GroEL/GroES-mediated folding of a protein too large to be encapsulated

The chaperonin GroEL binds nonnative proteins too large to fit inside the p roductive GroEL-GroES cis cavity, but whether and how it assists their fold ing has remained unanswered. We have examined yeast mitochondrial aconitase , an 82 kDa monomeric Fe4S4 cluster-containing enzyme, observed to aggregat e in chaperonin-deficient mitochondria. We observed that aconitase folding both in vivo and in vitro requires both GroEL and GroES, and proceeds via m ultiple rounds of binding and release. Unlike the folding of smaller substr ates, however, this mechanism does not involve cis encapsulation but, rathe r, requires GroES binding to the trans ring to release nonnative substrate, which likely folds in solution. Following the phase of ATP/ GroES-dependen t refolding, GroEL stably bound apoaconitase, releasing active holoenzyme u pon Fe4S4 co-factor formation, independent of ATP and GroES.