Reconstitution of the metal-tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1-ATPase

Yuichi Someya, Yoshinori Moriyama, Masamitsu Futai, Tetsuo Sawai, Akihito Yamaguchi

Research output: Contribution to journalArticle

8 Citations (Scopus)

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 Iq 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% n-octyl-β-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.

Original languageEnglish
Pages (from-to)72-76
Number of pages5
JournalFEBS Letters
Volume374
Issue number1
DOIs
Publication statusPublished - Oct 23 1995

Keywords

  • Antiporter
  • H-ATPase
  • Proteoliposome
  • Reconstitution
  • Tetracycline
  • Tetracycline/H antiporter

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

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