Functional analysis of membranous F o-a subunit of F 1F o-ATP synthase by in vitro protein synthesis

Yutetsu Kuruma, Toshiharu Suzuki, Sakurako Ono, Masasuke Yoshida, Takuya Ueda

研究成果: Article査読

39 被引用数 (Scopus)

抄録

The a subunit of F 1F o (F 1F o-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H +-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F o a-less F 1F ocomplex in Escherichia coli membrane vesicles and liposomes. The resulting F 1F o has a H +-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F 1F o. By using this procedure, we analysed five mutations of F 1F o, where the conserved residues in the a subunit (Asn 90, Asp 112, Arg 169, Asn 173 and Gln 217) were individually replaced with alanine. All of themutant F o a subunits were successfully incorporated into F 1F o, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F 1F o. The N173A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn 90 is located in the middle of putative second transmembrane helix and likely to play an important role in H +-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

本文言語English
ページ(範囲)631-638
ページ数8
ジャーナルBiochemical Journal
442
3
DOI
出版ステータスPublished - 2012 3 15
外部発表はい

ASJC Scopus subject areas

  • 生化学
  • 分子生物学
  • 細胞生物学

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