title: Subunit G of the vacuolar proton pump. Molecular characterization and functional expression.
abstract: The vacuolar type proton pump of clathrin-coated vesicles has a multisubunit ATP hydrolytic center that is peripheral to the membrane. Polypeptides present in this domain include the well characterized subunits A, B, C, D, E, and F; SFD, a dimer composed of 50- and 57-kDa polypeptides; and polypeptides termed G and H. Of these, subunits A, B, C, and E have been shown to be necessary but not sufficient for significant ATPase activity; in addition, either polypeptide G or H is also required for ATP hydrolysis (Xie, X.-S. (1996) J. Biol. Chem. 271, 30980-30985). In this study, the polypeptides G and H were purified and directly sequenced. Subsequent molecular analysis has revealed that these proteins are isoforms, which we designate G1 and G2. The cDNAs encoding the rat and bovine brain and chicken osteoclast forms of G1 have been cloned. The open reading frames of the rat and bovine clones encode hydrophilic proteins of 118 amino acids that differ at only five residues; bovine G1 has 36% identity with VMA10, a component of the proton channel of yeast. Northern blot analysis revealed a 1. 0-kilobase pair transcript encoding G1 in bovine brain, kidney, heart, and spleen. The cDNA encoding bovine polypeptide H was cloned and sequenced, revealing this protein to be 64% identical to G1, constituting isoform G2. In Northern blot analysis, a single 1. 7-kilobase pair transcript hybridized with a probe to G2 in brain, but not in heart, kidney, or spleen. An antibody against a bovine G1-specific domain reacts with V pump from bovine brain, kidney, and chromaffin granule, whereas an anti-G2 antibody reacts only with proton pump from brain. The bovine forms of G1 and G2 were subsequently expressed in Escherichia coli and Sf9 cells, respectively, and purified to homogeneity. Reconstitution of ATP hydrolysis was achieved by combination of recombinant subunits A, B, C, and E with either recombinant G1 or G2, demonstrating the role of these isoforms in pump function.
authors.affiliation: Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9121, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: Crider
authors.forename: P
authors.initials: P
authors.lastname: Andersen
authors.forename: A E
authors.initials: AE
authors.lastname: White
authors.forename: Z
authors.initials: Z
authors.lastname: Zhou
authors.forename: X
authors.initials: X
authors.lastname: Li
authors.forename: J P
authors.initials: JP
authors.lastname: Mattsson
authors.forename: L
authors.initials: L
authors.lastname: Lundberg
authors.forename: D J
authors.initials: DJ
authors.lastname: Keeling
authors.forename: X S
authors.initials: XS
authors.lastname: Xie
authors.forename: D K
authors.initials: DK
authors.lastname: Stone
authors.forename: S B
authors.initials: SB
authors.lastname: Peng
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002966:Clathrin; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism*; D017931:DNA Primers; D015723:Gene Library; D007700:Kinetics; D046911:Macromolecular Substances; D018402:Manduca; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017494:Proton Pumps / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000378:metabolism*
chemical_list: D002966:Clathrin; D017931:DNA Primers; D046911:Macromolecular Substances; D010446:Peptide Fragments; D017494:Proton Pumps; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; D000252:Calcium-Transporting ATPases