Escherichia coli F1Fo-ATP synthase with a b/δ fusion protein allows analysis of the function of the individual b subunits

Abstract

The "stator stalk" of F1Fo-ATP synthase is essential for rotational catalysis as it connects the nonrotating portions of the enzyme. In Escherichia coli, the stator stalk consists of two (identical) b subunits and the δ subunit. In mycobacteria, one of the b subunits and the δ subunit are replaced by a b/δ fusion protein; the remaining b subunit is of the shorter b' type. In the present study, it is shown that it is possible to generate a functional E. coli ATP synthase containing a b/δ fusion protein. This construct allowed the analysis of the roles of the individual b subunits. The full-length b subunit (which in this case is covalently linked to δ in the fusion protein) is responsible for connecting the stalk to the catalytic F1 subcomplex. It is not required for interaction with the membrane-embedded Fo subcomplex, as its transmembrane helix can be removed. Attachment to Fo is the function of the other b subunit which in turn has only a minor (if any at all) role in binding to δ. Also in E. coli the second b subunit can be shortened to a b' type.

Keywords: ATP Synthase; Enzyme Catalysis; Enzyme Mechanisms; Membrane Proteins; Protein Assembly.

FIGURE 1.

Schematic representation of the b/δ fusion protein of M. vanbaalenii. The upper line shows the result of a secondary structure prediction. α-Helices are marked in red, β-sheets in blue. In the lower line the green portion indicates the segment corresponding to a b subunit, with the single transmembrane helix shown in cyan. The yellow portion indicates the part corresponding to δ.

FIGURE 2.

ATP-driven H⁺ pumping activity. ATP-driven proton pumping was monitored via quenching of the fluorescence of acridine orange (AO). At t = 0 s, the reaction was initiated by addition of 1 mm ATP. At t = 240 s, the reaction was stopped by addition of 1 μm carbonylcyanide m-chlorophenylhydrazone. The resulting traces can be grouped into four categories: no quenching for membranes obtained from strains expressing no ATP synthase (pUC118/DK8, as negative control), or (potentially) expressing (b^ΔN/δ), b^A79K/A128Dδ, (b^A79K/A128D/δ), and b^ΔN(b^A79K/A128D/δ) ATP synthase; approximately 25% quenching for membranes containing b(b^A79K/A128D/δ), b(b^{ΔN/A79K/A128D}/δ), and b^ΔC(b^{ΔN/A79K/A128D}/δ) enzyme; approximately 60% quenching for membranes with b^ΔC(b^ΔN/δ) ATP synthase; and >85% quenching for membranes containing (b/δ), b(b/δ), and b(b^ΔN/δ) enzyme as well as the positive (wild-type) control.

FIGURE 3.

Model of the possible arrangement of the transmembrane helices in E. coli ATP synthase. The figure shows helix 2 of 4 of the 10 c subunits in blue; helix 2 is located on the outside of the c ring. 4 of the 5 of the a subunits are shown in green; the transmembrane helices of the two b subunit are colored red. The view is from the cytoplasmic side, where the F₁ subcomplex is located. The arrow gives the direction of rotation of the c ring during ATP synthesis. The proposed arrangement of the helices is presented under “Discussion.”