| Title: | "The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export" |
| Author(s): | Ketchum CJ; Schmidt WK; Rajendrakumar GV; Michaelis S; Maloney PC; |
| Address: | "Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA" |
| ISSN/ISBN: | 0021-9258 (Print) 0021-9258 (Linking) |
| 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 (V(max) = 18 nmol/mg/min; K(m) 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 K(I) = 12 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(32)P]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" |
| Keywords: | ATP-Binding Cassette Transporters/chemistry/genetics/*metabolism Adenosine Triphosphate/*analogs & derivatives/*metabolism/pharmacokinetics Amino Acid Substitution Animals Azides/pharmacokinetics Cell Line Cell Membrane/metabolism Fungal Proteins/chemistr; |
| Notes: | "MedlineKetchum, C J Schmidt, W K Rajendrakumar, G V Michaelis, S Maloney, P C eng 1 F32 DK09995-01/DK/NIDDK NIH HHS/ DK48977/DK/NIDDK NIH HHS/ DK58029/DK/NIDDK NIH HHS/ GM51508/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 2001/06/05 J Biol Chem. 2001 Aug 3; 276(31):29007-11. doi: 10.1074/jbc.M100810200. Epub 2001 Jun 1" |
