Systems analyses reveal two chaperone networks with distinct functions in eukaryotic cells.

Molecular chaperones assist the folding of newly translated and stress-denatured proteins. In prokaryotes, overlapping sets of chaperones mediate both processes. In contrast, we find that eukaryotes evolved distinct chaperone networks to carry out these functions. Genomic and functional analyses indicate that in addition to stress-inducible chaperones that protect the cellular proteome from stress, eukaryotes contain a stress-repressed chaperone network that is dedicated to protein biogenesis. These stress-repressed chaperones are transcriptionally, functionally, and physically linked to the translational apparatus and associate with nascent polypeptides emerging from the ribosome. Consistent with a function in de novo protein folding, impairment of the translation-linked chaperone network renders cells sensitive to misfolding in the context of protein synthesis but not in the context of environmental stress. The emergence of a translation-linked chaperone network likely underlies the elaborate cotranslational folding process necessary for the evolution of larger multidomain proteins characteristic of eukaryotic cells.

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.