Unlike the GroEL homologs of eubacteria and mitochondria, oligomer preparations of the higher plant chloroplast chaperonin 60 (cpn60) consist of roughly equal amounts of two divergent subunits, ␣ and ␤. The functional significance of these isoforms, their structural organization into tetradecamers, and their interactions with the unique binary chloroplast chaperonin 10 (cpn10) have not been elucidated. Toward this goal, we have cloned the ␣ and ␤ subunits of the ch-cpn60 of pea (Pisum sativum), expressed them individually in Escherichia coli, and subjected the purified monomers to in vitro reconstitution experiments. In the absence of other factors, neither subunit (alone or in combination) spontaneously assembles into a higher order structure. However, in the presence of MgATP, the ␤ subunits form tetradecamers in a cooperative reaction that is potentiated by cpn10. In contrast, ␣ subunits only assemble in the presence of ␤ subunits. Although ␤ and ␣/␤ 14-mers are indistinguishable by electron microscopy and can both assist protein folding, their specificities for cpn10 are entirely different. Similar to the authentic chloroplast protein, the reconstituted ␣/␤ 14-mers are functionally compatible with bacterial, mitochondrial, and chloroplast cpn10. In contrast, the folding reaction mediated by the reconstituted ␤ 14-mers is only efficient with mitochondrial cpn10. The ability to reconstitute two types of functional oligomer in vitro provides a unique tool, which will allow us to investigate the mechanism of this unusual chaperonin system.

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