Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily.
STE6, the yeast a-factor transporter, is a member of the ATP binding cassette protein superfamily, which also includes the mammalian multidrug resistance protein and the cystic fibrosis gene product. These proteins contain two homologous halves, each with six membrane spanning segments and a predicted ATP nucleotide binding domain. To assess the importance of the two halves of STE6, and to examine the functional significance of residues conserved among members of the ATP binding cassette superfamily, we introduced mutations into the nucleotide binding domains of STE6. Our analysis demonstrates that both halves of STE6 are critical for function and that some, but not all, mutations analogous to those known to result in cystic fibrosis impair STE6 activity. To examine further the functional contribution of each half of the STE6 protein, we severed the STE6 coding sequence and expressed the two halves of the transporter as separate polypeptides. Whereas 'half-molecules' are unable to provide transport function individually, co-expression of both half-molecules in the same cell leads to functional reconstitution of STE6-mediated a-factor transport.
Berkower
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Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Michaelis
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D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids; D011233:Precipitin Tests; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D005656:Fungal Proteins; D000255:Adenosine Triphosphate