Study of second-site suppression in the pheP gene for the phenylalanine transporter of Escherichia coli.
Site-directed mutagenesis was used to investigate a region of the PheP protein corresponding to the postulated consensus amphipathic region (CAR) in the GabP protein. Whereas some critical residues are conserved in both proteins, there are major differences between the two proteins which may reflect different functions for this region. Replacement of R317, Y313, or P341 by a number of other amino acids destroyed the PheP function. An R317E-E234R double mutant exhibited low levels of PheP transport activity, indicating that there is a possible interaction between these two residues in the wild-type protein. E234 is highly conserved in members of the superfamily of amino acid-polyamine-organocation transporters and also is critical for PheP function in the wild-type protein. Second-site suppressors were isolated for mutants with mutations in E234, Y313, R317, and P341. Most suppressor mutations were found to cluster towards the extracellular face of spans III, IX, and X. Some mutations, such as changes at M116, were able to suppress each of the primary changes at positions E234, Y313, R317, and P341 but were unable to restore function to a number of other primary mutants. The possible implications of these results for the tertiary structure of the protein are discussed.
Pi
Jing
J
Department of Microbiology and Immunology, The University of Melbourne, Victoria 3010, Australia.
Chow
H
H
Pittard
A J
AJ
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D016384:Consensus Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D005798:Genes, Bacterial; D018698:Glutamic Acid / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D027361:Organic Anion Transporters / Q000235:genetics; D010649:Phenylalanine / Q000378:metabolism; D011392:Proline / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D026921:Amino Acid Transport Systems, Neutral; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D050485:GABA Plasma Membrane Transport Proteins; D027361:Organic Anion Transporters; C513601:gabP protein, E coli; C069320:phenylalanine-specific permease, E coli; D018698:Glutamic Acid; D014443:Tyrosine; D010649:Phenylalanine; D011392:Proline