Functional analysis of conserved cysteine residues in the catalytic subunit of the yeast vacuolar H+-ATPase

Lincoln Taiz, Hannah Nelson, Keith Maggert, Louis Morgan, Brad Yatabe, Saundra Lee Taiz, Bernard Rubinstein, Nathan Nelson

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

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 languageEnglish (US)
Pages (from-to)329-334
Number of pages6
JournalBBA - Biomembranes
Volume1194
Issue number2
DOIs
StatePublished - Sep 14 1994
Externally publishedYes

Keywords

  • ATPase
  • Cysteine
  • H-
  • N-Ethylmaleimide
  • Proton pump
  • vacuolar
  • Vacuole

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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