Mutational analysis of the non-homologous region of subunit A of the yeast V-ATPase.
Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit beta of the F(1)F(0) ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit beta. To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.
Shao
Elim
E
Department of Physiology, Tufts University School of Medicine, Boston Massachusetts 02111, USA.
Nishi
Tsuyoshi
T
Kawasaki-Nishi
Shoko
S
Forgac
Michael
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases