Reconstitution of the Fo complex of Escherichia coli ATP synthase from isolated subunits. Varying the number of essential carboxylates by co-incorporation of wild-type and mutant subunit c after purification in organic solvent.

Subunit c of the Escherichia coli F1F0-ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent-solubilized F0 subunits a and b to form a functionally active H+ channel. The rates of H+ uptake by the proteoliposomes containing the reconstituted F0 complex were comparable to those observed with native F0 reconstituted without subunit dissociation. The F0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of the F1-ATPase sector of the complex. Reconstitution of D61N and D61G mutant c subunits with wild-type subunits a and b produced an inactive F0. Hybrid F0 complexes, formed with mixtures of wild-type and D61N or D61G mutant c subunits, were also prepared. Formation of an active F0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e. active F0 formation was gradually disrupted as the mutant/wild-type ratio was increased from 0.05 to 0.2. The hybrid reconstitution studies support a model where inactivation of one of the 9-12 c subunits found in F0 is sufficient to abolish activity.