Membrane topology of the multidrug transporter MdfA: complementary gene fusion studies reveal a nonessential C-terminal domain.
The hydrophobicity profile and sequence alignment of the Escherichia coli multidrug transporter MdfA indicate that it belongs to the 12-transmembrane-domain family of transporters. According to this prediction, MdfA contains a single membrane-embedded charged residue (Glu26), which was shown to play an important role in substrate recognition. To test the predicted secondary structure of MdfA, we analyzed complementary pairs of hybrids of MdfA-PhoA (alkaline phosphatase, functional in the periplasm) and MdfA-Cat (chloramphenicol acetyltransferase, functional in the cytoplasm), generated in all the putative cytoplasmic and periplasmic loops of MdfA. Our results support the 12-transmembrane topology model and the suggestion that except for Glu26, no other charged residues are present in the membrane domain of MdfA. Surprisingly, by testing the ability of the truncated MdfA-Cat and MdfA-PhoA hybrids to confer multidrug resistance, we demonstrate that the entire C-terminal transmembrane domain and the cytoplasmic C terminus are not essential for MdfA-mediated drug resistance and transport.
Adler
Julia
J
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Bibi
Eitan
E
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000254:growth & development; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C118438:Mdfa protein, E coli; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D000469:Alkaline Phosphatase