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• W2088142634 abstract "We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a2, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a1) we described (Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262–17266). Sequence comparison indicates that the a2 isoform is the bovine homologue of a 116-kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.-K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137–1144). The bovine a1 and a2 isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6–8 transmembrane regions. Northern blot analysis demonstrates that isoform a2 is highly expressed in lung, kidney, and spleen. To determine the possible role of the a2 isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (VO) containing isoform a2. This VO conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V1) of vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50–57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the 116-kDa polypeptide functions as part of the proton channel of V-ATPases. We have identified a cDNA encoding an isoform of the 116-kDa subunit of the bovine vacuolar proton translocating ATPase. The predicted protein sequence of the new isoform, designated a2, consists of 854 amino acids with a calculated molecular mass of 98,010 Da; it has approximately 50% identity to the original isoform (a1) we described (Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol. Chem. 269, 17262–17266). Sequence comparison indicates that the a2 isoform is the bovine homologue of a 116-kDa polypeptide identified in mouse as an immune regulatory factor (Lee, C.-K., Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137–1144). The bovine a1 and a2 isoforms share strikingly similar structures with hydrophilic amino-terminal halves that are composed of more than 30% charged residues and hydrophobic carboxyl-terminal halves that contain 6–8 transmembrane regions. Northern blot analysis demonstrates that isoform a2 is highly expressed in lung, kidney, and spleen. To determine the possible role of the a2 isoform in vacuolar proton pump function, we purified from bovine lung a vacuolar pump proton channel (VO) containing isoform a2. This VO conducts bafilomycin-sensitive proton flow after reconstitution and acid activation, and supports proton pumping activity after assembly with the catalytic sector (V1) of vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50–57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the 116-kDa polypeptide functions as part of the proton channel of V-ATPases. vacuolar-type proton translocating ATPase polyoxyethylene 9-lauryl ether 2-(N-morpholino)ethanesulfonic acid polyacrylamide gel electrophoresis sub-58-kDa doublet (50- and 57-kDa polypeptides required for function of V-ATPase) polymerase chain reaction kilobase. Vacuolar-type proton translocating ATPases (V-ATPases)1 are widely distributed in eukaryotic cells where they are found in most organelles. In addition, these proton pumps are localized to plasma membranes of epithelia, macrophages, and specialized polarized cells. V-ATPases have been shown to control osteoclast-mediated bone reabsorption and renal acidification and are thereby involved in the pathogenesis of osteoporosis and the systemic acidosis of uremia (1Stone D.K. Xie X.S. Kidney Int. 1988; 33: 403-413Abstract Full Text PDF Scopus (46) Google Scholar, 2Alpern R.A. Stone D.K. Rector F.C. Brenner B.M. Rector F.C. The Kidney. Sanders Press, Philadelphia1990: 318-380Google Scholar, 3Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar). Key to understanding the regulation of these diversely distributed proton pumps is a basic investigation of the structure of the enzymes and delineation of the roles of the individual subunits in pump function. The primary structures of V pump subunits, as well as the overall subunit composition and quartenary structures of the holoenzyme, are highly conserved through species as evolutionarily diverse as Archaebacteria, Caenorhabditis elegans, andHomo sapiens. In its simplest form, the V pump ofEnterococcus hirae is composed of 11 subunits, some of which are homologues of the subunits of the V-type proton pump of clathrin-coated vesicles of bovine brain (4Takase K. Kakinuma S. Yamato I. Konishi K. Igarashi K. Kakinuma Y. J. Biol. Chem. 1994; 269: 11037-11044Abstract Full Text PDF PubMed Google Scholar). Structurally, V-ATPases resemble F1FO-type ATP synthases in that they are complex hetero-oligomers with two functional domains: an ATP-hydrolytic sector (V1 or VC) that is peripheral to the membrane, and a transmembranous proton channel (VO or VB). The V1 domain of the V pump of clathrin-coated vesicles consists of essential, core subunits of 70, 58, 40, 34, 33, 14, and 10 kDa, designated A, B, C, D, E, G, and F, respectively. In addition, a key regulatory element, the sub-58-kDa doublet (SFD), consists of polypeptides of 57 and 50 kDa, activates V1 and functionally couples ATP hydrolysis to proton flow through the transmembranous sector, VO (5Xie X.-S. Crider B.P. Ma Y.-M. Stone D.K. J. Biol. Chem. 1994; 269: 25809-25815Abstract Full Text PDF PubMed Google Scholar, 6Zhou Z. Peng S.-B. Crider B.P. Slaughter C. Xie X.-S. Stone D.K. J. Biol. Chem. 1998; 278: 5878-5884Abstract Full Text Full Text PDF Scopus (33) Google Scholar). Separation of V1 from VO results in marked changes in the functions of these two domains. Although native holoenzyme hydrolyzes MgATP at a rate 3-fold higher than CaATP, isolated V1 hydrolyzes ATP only in the presence of Ca2+; Mg2+, in fact, inhibits ATP hydrolysis catalyzed by V1 (7Xie X.-S. Stone D.K. J. Biol. Chem. 1988; 263: 9859-9867Abstract Full Text PDF PubMed Google Scholar). In addition, the proton channel, VO, is closed after separation from V1, and requires incubation at an acidic pH to restore proton flow, which is inhibitable by bafilomycin A1, a V-ATPase specific inhibitor (8Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17379-17381Abstract Full Text PDF PubMed Google Scholar). Although the subunit composition of V1 is now well defined, there are conflicting reports regarding the components of VO. All investigators find a 17-kDa polypeptide (subunit c), as well as a 39-kDa subunit in VO preparations. In addition, the VO component of the proton pump of clathrin-coated vesicles contains a 116-kDa polypeptide, and a polypeptide of this mass, or a smaller homologue, has been demonstrated in most V-pump preparations. The function of the 116-kDa subunit is not defined, but its predicted structure consists of 6–8 transmembranous sectors, suggesting that it may function similar to subunit a of FO. Additional structural complexity exists in V-ATPases in the form of subunit isoforms. Two forms of subunit A (9Puopolo K. Kumamoto C. Adachi I. Forgac M. J. Biol. Chem. 1991; 266: 24564-24572Abstract Full Text PDF PubMed Google Scholar, 10Peng S.-B. Zhang Y. Crider B.P. White A.E. Fried V.A. Stone D.K. Xie X.-S. J. Biol. Chem. 1994; 269: 27778-27782Abstract Full Text PDF PubMed Google Scholar, 11van Hille B. Richener H. Evans D.B. Green J.R. Bilbe G. J. Biol. Chem. 1993; 268: 7075-7080Abstract Full Text PDF PubMed Google Scholar) and subunit B (12Südhof T.C. Fried V.A. Stone D.K. Johnston P.A. Xie X.-S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6067-6071Crossref PubMed Scopus (81) Google Scholar, 13Nelson R.D. Guo X.-L. Masood K. Brown D. Kalkbrenner M. Gluck S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3541-3545Crossref PubMed Scopus (192) Google Scholar) have been identified. Subunit G, a recently identified subunit that is required for ATP hydrolysis, has two isoforms that differ in tissue distribution and function (14Crider B.P. Andersen P. White A.E. Zhou Z. Li X. Mattsson J.P. Lundberg L. Keeling D.J. Xie X.-S. Stone D.K. Peng S.-B. J. Biol. Chem. 1997; 272: 10721-10728Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Most recently, we have demonstrated that the 50- and 57-kDa polypeptides of SFD are isoforms resulting through alternative mRNA splicing (7Xie X.-S. Stone D.K. J. Biol. Chem. 1988; 263: 9859-9867Abstract Full Text PDF PubMed Google Scholar). In addition, three forms of the c subunit of VO have been shown to be required for V-pump function in yeast (15Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anrakau Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The 116-kDa subunit of VO, the subject of this investigation, appears to have the greatest degree of isoform diversity of all V-pump components. This diversity arises through two mechanisms. First, alternative splicing of mRNA results in two forms of the subunit prevalent in brain (a1 isoform). This alternative splicing results in changes within a predicted protease sensitivity motif (PEST site, a region enriched in proline, glutamic acid, serine, or threonine residues), implying differences in the biological half lives of the two isoforms (16Perin M.S. Fried V.A. Stone D.K. Xie X.-S. Südhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar, 17Peng S.-B. Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17262-17266Abstract Full Text PDF PubMed Google Scholar). Second, higher organisms have separate genes that encode distinct isoforms of the 116-kDa subunit. In yeast, two such genes, designated VPH1 (20Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar) and STV1 (21Bauerle C. Ho M.-N. Lindorfer M.A. Stevens T.H. J. Biol. Chem. 1993; 268: 12749-12757Abstract Full Text PDF PubMed Google Scholar), encode proteins with amino acid sequence homology to the mammalian 116-kDa polypeptide. Cumulative evidence suggests that three separate genes encode forms of the 116-kDa subunit in mammalian species. In addition to the a1 isoform of bovine brain, a related homologue has been identified in murine T cells, and a third form in human osteoclasts. Although interspecies comparison of primary sequence complicates this point, it is notable that the sequence divergence between these bovine, murine, and human forms of the 116-kDa subunit greatly exceeds the divergence observed in all other pump subunits. Moreover, recent experiments have demonstrated three distinct genes encoding 116-kDa isoforms in chicken. 2J. Mattsson, X. Li, S. B. Peng, P. Andersen, B. Crider, L. Lundberg, D. K. Stone, and D. Keeling, manuscript in preparation. Of these putative forms of the 116-kDa subunit, the isoform isolated from murine T cells has not been identified as a V pump component. In fact, the cDNA encoding this subunit was isolated by a strategy designed to identify novel immune regulatory factors. To investigate the function of this isoform and to determine its relationship to V pump function, we have cloned and sequenced the cDNA encoding this isoform of the 116-kDa subunit. It shares only 50% identity to the 116-kDa subunit of bovine brain that we described previously, but has 91.6% identity to the 116-kDa isoform of murine T cells. The two polypeptides have strikingly similar structure, with hydrophilic amino-terminal halves that are composed of >30% charged residues and hydrophobic carboxyl-terminal halves that contain 6–8 transmembrane regions. The new isoform, designated a2, copurifies with vacuolar proton channel from lung. Reconstitution experiments demonstrate that it is associated with functional VO, indicating that it is a genuine isoform of the 116-kDa subunit of V pumps. Restriction enzymes, T4 DNA ligase, and a nick translation kit for DNA probe labeling were purchased from Boehringer Mannheim; the GeneAmp polymerase chain reaction (PCR) reagent kit withThermus aquaticus Taq DNA polymerase and DNA sequencing materials and reagents were from Perkin-Elmer; a TA cloning kit containing vector, pCR 2.1, and DNA ligase were from Invitrogen;Escherichia coli strains XLI-Blue-MRF′ and XLOLR and helper phage R408 were from Stratagene; radioactive materials and an ECL kit for Western blot analysis were from Amersham Pharmacia Biotech; nitrocellulose membranes for plaque lift were from Millipore Corp., and chemicals for SDS-PAGE were from Bio-Rad. A bovine brain cDNA library in λZAP was the kind gift of Dr. Richard A. F. Dixon (The University of Texas Health Science Center at Houston). All other reagents were from Sigma. Two oligonucleotide primers, 5′-TCICC(G/A)AACATIACIGC(G/A)AA-3′ and 5′-AAGTG(C/T)(C/T)TIATIGCIGA(A/G)GTITGGTG-3′, were designed and synthesized in accord with two regions of protein sequences of 116-kDa subunits of vacuolar ATPases that are highly conserved in all species (16Perin M.S. Fried V.A. Stone D.K. Xie X.-S. Südhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar, 17Peng S.-B. Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17262-17266Abstract Full Text PDF PubMed Google Scholar, 18Li Y.-P. Chen W. Stashenko P. Biochem. Biophys. Res. Commun. 1996; 218: 813-821Crossref PubMed Scopus (81) Google Scholar, 19Lee C.-K. Ghoshal K. Beaman K.D. Mol. Immunol. 1990; 27: 1137-1144Crossref PubMed Scopus (48) Google Scholar, 20Manolson M.F. Wu B. Proteau D. Taillon B.E. Roberts B.T. Hoyt M.A. Jones E.W. J. Biol. Chem. 1994; 269: 14064-14074Abstract Full Text PDF PubMed Google Scholar, 21Bauerle C. Ho M.-N. Lindorfer M.A. Stevens T.H. J. Biol. Chem. 1993; 268: 12749-12757Abstract Full Text PDF PubMed Google Scholar). Deoxyinosine (I) was used in the third position of some codons with a degeneracy of two or more. λZAP phage DNA from amplified bovine brain cDNA library was purified by standard procedure (22Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and used as a template for PCR, which was performed with 20 pmol of each primer and 1 μg of purified λZAP DNA. PCR products of 0.3 kb were cloned into pCR 2.1 vector using a TA cloning kit from Invitrogen. The positive colonies from TA cloning were analyzed by DNA sequencing, and a 0.3-kb insert was excised with EcoRI digestion, purified by preparative agarose gel electrophoresis, and used to screen a bovine brain cDNA library. A bovine brain cDNA library in λZAP (insert size, >2.0 kb), transfected intoE. coli, XLI-Blue-MRF′, was screened with the 0.3-kb PCR product that had been labeled with [α-32P]dCTP by nick translation. Screening of 2 × 106 individual phages was performed using a double-lift procedure wherein plaques were transferred to the nitrocellulose membranes for 5 min in each lift. The membranes were prehybridized for at least 4 h at 60 °C in a solution containing 5× SSC, 5× Denhardt's solution, 0.1 mg/ml of sheared salmon sperm DNA, and 0.1% SDS. Hybridization was performed at 50 °C overnight with the same solution plus labeled probe, which was added at a concentration of 5–10 × 105 cpm/ml of hybridization solution. The membranes were then washed for 15 min at room temperature first with 2× SSC and 0.1% SDS, then with 0.5× SSC and 0.1% SDS, and finally for 30 min at 55 °C with 0.1× SSC and 0.1% SDS. Autoradiography was performed with an intensifying screen at −80 °C for 24–48 h. Duplicate positive clones were cored and rescreened through one or more cycles until purified colonies were obtained. Inserts of all positive clones were excised and cloned into pBluescript with the helper phage R408, as described (10Peng S.-B. Zhang Y. Crider B.P. White A.E. Fried V.A. Stone D.K. Xie X.-S. J. Biol. Chem. 1994; 269: 27778-27782Abstract Full Text PDF PubMed Google Scholar). Plasmid DNA was prepared by alkaline lysis, and DNA sequencing reactions were performed using a Model 377 ABI PRISM DNA sequencer and the manufacturer's reagents. All positive clones were sequenced in both orientations using M13 reverse, M13 (−21), and sequence-specific oligonucleotides as primers. DNA and protein data base analysis was performed using PC/GENE-based programs. Poly (A+) RNA (2 μg/lane) from different bovine tissues was denatured and fractionated by 1% formaldehyde-agarose gel electrophoresis, and transferred to a Zeta-probe blotting membrane (Bio-Rad). After baking at 80 °C in a vacuum oven for 1 h, the membrane was prehybridized for 4 h at 50 °C in a solution consisting of 50% formamide, 1.5× saline/sodium phosphate/EDTA, 1% SDS, 0.5% nonfat dry milk, and 0.5 mg/ml of denatured salmon sperm DNA. A 0.6-kb cDNA fragment encoding the NH2-terminal portion of isoform a2was labeled with [α-32P]dCTP by nick translation and added to the hybridization solution at a concentration of 1 × 106 cpm/ml of solution. Hybridization was then carried out at 50 °C overnight. The membrane was sequentially washed for 15 min at room temperature with 2× SSC and 0.1% SDS, 0.5× SSC and 0.1% SDS, and 0.1× SSC and 0.1% SDS, respectively. A final wash was carried out at 60 °C for 30 min with 0.1× SSC plus 0.1% SDS, and autoradiography was performed with an intensifying screen at −80 °C for 3–5 days. Isoform a1-specific (CVLRRQYLRRKHLGT) and a2-specific (CGTIPSFMNTIPTKET) peptides were synthesized based upon the deduced protein sequences, coupled to keyhole limpet hemocyanin, and utilized for immunization of a New Zealand White rabbits to generate polyclonal antibodies, as described (23Peng S.-B. Crider B.P. Tsai S.-J. Xie X.-S. Stone D.K. J. Biol. Chem. 1996; 271: 3324-3327Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). The preparations of antibodies directed against the 70-kDa (subunit A) and 39-kDa subunits have been previously reported. For Western blot analysis, protein samples were separated by 11% SDS-PAGE and transferred electrophoretically to nitrocellulose filters. Immunodetection was performed using immune serum at a 1:5000 dilution and an Amersham Pharmacia Biotech ECL Western blotting system. Microsomes were prepared from bovine lung by using the buffer solution and initial steps used to prepare clathrin-coated vesicles from bovine brain. Briefly, bovine lung (1 kg) was homogenized in a Waring blender in 2 liters of Buffer A, consisting of 100 mm MES (pH 6.5), 3 mm azide, 1 mmEGTA, and 0.5 mm MgCl2. The homogenate was centrifuged at 3000 × g for 20 min, and the resulting supernatant was centrifuged at 180,000 × g for 1 h. The final crude, microsomal pellet was utilized for isolation of vacuolar proton pump and VO, as follows. Membrane pellet (5 ml) was resuspended in 20 ml of Buffer A with 1% C12E9 (polyoxyethylene 9-lauryl ether) and incubated on ice for 30 min. After centrifugation at 180,000 ×g for 1 h, the resulting pellet was resuspended in Buffer A containing 1% sodium cholate. Centrifugation was repeated, and the final pellet was resuspended in 3 ml of 1% Zwittergent 3–16, and incubated at room temperature for 1 h. After centrifugation at 180,000 × g for 1 h, the supernatant (3 ml) was loaded on two 13-ml, linear (15–30%) glycerol gradients prepared in Buffer G, consisting of 20 mm Tris-HCl (pH 7.5), 0.05% C12E9, 5 mm dithiothreitol, and 0.5 mm EDTA. After centrifugation at 180,000 ×g for 22 h at 4 °C, fractions of 1 ml were harvested from the bottom of the tube, subjected to SDS-PAGE, Western blot, and proton pumping and/or proton channel activity analysis. For further purification, the peak fractions were combined, concentrated with a Millipore Ultrafree-MC centrifugal filter unit, and separated by a second 15–30% glycerol gradient centrifugation that was performed as described above. Clathrin-coated vesicles were prepared from batches of 30 bovine brains, and V pump was solubilized with 1% C12E9 and purified to a specific activity of 14–16 μmol of Pi × mg protein−1 × min−1, as described (24Xie X.S. Stone D.K. J. Biol. Chem. 1986; 261: 2492-2495Abstract Full Text PDF PubMed Google Scholar). V1 and SFD were prepared as reported (7Xie X.-S. Stone D.K. J. Biol. Chem. 1988; 263: 9859-9867Abstract Full Text PDF PubMed Google Scholar, 5Xie X.-S. Crider B.P. Ma Y.-M. Stone D.K. J. Biol. Chem. 1994; 269: 25809-25815Abstract Full Text PDF PubMed Google Scholar). Reconstitutions of vacuolar proton pump and proton channel (VO) were performed by the freeze-thaw, cholate-dilution method using liposomes prepared from pure lipids. Stock solution of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol at a mass ratio of 4.32:2.76:0.2:2.7 were prepared as described (25Xie X.-S. Tsai S.-J. Stone D.K. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8913-8917Crossref PubMed Scopus (32) Google Scholar). Typically, 100 μl (0.5–0.8 mg of protein/ml) of purified proton pump or VO, was mixed with 3 mg of liposomes and 26 μl of reconstitution buffer consisting of 6% sodium cholate, 30% glycerol, 600 mm KCl, 6 μl of 600 mm dithiothreitol, and 60 mm MgCl2. The mixture was frozen in liquid N2 for 2 min and then incubated at room temperature for 1 h. Proteoliposomes were diluted with 6 ml of 150 mm KCl, 20 mm Tricine (pH 7.5), 3 mm MgCl2, and 0.5 mm EDTA (dilution buffer); concentrated by centrifugation at 110,000 × gfor 1 h at 15 °C; and then resuspended with 50 μl of dilution buffer for proton transport assays described below. Acid activation of the latent proton conductance of reconstituted VO was performed as described (5Xie X.-S. Crider B.P. Ma Y.-M. Stone D.K. J. Biol. Chem. 1994; 269: 25809-25815Abstract Full Text PDF PubMed Google Scholar), by incubation of 5 μl of proteoliposomes containing VO with 2 μl of 0.5 m MES (pH 5.0) for 1 h on ice. Proteoliposome acidification was assessed by the measurement of acridine orange quenching in an SLM-Amino DW2-C dual wavelength spectrophotometer as ΔA492–540 (24Xie X.S. Stone D.K. J. Biol. Chem. 1986; 261: 2492-2495Abstract Full Text PDF PubMed Google Scholar, 26Gluck S. Kelly S. Al-Awqati Q. J. Biol. Chem. 1982; 257: 9230-9233Abstract Full Text PDF PubMed Google Scholar). Proteoliposomes containing proton pump or VO were diluted into 1.6 ml of assay buffer, consisting of 150 mm NaCl, 30 mm sodium-Tricine (pH 7.5), 3 mm MgCl2, 0.5 mm EDTA, and 6 μm acridine orange. Reactions were initiated by the addition of ATP (final concentration, 1.3 mm) and/or valinomycin (1 μm), and were terminated by the addition of 1.6 μg of the proton ionophore bis(hexafluoroacetonyl) acetone, as indicated in the legends to the figures. Holoenzyme was reassembled with 3 pmol each of V1, VO, and SFD, which were mixed with 280 μg of lipid and 10 mm MES. The mixture was then incubated overnight at room temperature. The reassembled holoenzyme was reconstituted into liposomes for assessment of proton pumping as described above. Two degenerate primers, designed to match two conserved regions of the 116-kDa subunits, were used in polymerase chain reactions to generate 0.3-kb DNA fragments from a bovine brain cDNA library, as described under “Experimental Procedures.” The PCR products were cloned into pCR 2.1 vector using a commercial TA cloning kit (Invitrogen), and 25 positive clones were selected and sequenced. Among them, two groups of DNA sequences were obtained. The sequences of the first group (23 clones) exactly matched the a1 isoform sequence we have described (17Peng S.-B. Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17262-17266Abstract Full Text PDF PubMed Google Scholar). The remaining two clones had sequences that were identical to one another, but they encoded a protein with a predicted amino acid sequence different from that of the a1 isoform of the 116-kDa polypeptide. The 0.3-kb insert from the second group of TA clones was used to screen a bovine brain cDNA library in λZAP. 2 × 106individual bacteriophages were screened, yielding six positive clones, designated B2-2, B9-1, B17-1, B26-1, B31-1, and B34-1. DNA sequencing demonstrated that clone B31-1 contained the full coding region, clone B2–2 lacked coding region for 9 amino acids at the 5′-end, and the other clones had inserts of 1.5–2.5 kb. All of the clones had identical sequences in overlapping regions. The full sequence of clone B31–1 includes a 2565-base pair open reading frame and untranslated regions of 174 and 1703 base pairs at the 5′- and 3′-ends, respectively Translation of the open reading frame of clone B31–1 predicts an 854-amino acid polypeptide with calculated molecular mass of 98,010 Da, which is close to the mass of 96,301 Da of isoform a1. Three potential N-glycosylation sites are present at residues 43, 157, and 505. The calculated isoelectric point is 5.89. This isoform shares 50% identity with the a1 isoform that we described previously (16Perin M.S. Fried V.A. Stone D.K. Xie X.-S. Südhof T.C. J. Biol. Chem. 1991; 266: 3877-3881Abstract Full Text PDF PubMed Google Scholar, 17Peng S.-B. Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17262-17266Abstract Full Text PDF PubMed Google Scholar). Kyte-Doolittle (27Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-123Crossref PubMed Scopus (17296) Google Scholar) analysis reveals that the two isoforms have strikingly similar structures, with two characteristic domains: a hydrophilic amino-terminal half that is composed of more than 30% charged residues, and a highly conserved and hydrophobic carboxyl-terminal half that contains 6–8 transmembrane regions. Data base searches demonstrated that isoform a2 shares 91.6% identity at the amino acid level with mouse J6B7, a putative immune regulatory protein from T cells (19Lee C.-K. Ghoshal K. Beaman K.D. Mol. Immunol. 1990; 27: 1137-1144Crossref PubMed Scopus (48) Google Scholar) (Fig.1). As shown, particularly high levels of conservation were observed in predicted transmembranous sectors. The tissue distribution of mRNA encoding the a2 isoform was investigated by Northern blot analysis. Although two transcripts of approximate sizes of 3.4 and 5.4 kb were detected in all tissues (brain, heart, kidney, lung, and spleen), the absolute and relative abundances differed between tissues. High levels of expression were found in the kidney, lung and spleen, whereas the brain had very low levels of a2 transcripts (Fig.2). To determine the relationship of the cloned cDNA to the 116-kDa component of vacuolar proton pump, we generated isoform a1-, and a2-specific, anti-peptide antibodies based on predicted amino acid sequence. As shown in Fig.3, lane 3, the a2-specific antibody reacts with a minor portion of the 116-kDa band of highly purified bovine brain vacuolar proton pump, indicating the presence of isoform a2 in V-ATPase complex. The same enzyme reacts heavily with isoform a1 specific, anti-peptide antibody (Fig. 3, lane 2). This suggests that isoform a1 is the major form and a2 is the minor form of the vacuolar proton pump in the brain, which is in good accord with the results of Northern blot analysis we obtained in this study (Fig. 2) and in previous investigations (17Peng S.-B. Crider B.P. Xie X.-S. Stone D.K. J. Biol. Chem. 1994; 269: 17262-17266Abstract Full Text PDF PubMed Google Scholar). Whereas these findings are highly suggestive that the a2 isoform is present in subpopulation V-type proton pumps in bovine brain, the co-purification of the a1and a2 isoforms precluded any direct investigation of whether the a2 isoform was associated with a functional proton pump. We therefore sought to find an alternative tissue source highly enriched in the a2 isoform. As demonstrated by Northern blot analysis in Fig. 2, mRNA for isoform a2is present in high copy number in lung. We therefore attempted to isolate proton pump from bovine lung using the solubilization and purification procedure we developed for the V-type proton pump of clathrin-coated vesicles of bovine brain. Microsomes were prepared from freshly harvested bovine lung by homogenization and a differential centrifugation. For comparative purposes, freshly harvested bovine brain was processed identically. However, testing of V-pump activities in the two microsomal preparations revealed that the vesicles from lung had only 150 the specific activity of the vesicles from bovine brains, as assessed by ATP generated acridine orange quenching. To determine whether this relatively low activity was due to an intrinsic proteolysis of the pump from lung, we tested several different solutions for microsomal preparation. These included variances in pH from 6.5 to 7.5 and inclusion of proteinase inhibitors. The same results, however, were obtained. Moreover, C12E9, the detergent routinely utilized for the solubilization of the V pump of clathrin-coated vesicles, was ineffective in solubilizing bafilomycin-sensitive ATPase activity from lung microsomes. Numerous detergents were tested and we ultimately determined that the V-ATPase of lung was optimally solubilized with Zwittergent 3–16. Subsequently, purification of intact V pump was attempted by our standard protocol, which includes hydroxylapatite chromatography, and glycerol gradient centrifugation. Repeated attempts at purification, however, resulted in minuscule amounts of pump that migrated to the usual position in glycerol gradient centrifugation (data not shown). Instead, Western blot analysis using an anti-a2 isoform antibody rev" @default.
• W2088142634 created "2016-06-24" @default.
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• W2088142634 creator A5004078949 @default.
• W2088142634 creator A5018223304 @default.
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• W2088142634 date "1999-01-01" @default.
• W2088142634 modified "2023-09-30" @default.
• W2088142634 title "Identification and Reconstitution of an Isoform of the 116-kDa Subunit of the Vacuolar Proton Translocating ATPase" @default.
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