Membrane Topology of the Multidrug Transporter MdfA: Complementary Gene Fusion Studies Reveal a Nonessential C-Terminal Domain

Abstract

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.

Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

“…This pattern supports the topological model of MdfA (18,19), since the moderately or highly reactive mutants are found within the putative loops or at the edges of transmembrane domains. Another group of mutants, including Cys-21 (a single native cysteine in otherwise Cys-less MdfA), V23C, E26C, G187C, V231C, and L268C were much less reactive with NEM.…”

“…After examination of drug resistance activity of single cysteine mutants expressed from pT7-5/mdfA-His 6 (low expression system), the mutants were subcloned into pT7-5/pAra/mdfA-His 6 plasmid. This configuration enables high levels of MdfA expression in a tightly controlled manner, with arabinose as the inducer (19).…”

“…A model for MdfA secondary structure was constructed based on the hydropathy profile, distribution of positively charged residues, and gene fusion analysis (18,19). Amino acid residues that were isolated by the genetic screens are circled, and their clustering in two regions is illustrated.…”

“…Transport experiments show that MdfA is driven by the proton electrochemical gradient and functions as a drug/proton antiporter (17,15). As predicted from the hydropathy plot of the protein and gene fusion analysis, the putative 12 transmembrane domains (TMs) of the protein contain at least one membrane-embedded charged amino acid residue, Glu-26, in the middle of the TM1 (18,19) (see Fig. 1).…”

The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions

“…Previous studies have proposed that MdfA is a drug͞ proton antiporter (12). As predicted from the hydropathy plot of the protein and confirmed by gene-fusion studies, the putative 12 transmembrane regions of MdfA have only one charged amino acid residue, glutamate at position 26, which is embedded in the membrane, in the middle of putative transmembrane segment 1 (13). As demonstrated by mutational analysis, the amino acid residue at this position is important for substrate recognition and binding by MdfA but not for proton translocation (ref.…”