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
Dmitriev, O.Y.; Altendorf, K.; Fillingame, R.H.
European Journal of Biochemistry 233(2): 478-483
Subunit c of the Escherichia coli F-1F-0-ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent-solubilized F-o subunits a and b to form a functionally active H+ channel. The rates of H+ uptake by the proteoliposomes containing the reconstituted F-0 complex were comparable to those observed with native F-0 reconstituted without subunit dissociation. The F-0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of the F-1-ATPase sector of the complex. Reconstitution of D61N and D61G mutant c subunits with wild-type subunits a and b produced an inactive F-0. Hybrid F-0 complexes, formed with mixtures of wild-type and D61N or D61G mutant c subunits, were also prepared. Formation of an active F-0 was prevented by addition of relatively small proportions of D61N or D61G mutant c subunits, i.e. active F-0 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 F-0 is sufficient to abolish activity.