Gg. Chen et At. Jagendorf, CHLOROPLAST MOLECULAR CHAPERONE ASSISTED REFOLDING AND RECONSTITUTIONOF AN ACTIVE MULTISUBUNIT COUPLING FACTOR CF1 CORE, Proceedings of the National Academy of Sciences of the United Statesof America, 91(24), 1994, pp. 11497-11501
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.