The transmembrane domain of subunit b of the Escherichia coli F1FO ATP synthase is sufficient for H+‐translocating activity together with subunits a and c
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Media type:
E-Article
Title:
The transmembrane domain of subunit b of the Escherichia coli F1FO ATP synthase is sufficient for H+‐translocating activity together with subunits a and c
Description:
<jats:p>Subunit <jats:italic>b</jats:italic> is indispensable for the formation of a functional H<jats:sup>+</jats:sup>‐translocating F<jats:sub>O</jats:sub> complex both <jats:italic>in vivo</jats:italic> and <jats:italic>in vitro</jats:italic>. Whereas the very C‐terminus of subunit <jats:italic>b</jats:italic> interacts with F<jats:sub>1</jats:sub> and plays a crucial role in enzyme assembly, the C‐terminal region is also considered to be necessary for proper reconstitution of F<jats:sub>O</jats:sub> into liposomes. Here, we show that a synthetic peptide, residues 1–34 of subunit <jats:italic>b</jats:italic> (<jats:italic>b</jats:italic><jats:sub>1−34</jats:sub>) [Dmitriev, O., Jones, P.C., Jiang, W. & Fillingame, R.H. (1999) <jats:italic>J. Biol. Chem.</jats:italic><jats:bold>274</jats:bold>, 15598–15604], corresponding to the membrane domain of subunit <jats:italic>b</jats:italic> was sufficient in forming an active F<jats:sub>O</jats:sub> complex when coreconstituted with purified <jats:italic>ac</jats:italic> subcomplex. H<jats:sup>+</jats:sup> translocation was shown to be sensitive to the specific inhibitor <jats:italic>N,N′</jats:italic>‐dicyclohexylcarbodiimide, and the resulting F<jats:sub>O</jats:sub> complexes were deficient in binding of isolated F<jats:sub>1</jats:sub>. This demonstrates that only the membrane part of subunit <jats:italic>b</jats:italic> is sufficient, as well as necessary, for H<jats:sup>+</jats:sup> translocation across the membrane, whereas the binding of F<jats:sub>1</jats:sub> to F<jats:sub>O</jats:sub> is mainly triggered by C‐terminal residues beyond Glu34 in subunit <jats:italic>b</jats:italic>. Comparison of the data with former reconstitution experiments additionally indicated that parts of the hydrophilic portion of the subunit <jats:italic>b</jats:italic> dimer are not involved in the process of ion translocation itself, but might organize subunits <jats:italic>a</jats:italic> and <jats:italic>c</jats:italic> in F<jats:sub>O</jats:sub> assembly. Furthermore, the data obtained functionally support the monomeric NMR structure of the synthetic <jats:italic>b</jats:italic><jats:sub>1−34</jats:sub>.</jats:p>