RECONSTITUTION OF THE METAL-TETRACYCLINE/H+ ANTIPORTER OF ESCHERICHIA-COLI IN PROTEOLIPOSOMES INCLUDING F0F1-ATPASE

The tetracycline resistance gene (tetA) was cloned downstream of the lac promoter, When expression of the tetA gene in E. coli cells carrying the lac I-q gene was induced with isopropyl beta-D-thiogalactopyranoside, the tetracycline resistance protein (TetA) was overproduced, amounting to about 30% of the integral cytoplasmic membrane protein, Essentially pure TetA protein could be obtained by solubilization with 1.25% n-octyl-beta-D-glucopyranoside and one-step purification by DEAE Sepharose CL-6B column chromatography. The TetA protein was incorporated into proteoliposomes with F0F1-ATPase. The proteoliposomes exhibited [H-3]tetracycline transport dependent on ATP hydrolysis, The specific activity was about 2 nmol/mg protein/min. The proteoliposomes also showed HC efflux coupled with tetracycline influx. Tetracycline/H+ antiport by proteoliposomes reconstituted with the Ser-65 --> Cys mutant TetA protein was inhibited by N-ethylmaleimide. These results proved for the first time that the tetracycline/H+ antiport is only mediated by the TetA protein.