The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export.
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.
Ketchum
C J
CJ
Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Schmidt
W K
WK
Rajendrakumar
G V
GV
Michaelis
S
S
Maloney
P C
PC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000493:pharmacokinetics; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000493:pharmacokinetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005838:Genotype; D006023:Glycoproteins; D006868:Hydrolysis; D007700:Kinetics; D000072235:Mating Factor; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D001386:Azides; D005656:Fungal Proteins; D006023:Glycoproteins; D010455:Peptides; D010675:Pheromones; D011994:Recombinant Proteins; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000072235:Mating Factor; D000255:Adenosine Triphosphate