RECONSTITUTION OF THE F-0 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 F-0 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.