Abstract
Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE.
Highlights
The question of multiple drug recognition has been addressed with several approaches
According to the secondary structure model of EmrE [31, 32], Tyr-4 and -6 are predicted to be located at the beginning of transmembrane segment (TM)1 facing the cytoplasm [33]; Tyr-53 is located in loop 2 connecting TM2 and TM3, whereas the other two tyrosine residues are located in the transmembrane regions Tyr-40 in TM 2 and Tyr-60 in TM 3
We characterized the role of tyrosine residues in the function of EmrE with a set of mutants constructed using site-directed mutagenesis
Summary
Bacterial Strains and Plasmids—E. coli DH5␣ (Invitrogen) and TA15 [25] strains were used throughout this work. The unbound material was discarded, and the His-tagged protein bound to beads was washed twice with SDS-urea buffer. Membranes, solubilized in 0.8% DDM-Na buffer, were added to the washed beads and incubated at 4 °C for 1 h. The unbound material was discarded, and the His-tagged protein bound to beads was washed twice with 0.08% DDM-Na buffer. The bead fraction was incubated for 15 min at room temperature with 450 l of 0.08% DDM-Na. buffer containing 150 mM imidazole to release the His-tagged protein and [3H] TPPϩ bound to it from the beads. The protein was eluted from the beads using a buffer containing 200 mM -mercaptoethanol, 100 mM Tris-HCl, pH 6.8, 4% SDS, 40% glycerol, 0.2% bromphenol blue, and 450 mM imidazole and analyzed by SDS-PAGE.
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