Mutational analysis of subunit G (Vma10p) of the yeast vacuolar H+-ATPase.
The G subunit of V-ATPases is a soluble subunit that shows homology with the b subunit of F-ATPases and may be part of the "stator" stalk connecting the peripheral V(1) and membrane V(0) sectors. When the N-terminal half of the G subunit is modeled as an alpha helix, most of the conserved residues fall on one face of the helix (Hunt, I. E., and Bowman, B. J. (1997) J. Bioenerg. Biomembr. 29, 533-540). We probed the function of this region by site-directed mutagenesis of the yeast VMA10 gene. Stable G subunits were produced in the presence of Y46A and K55A mutations, but subunit E was destabilized, resulting in loss of the V-ATPase assembly. Mutations E14A and K50A allowed wild-type growth and assembly of V-ATPase complexes, but the complexes formed were unstable. Mutations R25A and R25L stabilized V-ATPase complexes relative to wild-type and partially inhibited disassembly of V(1) from V(0) in response to glucose deprivation even though the mutant enzymes were fully active. A 2-amino acid deletion in the middle of the predicted N-terminal helix (DeltaQ29D30) allowed assembly of a functional V-ATPase. The results indicate that, although the N-terminal half of the G subunit is essential for V-ATPase activity, either this region is not a rigid helix or the presence of a continuous, conserved face of the helix is not essential.
Charsky
C M
CM
Department of Biochemistry and Molecular Biology, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA.
Schumann
N J
NJ
Kane
P M
PM
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D025262:Vacuolar Proton-Translocating ATPases; D015003:Yeasts / Q000201:enzymology
D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases