Studies on the Bacterial Tetracyclin/H^+ Antiport Mechanisms using Site-directed Mutagenesis

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Bacterial tetracycline resistance is based on the active efflux of the drug. We revealed that the tetracycline resistance protein (TetA) is a 1:1 antiporter of Mg^<2+>-tetracycline chelation complex and proton at late 1980's.In this study, we established many site-directed mutants of the transposon Tn10- encoded TetA protein and performed the identification of the functionally essential residues in its transport function. As a result, we revealed some important principles common to the other membrane transport proteins as follows:1) All of the three acidic residues and one basic residue located in the putative transmembrane region of the TetA protein are essential for its transport function. In contrast, among eight conserved acidic residues in the hydrophilic region, only one residue, Asp66, is essential. Out of nine conserved basic residues in the hydrophilic region, one residue, Asp70, is essential for the transport function and one residue, Asp31, is essential for incorporation of the protein into the membrane.2) Functionally important residues in the transmembrane region are arranged on vertical stripes in the transmembrane alpha-helices, indicating the presence of the channel-like structure.3) There are two hydrophilic loops comprising highly conserved residues, which are likely to play as a gate for substrate translocation.On the basis of these observations, we claimed the gating-channel model for tetracycline/H^+ antiport.