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

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)631-638
Number of pages8
JournalBiochemical Journal
Volume442
Issue number3
DOIs
Publication statusPublished - 2012 Mar 15
Externally publishedYes

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Functional analysis
Adenosine Triphosphate
Escherichia coli
Hydrolysis
Proteins
Liposomes
Alanine
Peptide Hydrolases
Membranes
Mutation
In Vitro Techniques

Keywords

  • A subunit
  • Cell-free protein synthesis
  • F F -ATP synthase (F F )
  • H -pump
  • Membrane protein
  • Proteoliposome

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Functional analysis of membranous F o-a subunit of F 1F o-ATP synthase by in vitro protein synthesis. / Kuruma, Yutetsu; Suzuki, Toshiharu; Ono, Sakurako; Yoshida, Masasuke; Ueda, Takuya.

In: Biochemical Journal, Vol. 442, No. 3, 15.03.2012, p. 631-638.

Research output: Contribution to journalArticle

Kuruma, Yutetsu ; Suzuki, Toshiharu ; Ono, Sakurako ; Yoshida, Masasuke ; Ueda, Takuya. / Functional analysis of membranous F o-a subunit of F 1F o-ATP synthase by in vitro protein synthesis. In: Biochemical Journal. 2012 ; Vol. 442, No. 3. pp. 631-638.
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