RECONSTITUTION OF THE F-0 COMPLEX OF ESCHERICHIA-COLI ATP SYNTHASE FROM ISOLATED SUBUNITS - VARYING THE NUMBER OF ESSENTIAL CARBOXYLATES BYCO-INCORPORATION OF WILD-TYPE AND MUTANT SUBUNIT-C AFTER PURIFICATIONIN ORGANIC-SOLVENT

Subunit c of the Escherichia coli F1F0-ATPase, purified in chloroform/ methanol (2:1), was reconstituted with detergent-solubilized F-0 subun its a and b to form a functionally active H+ channel. The rates of Huptake by the proteoliposomes containing the reconstituted F-0 complex were comparable to those observed with native F-0 reconstituted witho ut subunit dissociation. The F-0 reconstituted from purified subunits was also shown to form an active ATP-driven H+ pump upon binding of th e F-1-ATPase sector of the complex. Reconstitution of D61N and D61G mu tant 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 o r D61G mutant c subunits, were also prepared. Formation of an active F -0 was prevented by addition of relatively small proportions of D61N o r D61G mutant c subunits, i.e. active F-0 formation was gradually disr upted as the mutant/wild-type ratio was increased from 0.05 to 0.2. Th e hybrid reconstitution studies support a model where inactivation of one of the 9-12 c subunits found in F-0 is sufficient to abolish activ ity.