The Yeast a-factor Transporter Ste6p, a Member of the ABC Superfamily, Couples ATP Hydrolysis to Pheromone Export

Christian J. Ketchum, Walter K. Schmidt, Garnepudi V. Rajendrakumar, Susan Michaelis, Peter C Maloney

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (Vmax = 18 nmol/mg/min; Km for MgATP = 0.2 mM) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent KI = 12 μM). Modest stimulation (∼40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[α-32P] ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a-factor transport, which in turn promotes mating.

Original languageEnglish (US)
Pages (from-to)29007-29011
Number of pages5
JournalJournal of Biological Chemistry
Volume276
Issue number31
DOIs
StatePublished - Aug 3 2001

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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