Reconstitution of the metal-tetracycline/H+antiporter ofEscherichia coliin proteoliposomes including F0F1-ATPase

Abstract

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 β‐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% and one‐step purification by DEAE Sepharose CL‐6B column chromatography. The TetA protein was incorporated into proteoliposomes with F₀F₁‐ATPase. The proteoliposomes exhibited [³H]tetracycline transport dependent on ATP hydrolysis. The specific activity was about 2 nmol/mg protein/min. The proteoliposomes also showed H⁺ 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.