Abstract
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.
Original language | English (US) |
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Pages (from-to) | 329-334 |
Number of pages | 6 |
Journal | BBA - Biomembranes |
Volume | 1194 |
Issue number | 2 |
DOIs | |
State | Published - Sep 14 1994 |
Externally published | Yes |
Keywords
- ATPase
- Cysteine
- H-
- N-Ethylmaleimide
- Proton pump
- vacuolar
- Vacuole
ASJC Scopus subject areas
- Biophysics
- Biochemistry
- Cell Biology