Chloroplast molecular chaperone-assisted refolding and reconstitution of an active multisubunit coupling factor CF1 core.
The chloroplast coupling factor 1 (CF1) is composed of five kinds of subunits with a stoichiometry of alpha 3 beta 3 gamma delta epsilon. Reconstitution of a catalytically active alpha 3 beta 3 gamma core from urea-denatured subunits at a physiological pH is reported here. A restoration of approximately 90% of the CF1 ATPase activity has been observed. The reconstitution was achieved by using subunits overexpressed 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 reconstitute the active CF1 core, as did the GroEL/GroES pair (E. coli chaperonin 60/10 homologues). Characteristics of the reconstituted ATPase were very close to those of the native complex, including methanol-reversible inhibition by the purified epsilon subunit of CF1 and sensitivity 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 subunits in the reconstitution mixture. Finally, a model for the assembly of the CF1 core alpha 3 beta 3 gamma structure is proposed.
Chen
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GG
Section of Plant Biology, Cornell University, Ithaca, NY 14853.
Jagendorf
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AT
D002736:Chloroplasts / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000378:metabolism; D010456:Peptides, Cyclic / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D011994:Recombinant Proteins
D046911:Macromolecular Substances; D018832:Molecular Chaperones; D010456:Peptides, Cyclic; D010940:Plant Proteins; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases; C003384:tentoxin