We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin and tubulin-based cytoskeleton. Consistent with prefoldin having a general role in chaperonin-mediated folding, we identify homologs in archaea, which have a class II chaperonin but contain neither actin nor tubulin. We show that by directing target proteins to chaperonin, prefoldin promotes folding in an environment in which there are many competing pathways for nonnative proteins.

Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood. We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y. Gao, J. 0. Thomas, R. L. Chow, G.-H. Lee, and N. J. Cowan, Cell 69:1043-1050, 1992). Here we show that the same chaperonin also forms a binary complex with unfolded a-or ,-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded a-or 13-tubulin polypeptides might exchange. Rather, two additional protein cofactors are required for the generation of properly folded a-or 13-tubulin, which is then competent for exchange into preexisting al-tubulin heterodimers. We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes. These data imply that actin and a-and 13-tubulin interact with the same site(s) on chaperonin complexes.There is compelling evidence that all of the information required for a given protein to assume its correct threedimensional structure is contained within its primary sequence (2, 24). Though this implies that the folding of proteins in vivo can occur spontaneously, it is now clear that in many cases folding is facilitated via interaction with one or more members of a class of proteins known as molecular chaperones or polypeptide chain-binding proteins (7,36). The majority of currently identified chaperones belong to one of two conserved families, exemplified by the stressinduced (though also constitutively expressed) heat shock proteins hsp70 and hsp60; the latter are also termed chaperonins (10,15,35). Both groups use the energy of ATP hydrolysis to release their bound substrate proteins and seem to act by stabilizing the conformation of folding intermediates, thereby preventing the formation of aberrant structures and directing the polypeptides toward correct folding, assembly, and translocation (5,16,17,21,25,38,46). Although hsp70 proteins and chaperonins share some common functional features, their roles are not interchangeable, and they are structurally distinct: while members of the hsp7O family act as monomers or dimers, chaperonins are multisubunit toroidal complexes (3-5, 8, 14, 16, 17, 22, 23, 33, 34, 44).The mechanisms involved in chaperonin-mediated protein folding are not well understood. In the case of the bacterial chaperonin (GroEL), there is evidence that the chaperonin can recognize secondary structure elements (28), which could result in the stabilization of conformational intermediates bound to the chaperonin (32). The folding reaction requires the hydrolysis of about 100 mol of ATP per mol of protein folded and may involve the ordered, st...

Chaperonins are ubiquitous multisubunit toroidal complexes that aid protein folding in an ATP-dependent manner. Current models of folding by the bacterial chaperonin GroEL depict its role as unfolding and releasing molecules that have misfolded, so that they can return to a potentially productive folding pathway in solution. Accordingly, a given target polypeptide might require several cycles of binding and ATP-driven release from different chaperonin complexes before reaching the native state. Surprisingly, cycling of a target protein does not guarantee its folding, and we report here that unfolded beta-actin or alpha-tubulin both form tight complexes when presented to either GroEL or its mitochondrial homologue, and both undergo cycles of release and rebinding upon incubation with ATP, but no native protein is produced. We conclude that different chaperonins produce distinctive spectra of folding intermediates.