Defining the TRiC/CCT interactome links chaperonin function to stabilization of newly made proteins with complex topologies.

Folding within the crowded cellular milieu often requires assistance from molecular chaperones that prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to folding the proteome remains elusive. Here we demonstrate that the eukaryotic chaperonin TRiC/CCT (TCP1-ring complex or chaperonin containing TCP1) has broad binding specificity in vitro, similar to the prokaryotic chaperonin GroEL. However, in vivo, TRiC substrate selection is not based solely on intrinsic determinants; instead, specificity is dictated by factors present during protein biogenesis. The identification of cellular substrates revealed that TRiC interacts with folding intermediates of a subset of structurally and functionally diverse polypeptides. Bioinformatics analysis revealed an enrichment in multidomain proteins and regions of beta-strand propensity that are predicted to be slow folding and aggregation prone. Thus, TRiC may have evolved to protect complex protein topologies within its central cavity during biosynthesis and folding.

Hsp110 cooperates with different cytosolic HSP70 systems in a pathway for de novo folding.

Molecular chaperones such as Hsp70 use ATP binding and hydrolysis to prevent aggregation and ensure the efficient folding of newly translated and stress-denatured polypeptides. Eukaryotic cells contain several cytosolic Hsp70 subfamilies. In yeast, these include the Hsp70s SSB and SSA as well as the Hsp110-like Sse1/2p. The cellular functions and interplay between these different Hsp70 systems remain ill-defined. Here we show that the different cytosolic Hsp70 systems functionally interact with Hsp110 to form a chaperone network that interacts with newly translated polypeptides during their biogenesis. Both SSB and SSA Hsp70s form stable complexes with the Hsp110 Sse1p. Pulse-chase analysis indicates that these Hsp70/Hsp110 teams, SSB/SSE and SSA/SSE, transiently associate with newly synthesized polypeptides with different kinetics. SSB Hsp70s bind cotranslationally to a large fraction of nascent chains, suggesting an early role in the stabilization of nascent chains. SSA Hsp70s bind mostly post-translationally to a more restricted subset of newly translated polypeptides, suggesting a downstream function in the folding pathway. Notably, loss of SSB dramatically enhances the cotranslational association of SSA with nascent chains, suggesting SSA can partially fulfill an SSB-like function. On the other hand, the absence of SSE1 enhances polypeptide binding to both SSB and SSA and impairs cell growth. It, thus, appears that Hsp110 is an important regulator of Hsp70-substrate interactions. Based on our data, we propose that Hsp110 cooperates with the SSB and SSA Hsp70 subfamilies, which act sequentially during de novo folding.