Reconstitution of the F 0 Complex of Escherichia coll ATP Synthase from Isolated Subunits

Abstract

Subunit c of the Escherichia coli F 1 F 0 ‐ATPase, purified in chloroform/methanol (2:1), was reconstituted with detergent‐solubilized F 0 subunits a and h to form a functionally active FT 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 FT 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.