CHLOROPLAST MOLECULAR CHAPERONE ASSISTED REFOLDING AND RECONSTITUTIONOF AN ACTIVE MULTISUBUNIT COUPLING FACTOR CF1 CORE

The chloroplast coupling factor 1 (CF1) is composed of five kinds of s ubunits with a stoichiometry of alpha(3) beta(3) gamma delta epsilon R econstitution of a catalytically active alpha(3) beta(3) gamma core fr om urea-denatured subunits at a physiological pH is reported here. A r estoration of approximately 90% of the CF1 ATPase activity has been ob served. The reconstitution was achieved by using subunits overexpresse d in Escherichia coli, purified, and combined in the presence of MgATP , K+, and a mixture of several chloroplast molecular chaperones at pH 7.5. The combination of chaperonin 60 and chaperonin 24 failed to reco nstitute the active CF1 core, as did the GroEL/GroES pair (E. coli cha peronin 60/10 homologues). Characteristics of the reconstituted ATPase were very close to those of the native complex, including methanol-re versible inhibition by the purified epsilon subunit of CF1 and sensiti vity to Inhibition by azide and by tentoxin. In reconstitution with a mixture of tentoxin-resistant and -sensitive beta subunits, the extent of inhibition by tentoxin depended on the proportion of sensitive sub units in the reconstitution mixture. Finally, a model for the assembly of the CF1 core alpha(3) beta(3) gamma structure is proposed.