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 Iq 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 [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.
Someya
Y
Y
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Moriyama
Y
Y
Futai
M
M
Sawai
T
T
Yamaguchi
A
A
D000595:Amino Acid Sequence; D017920:Antiporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active / Q000187:drug effects; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D008670:Metals; D008969:Molecular Sequence Data; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013752:Tetracycline / Q000378:metabolism*
D017920:Antiporters; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008670:Metals; D011510:Proteolipids; D013439:Sulfhydryl Reagents; C020485:proteoliposomes; C079051:tetA protein, Bacteria; D006859:Hydrogen; D006180:Proton-Translocating ATPases; D013752:Tetracycline