Mutagenesis Studies of the F1F0 ATP Synthase b Subunit Membrane Domain

A homodimer of b subunits constitutes the peripheral stalk linking the F₁ and F₀ sectors of the Escherichia coli ATP synthase. Each b subunit has a single-membrane domain. The constraints on the membrane domain have been studied by systematic mutagenesis. Replacement of a segment proximal to the cytoplasmic side of the membrane had minimal impact on F₁F₀ ATP synthase. However, multiple substitutions on the periplasmic side resulted in defects in assembly of the enzyme complex. These mutants had insufficient oxidative phosphorylation to support growth, and biochemical studies showed little F₁F₀ ATPase and no detectable ATP-driven proton pumping activity. Expression of the b_N2A,T6A,Q10A subunit was also oxidative phosphorylation deficient, but the b_N2A,T6A,Q10A protein was incorporated into an F₁F₀ complex. Single amino acid substitutions had minimal reductions in F₁F₀ ATP synthase function. The evidence suggests that the b subunit membrane domain has several sites of interaction contributing to assembly of F₀, and that these interactions are strongest on the periplasmic side of the bilayer.