Glutamate residues located within putative transmembrane helices are essential for TetA(P)-mediated tetracycline efflux

The tetA (P) gene from Clostridium perfringens encodes a unique membrane protein that is responsible for the active efflux of tetracycline from resistant cells, The novel TetA(P) protein has neither the typical structure nor the conserved motifs that are found in tetracycline efflux proteins from classes A through H or classes K and L. Site-directed mutagenesis of selected residues within TetA(P) was performed to elucidate their role in tetracycline efflux. Glutamate residues 52 and 59, negatively charged residues located within putative transmembrane helix 2, could not be replaced by either glutamine or aspartate and so were essential for tetracycline efflux. Replacement of Glu89, which was located at the end of helix 3, by aspartate but not by glutamine allowed TetA(P) function, indicating the importance of a carboxyl group at this position, After mutation of the Asp67 residue, located within cytoplasmic loop 1, no immunoreactive protein was detected, It is concluded that negatively charged residues that appear to be located within or near the membrane are important for the function of TetA(P).