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.

We describe the complete beta-tubulin folding pathway. Folding intermediates produced via ATP-dependent interaction with cytosolic chaperonin undergo a sequence of interactions with four proteins (cofactors A, D, E, and C). The postchaperonin steps in the reaction cascade do not depend on ATP or GTP hydrolysis, although GTP plays a structural role in tubulin folding. Cofactors A and D function by capturing and stabilizing beta-tubulin in a quasi-native conformation. Cofactor E binds to the cofactor D-beta-tubulin complex; interaction with cofactor C then causes the release of beta-tubulin polypeptides that are committed to the native state. Sequence analysis identifies yeast homologs of cofactors D (cin1) and E (pac2), characterized by mutations that affect microtubule function.

The production of native α/β tubulin heterodimer in vitro depends on the action of cytosolic chaperonin and several protein cofactors. We previously showed that four such cofactors (termed A, C, D, and E) together with native tubulin act on β-tubulin folding intermediates generated by the chaperonin to produce polymerizable tubulin heterodimers. However, this set of cofactors generates native heterodimers only very inefficiently from α-tubulin folding intermediates produced by the same chaperonin. Here we describe the isolation, characterization, and genetic analysis of a novel tubulin folding cofactor (cofactor B) that greatly enhances the efficiency of α-tubulin folding in vitro. This enabled an integrated study of α- and β-tubulin folding: we find that the pathways leading to the formation of native α- and β-tubulin converge in that the folding of the α subunit requires the participation of cofactor complexes containing the β subunit and vice versa. We also show that sequestration of native α-or β-tubulins by complex formation with cofactors results in the destabilization and decay of the remaining free subunit. These data demonstrate that tubulin folding cofactors function by placing and/or maintaining α-and β-tubulin polypeptides in an activated conformational state required for the formation of native α/β heterodimers, and imply that each subunit provides information necessary for the proper folding of the other.