Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H(+)-ATPase.
The A subunit of the yeast vacuolar ATPase contains three highly conserved cysteines: Cys-261, Cys-284, and Cys-538. Cys-261 is located within the nucleotide-binding P-loop. Each of the conserved cysteines, and one nonconserved cysteine, Cys-254, were altered to serine by site-directed mutagenesis, and the effects on growth at pH 7.5 were determined. The Cys-254-->Ser, Cys-261-->Ser and the double mutants all grew at pH 7.5 and contained nitrate- and bafilomycin-sensitive ATPase activity. However, the ATPase activities of the Cys-261-->Ser and the double mutants were insensitive to the sulfhydryl group inhibitor, N-ethylmaleimide, demonstrating that Cys-261 is the site of inhibition by N-ethylmaleimide. Changing either Cys-284 or Cys-538 to serine prevented growth at pH 7.5. Cys-284 and Cys-538 thus appear to be essential cysteine residues which are required either for assembly or catalysis.
Taiz
L
L
Biology Department, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Nelson
H
H
Maggert
K
K
Morgan
L
L
Yatabe
B
B
Taiz
S L
SL
Rubinstein
B
B
Nelson
N
N
D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine / Q000032:analysis*; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009566:Nitrates / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D012694:Serine
D009566:Nitrates; D012694:Serine; D006180:Proton-Translocating ATPases; D003545:Cysteine; D005033:Ethylmaleimide