Functional analysis of membranous F_o-a subunit of F₁F_o-ATP synthase by in vitro protein synthesis

The a subunit of F₁F_o (F₁F_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_oa-less F₁F_o complex in Escherichia coli membrane vesicles and liposomes. The resulting F₁F_o has a H⁺-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F₁F_o. By using this procedure, we analysed five mutations of F₁F_o, where the conserved residues in the a subunit (Asn⁹⁰, Asp¹¹², Arg¹⁶⁹, Asn¹⁷³ and Gln²¹⁷) were individually replaced with alanine. All of the mutant F_oa subunits were successfully incorporated into F₁F_o, showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in F₁F_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. Asn⁹⁰ 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.