Reconstitution of higher plant chloroplast chaperonin 60 tetradecamers active in protein folding

Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparat ions of the higher plant chloroplast chaperonin 60 (cpn60) consist of rough ly equal amounts of two divergent subunits, alpha and beta. The functional significance of these isoforms, their structural organization into tetradec amers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the alpha and beta subunits of the ch-cpn60 of pea (Pisum sativum), expressed t hem individually in Escherichia coli, and subjected the purified monomers t o in vitro reconstitution experiments. In the absence of other factors, nei ther subunit (alone or in combination) spontaneously assembles into a highe r order structure. However, in the presence of MgATP, the beta subunits for m tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, a subunits only assemble in the presence of beta subunits, Althou gh beta and alpha/beta 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are ent irely different. Similar to the authentic chloroplast protein, the reconsti tuted alpha/beta 14-mers are functionally compatible with bacterial, mitoch ondrial, and chloroplast cpn10, In contrast, the folding reaction mediated by the reconstituted beta 14-mers is only efficient with mitochondrial cpn1 0. The ability to reconstitute two types of functional oligomer in vitro pr ovides a unique tool, which will allow us to investigate the mechanism of t his unusual chaperonin system.