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.
Highlights
Vacuolar-type proton translocating ATPases (V-ATPases)1 are widely distributed in eukaryotic cells where they are found in most organelles
1 The abbreviations used are: V-ATPase, vacuolar-type proton translocating ATPase; C12E9, polyoxyethylene 9-lauryl ether; MES, 2-(Nmorpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SFD, sub-58-kDa doublet (50- and 57-kDa polypeptides required for function of V-ATPase); PCR, polymerase chain reaction; kb, kilobase
Materials—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 with Thermus 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-MRFЈ 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
Summary
V-ATPase, vacuolar-type proton translocating ATPase; C12E9, polyoxyethylene 9-lauryl ether; MES, 2-(Nmorpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SFD, sub-58-kDa doublet (50- and 57-kDa polypeptides required for function of V-ATPase); PCR, polymerase chain reaction; kb, kilobase. 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. Recent experiments have demonstrated three distinct genes encoding 116-kDa isoforms in chicken.2 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. 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. 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
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