNA labeling kit (Roche Molecular Biochemicals), T7 polymerase, and 1 μg of the plasmid pcM20BSK-A, linearized by SacI restriction. Hybridization was performed for 14 h at 68 °C in 50% formamide, 5× SSC buffer, 0.02% SDS, 0.1%N-laurylsarcosine, and 2% (w/v) blocking reagent (Roche Molecular Biochemicals) at a probe concentration of approximately 100 ng/ml. Stringency washing was carried out at 68 °C in low salt buffer (0.1× SSC buffer, 0.1% SDS). Labeled poly(A) RNA was detected by use of the chemiluminescent substrate CSPD® (Roche Molecular Biochemicals) according to the manufacturer's protocol. The membranes were exposed to a Kodak XAR 5 film for 30 min. Deglycosylation of the V-ATPase isolated from partially purified goblet cell apical membranes was performed in a buffer containing 50 mm Tris-HCl (pH 8.0) and 0.1% SDS. One hundred μg of V-ATPase were treated with 0.3 units of endoglycosidase F/N-glycosidase F (Roche Molecular Biochemicals) in 200 μl of incubation buffer for 12 h at 37 °C. The proteins were precipitated by trichloroacetic acid, subjected to SDS-PAGE, then blotted and stained with 10 μg ml−1 concanavalin A and 20 μg ml−1horseradish peroxidase modified according to Clegg (19Clegg J.C. Anal. Biochem. 1982; 127: 389-394Crossref PubMed Scopus (147) Google Scholar) and Hawkes (20Hawkes R. Anal. Biochem. 1982; 123: 143-146Crossref PubMed Scopus (283) Google Scholar). V-ATPase was purified according to Schweikl et al. (8Schweikl H. Klein U. Schindlbeck M. Wieczorek H. J. Biol. Chem. 1989; 264: 11136-11142Abstract Full Text PDF PubMed Google Scholar) and Wieczorek et al. (9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar), modified as follows. First, the whole midgut tissue was used for solubilization instead of partially purified goblet cell apical membranes. Second, the first steps of preparation were performed in the presence of 5 mmPefabloc® Sc (Biomol). Finally, centrifugation on the discontinuous sucrose density gradient was carried out in the presence of 0.2m KCl. 0.8 mg of purified V-ATPase was subjected to preparative SDS-PAGE and negative-stained by the precipitation of a white imidazole-zinc complex (21Ferreras M. Gavilanes J.G. Garcia-Segura J.M. Anal. Biochem. 1993; 213: 206-212Crossref PubMed Scopus (44) Google Scholar). The 20-kDa protein band was excised from the gel and shredded into small pieces, leading to a total volume of approximately 0.4 ml. Four hundred μl of a buffer consisting of 0.2 m sodium acetate (pH 5.2), 0.2% Triton X-100, and 5 mm Pefabloc® Sc were added to one-half of the shredded gel, and 400 μl of the same buffer, in addition containing 3 milliunits of N-glycosidase A (Roche Molecular Biochemicals), to the second half. Both samples were incubated under gentle agitation for 36 h at 37 °C. The eluates obtained after separation from the gel pieces were precipitated in trichloroacetic acid and washed in acetone. Dried pellets were resuspended in Laemmli buffer and subjected to SDS-PAGE. After Western blotting on a nitrocellulose membrane (BA85), the protein bands were either immunostained with the monoclonal antibody 224-3 (22Sumner J.P. Dow J.A.T. Earley F.G. Klein U. Jäger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) or silver-stained using the kit from Amersham Pharmacia Biotech. The putative coding region together with the complete 3′-untranslated region of the 20-kDa protein was amplified by PCR. The 27-mer upstream primer started with a 6-base nonsense sequence followed by two cytosine residues and the first 19 bases of the coding sequence (5′-TATCAGCCATGGGTGCTTCCTTTGTCC-3′; the generatedNcoI site is underlined) and the 22-mer downstream primer (5′-GTAATACGACTCACTATAGGGC-3′) corresponding to the T7 site of pBluescript SK(−). The PCR product was digested with NcoI at the 5′ end and with KpnI at the 3′ end, purified by gel electrophoresis in 0.75% agarose, and extracted from the gel using the QIAquick gel extraction kit from Qiagen. The fragment was cloned into the NcoI/KpnI-predigested translation vector pSPUTK (Stratagene) and transformed into competent E. coliXL1-Blue. The resulting plasmid pM9.7SPK was prepared using the QIAprep Spin Miniprep Kit from Qiagen; residual RNase was destroyed by a treatment with proteinase K followed by phenol/chloroform extraction and ethanol precipitation. Coupled in vitrotranscription/translation with SP6 polymerase and [35S]methionine (20 μCi; Amersham Pharmacia Biotech) was performed using the TNT-rabbit reticulocyte system from Promega. After SDS-PAGE and Coomassie Blue staining, the gel was incubated in enhancer solution (EN3HANCE, DuPont) for 1 h, washed in cold water for 1 h, and dried on Whatman filter paper. [35S]Methionine-labeled protein bands were visualized by phosphorimaging. Midguts from fifth instar larvae were homogenized with a glass Teflon homogenizer in a buffer containing 0.32 m sucrose, 0.02 m Tris-HCl (pH 7.6), 1 mm EGTA, 3 mm MgCl2, and 5 mm Pefabloc® Sc. After filtration through cotton gauze the homogenate was centrifuged at 700 × g for 10 min at 4 °C. The resulting supernatant was again centrifuged at 7000 × g for 10 min at 4 °C. The microsomal pellet was obtained by spinning the 7000 × g supernatant at 100,000 × g for 100 min at 4 °C. It was resuspended in a buffer containing 0.02 m Tris-HCl (pH 7.6), 1 mm EGTA, 3 mm MgCl2, and 5 mm Pefabloc® Sc, layered onto a discontinuous 20–60% (w/w) sucrose density gradient (10% steps), and centrifuged overnight in a swing-out rotor (SW41Ti, Beckman) at 25,000 rpm and at 4 °C. Fractions obtained were spun down and frozen in liquid nitrogen. Purification of goblet cell apical membranes was performed according to Wieczorek et al. (9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar). Isolation of V-ATPase from partially and from highly purified goblet cell apical membranes of the M. sexta larval midgut as well as from Malpighian tubules, protein determination by Amido Black, SDS-PAGE, [14C]DCCD labeling, Western blotting, and immunostaining were performed as described previously (8Schweikl H. Klein U. Schindlbeck M. Wieczorek H. J. Biol. Chem. 1989; 264: 11136-11142Abstract Full Text PDF PubMed Google Scholar, 9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar, 23Wieczorek H. Putzenlechner M. Zeiske W. Klein U. J. Biol. Chem. 1991; 266: 15340-15347Abstract Full Text PDF PubMed Google Scholar, 24Lepier A. Gräf R. Azuma M. Merzendorfer H. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 8502-8508Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). V-ATPase was assayed as enzyme activity sensitive to 1 μm bafilomycin B1 (Fluka) according to Wieczorek et al. (9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar). The activities of NADPH-cytochrome c reductase and glucose-6-phosphatase were determined following modified protocols (25Williams C.H. Kamin H. J. Biol. Chem. 1996; 237: 587-595Google Scholar, 26Aronson N.N. Touster O. Methods Enzymol. 1974; 31: 90-102Crossref PubMed Scopus (512) Google Scholar). To inhibit unspecific hydrolysis of glucose 6-phosphate by alkaline phosphatases, 1 mm levamisol was added to the assay mixtures. Cytoplasmic expression of the 9.7-kDa polypeptide as an MBP fusion protein inE. coli was performed according to Gräf et al. (27Gräf R. Lepier A. Harvey W.R. Wieczorek H. J. Biol. Chem. 1994; 269: 3767-3774Abstract Full Text PDF PubMed Google Scholar) using the pMal-c2 expression system from New England Biolabs. The vector that was obtained by inserting the coding sequence and the 3′-untranslated region of pcM20BSK-A into the multiple cloning site of pMal-c2 was named pM9.7Mal-c2. In immunoblots of V-ATPase isolated from highly purified goblet cell apical membranes, a polyclonal antiserum to the M. sexta V-ATPase revealed a prominent protein band with an apparent molecular mass of ∼20 kDa (lane 1 in Fig.1, see also Ref. 28Wieczorek H. J. Exp. Biol. 1992; 172: 335-343Crossref PubMed Google Scholar). The polypeptide could also be visualized by silver staining (lane 2 in Fig.1; see also Ref. 22Sumner J.P. Dow J.A.T. Earley F.G. Klein U. Jäger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), whereas staining with Amido Black (lane 3 in Fig. 1) as well as with Coomassie Blue (not shown) usually did not reveal detectable amounts of polypeptide. In immunoblots of V-ATPase purified from either midgut goblet cell apical membranes or Malpighian tubule brush border membranes, the 20-kDa polypeptide was recognized by the monoclonal antibody 224-3 (lanes 4 and 5 in Fig. 1, respectively), which is specific for this polypeptide but also shows slight cross-reactivity to subunit B (22Sumner J.P. Dow J.A.T. Earley F.G. Klein U. Jäger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar). Since the 20-kDa polypeptide was isolated, together with known V-ATPase subunits, from two different types of highly purified plasma membranes originating from two different tissues, midgut and Malpighian tubules, it appears to be a constitutive subunit of the insect V-ATPase. Two previous experiments already had indicated that the 20-kDa polypeptide may be a member of the Vo complex. First, it remained in the membrane fraction after peripheral subunits were stripped off by chaotropic iodide (24Lepier A. Gräf R. Azuma M. Merzendorfer H. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 8502-8508Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Second, its relative amount in goblet cell apical membranes was enriched during moult when the V1 complex was released from the membrane (22Sumner J.P. Dow J.A.T. Earley F.G. Klein U. Jäger D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 29Gräf R. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 20908-20913Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). If the 20-kDa polypeptide is a Vo subunit, it should be present in the free Vo complex together with the established M. sexta Vo subunits c (5Dow J.A.T. Goodwin S.F. Kaiser K. Gene (Amst.). 1992; 122: 355-360Crossref PubMed Scopus (38) Google Scholar) and M40 (6Merzendorfer H. Harvey W.R. Wieczorek H. FEBS Lett. 1997; 411: 239-244Crossref PubMed Scopus (21) Google Scholar). Partially purified goblet cell apical membranes from starving larvae turned out to be a good source for the isolation of the Vo complex because, as in membranes from moulting larvae, they contain enriched free Vo complexes from which the V1 complexes have been detached. 3M. Huss, H. Merzendorfer, W. R. Harvey, and H. Wieczorek, manuscript in preparation. After solubilization of goblet cell apical membranes and zonal centrifugation in a discontinuous sucrose density gradient (8Schweikl H. Klein U. Schindlbeck M. Wieczorek H. J. Biol. Chem. 1989; 264: 11136-11142Abstract Full Text PDF PubMed Google Scholar, 9Wieczorek H. Cioffi M. Klein U. Harvey W.R. Schweikl H. Wolfersberger M.G. Methods Enzymol. 1990; 192: 608-616Crossref PubMed Scopus (67) Google Scholar), the two established Vo subunits c and M40 as well as the putative novel 20-kDa subunit were found not only in the upper 30% fraction as part of the remaining V1Vo holoenzyme but also in the upper 20% fraction as part of the integral Vo complex (Fig.2). The strictly similar distribution of the 20-kDa polypeptide and the Vo subunits c and M40 indicates that the novel polypeptide is a member of the Vocomplex. The 20-kDa polypeptide was isolated by SDS-PAGE, and its N-terminal protein sequence was determined to be(M/G)AXFVPITVF(L/T)ILXGXVGI. The first 10 amino acids (underlined) were chosen to design a codon-optimized, degenerate primer pM20-deg, assuming that the initial amino acid was methionine and that the third one was possibly phenylalanine (see "Experimental Procedures"). PCR using theM. sexta λ-Zap II cDNA library as a template resulted in the specific amplification of a 750-bp fragment that was obtained only in the presence of both primers, pM20-deg and pT7 (Fig.3). Sequencing of the cloned PCR fragment revealed that the correct cDNA encoding the 20-kDa protein had been amplified; the sequence and position of the deduced N-terminal amino acids perfectly matched with those that were determined from protein sequencing at positions 11–19, a section that could not be attributed to the upstream primer. To obtain a full-length cDNA clone, the M. sexta λ-Zap II cDNA library was screened by hybridization with a digoxigenin-11-dUTP-labeled probe corresponding to the PCR fragment that was related to the 20-kDa polypeptide. After three screening steps, two independent cDNA clones were isolated and turned out to be identical in their nucleotide sequences. The cDNA sequence comprised 765 base pairs and was terminated by a poly(A) tail of 31 bp (Fig. 4). Unexpectedly, the open reading frame which corresponded to the data obtained from protein sequencing was only 264 bp in length, encoding a hydrophobic protein of 88 amino acids with a calculated molecular mass of 9.67 kDa and an isoelectric point at pH 9.04, determined according to Skoog and Wichman (30Skoog B. Wichman A. Trends Anal. Chem. 1986; 5: 82Crossref Scopus (183) Google Scholar). Prediction of hydropathic properties and secondary structure based on different algorithms (31Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17168) Google Scholar, 32Chou P.Y. Fasman G.D. Adv. Enzymol. Relat. Areas Mol. Biol. 1978; 47: 45-148PubMed Google Scholar, 33Levin J.M. Robson B. Garnier J. FEBS Lett. 1986; 205: 303-308Crossref PubMed Scopus (262) Google Scholar, 34Deleage G. Roux B. Protein Eng. 1987; 1: 289-294Crossref PubMed Scopus (319) Google Scholar, 35Rost B. Casadio R. Fariselli P. Sander C. Protein Sci. 1995; 4: 521-533Crossref PubMed Scopus (643) Google Scholar) showed a high probability for one membrane-spanning α-helix in the range of amino acid positions 40–60. A second predicted hydrophobic α-helix within the first 20 amino acids may possibly be too short to span the membrane. The C terminus was more hydrophilic than the N terminus and is likely to be located at the extracellular surface since, according to the PROSITE data base, it contains two potential glycosylation sites at positions 68–70 and 84–86 (Fig. 4). Although the protein deduced from cDNA cloning was only approximately half the size of the 20-kDa polypeptide identified by SDS-PAGE, four findings suggest that the 20-kDa protein was encoded by the open reading frame. First, the deduced N-terminal 19 amino acids were in entire agreement with the data obtained from sequencing of the 20-kDa protein (Fig. 4), except for the missing N-terminal methionine in the mature protein. Moreover, the initiator ATG (nucleotide positions 64–66) of the open reading frame was embedded in a sequence environment that is similar to that of other cloned cDNAs encoding V-ATPase subunits from M. sexta (5Dow J.A.T. Goodwin S.F. Kaiser K. Gene (Amst.). 1992; 122: 355-360Crossref PubMed Scopus (38) Google Scholar, 14Gräf R. Novak F.J.S. Harvey W.R. Wieczorek H. FEBS Lett. 1992; 300: 119-122Crossref PubMed Scopus (28) Google Scholar, 24Lepier A. Gräf R. Azuma M. Merzendorfer H. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 8502-8508Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 27Gräf R. Lepier A. Harvey W.R. Wieczorek H. J. Biol. Chem. 1994; 269: 3767-3774Abstract Full Text PDF PubMed Google Scholar, 36Novak F.J.S. Gräf R. Waring R.B. Wolfersberger M.G. Wieczorek H. Harvey W.R. Biochim. Biophys. Acta. 1992; 1132: 67-71Crossref PubMed Scopus (35) Google Scholar) and matched closely the Kozak consensus sequence for translational initiation by eucaryotic ribosomes (37Kozak M. J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2809) Google Scholar). Second, cDNA sequencing was performed three times with cDNAs cloned independently, each in both directions, rendering unlikely sequencing mistakes or cloning artifacts leading to a frameshift. Even if nucleotides were deleted or inserted, the resulting reading frames would not increase substantially the molecular masses of the corresponding polypeptides. Third, the initial PCR performed on the λ-Zap II DNA resulted in only one PCR fragment of the indicated length even at relatively low stringency. Thus, a possible splice variant encoding an expanded open reading frame seems not to exist in the cDNA library. This conclusion was supported by Northern blots of poly(A) RNA isolated from midgut and Malpighian tubules; in both cases only one mRNA species of approximately 800 bp in length was observed (Fig.5). Moreover, this experiment placed the identified mRNA precisely in those tissues in which the putative 20-kDa V-ATPase subunit was expressed (Fig. 1). Fourth and finally, both the deduced 9.7-kDa protein and the 20-kDa polypeptide appear to be hydrophobic membrane proteins. To supply direct and definitive evidence that the cloned cDNA encoded the 20-kDa polypeptide, we expressed the recombinant 9.7-kDa protein as a fusion protein in E. coli. After SDS-PAGE the fusion protein was blotted and immunostained with the monoclonal antibody 224-3. In contrast to the unfused maltose-binding protein (MBP) with a molecular mass of 42.7 kDa, the 9.7-kDa/MBP-fusion protein had an electrophoretic mobility corresponding to 53 kDa and was immunoreactive to the 20-kDa-specific, monoclonal antibody 224-3 (see Fig. 7, lanes 5 and 6). A BLASTP search revealed that the deduced M. sexta amino acid sequence was 46% identical and 69% similar to the 9.2-kDa membrane sector-associated protein of V-ATPase subun