title: Functional analysis of membranous Fo-a subunit of F1Fo-ATP synthase by in vitro protein synthesis.
abstract: The a subunit of F(1)F(o) (F(1)F(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(1)F(o) complex in Escherichia coli membrane vesicles and liposomes. The resulting F(1)F(o) has a H(+)-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F(1)F(o). By using this procedure, we analysed five mutations of F(1)F(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 the mutant F(o) a subunits were successfully incorporated into F(1)F(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(1)F(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.
authors.affiliation: Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa-shi, Chiba, Japan.
authors.forename: Yutetsu
authors.initials: Y
authors.lastname: Kuruma
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Sakurako
authors.initials: S
authors.lastname: Ono
authors.forename: Masasuke
authors.initials: M
authors.lastname: Yoshida
authors.forename: Takuya
authors.initials: T
authors.lastname: Ueda
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D014176:Protein Biosynthesis / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases