Functional asymmetry of the two nucleotide binding domains in the ABC transporter Ste6.
The yeast a-factor transporter Ste6 is a member of the ABC transporter family and is closely related to human MDR1. We constructed a set of 26 Ste6 mutants using a random mutagenesis approach. Cell fractionation experiments demonstrated that most of the mutants, with the notable exception of those with alterations in TM1, are transported to the plasma membrane, the presumptive site of action of Ste6. Trafficking, therefore, does not seem to be affected in most of the mutants. To identify regions in Ste6 that interact with the ABC transporter "signature motif" (LSGGQ) we screened for intragenic revertants of the LSGGQ mutant M68 (S507N). Suppressor mutations were identified in TM12 and upstream of TM6. Surprisingly, these mutations also suppressed the Walker A mutation G397D, which should be defective in ATP-binding and hydrolysis at NBD1. Photoaffinity labeling experiments with 8-azido-[alpha-32P]ATP showed that ATP binding at NBD2 is reduced by the suppressor mutation in TM12. The experiments further suggest that the two NBDs of Ste6 are not equivalent and affect each other's ability to bind and hydrolyze ATP.
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Germany.
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D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D000345:Affinity Labels; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001386:Azides / Q000493:pharmacokinetics; D001665:Binding Sites; D002462:Cell Membrane / Q000502:physiology; D017124:Conserved Sequence; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006023:Glycoproteins; D007700:Kinetics; D000072235:Mating Factor; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000235:genetics / Q000502:physiology; D010675:Pheromones / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D005656:Fungal Proteins; D006023:Glycoproteins; D010455:Peptides; D010675:Pheromones; C489277:STE6 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000072235:Mating Factor; D000255:Adenosine Triphosphate