title: The presence of the alternatively spliced A2 cassette in the vacuolar H+-ATPase subunit A prevents assembly of the V1 catalytic domain.
abstract: Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit. The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.
authors.affiliation: Department of Cell Biology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT, USA.
authors.forename: N
authors.initials: N
authors.lastname: Hernando
authors.forename: P
authors.initials: P
authors.lastname: David
authors.forename: M
authors.initials: M
authors.lastname: Tarsio
authors.forename: M
authors.initials: M
authors.lastname: Bartkiewicz
authors.forename: W C
authors.initials: WC
authors.lastname: Horne
authors.forename: P M
authors.initials: PM
authors.lastname: Kane
authors.forename: R
authors.initials: R
authors.lastname: Baron
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D002645:Chickens; D016384:Consensus Sequence; D005656:Fungal Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D016415:Sequence Alignment; D025262:Vacuolar Proton-Translocating ATPases
chemical_list: D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases