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Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

Summary: Life is resilient because living systems are able to respond to elevated temperatures with an ancient gene expression program called the heat shock response (HSR). In yeast, the transcription of hundreds of genes is upregulated at stress temperatures. Besides stress protection conferred by chaperones, the function of the majority of the upregulated genes under stress has remained enigmatic. We show that those genes are required to directly counterbalance increased protein turnover at stress temperatures and to maintain the metabolism. This anaplerotic reaction together with molecular chaperones allows yeast to efficiently buffer proteotoxic stress. When the capacity of this system is exhausted at extreme temperatures, aggregation processes stop translation and growth pauses. The emerging concept is that the HSR is modular with distinct programs dependent on the severity of the stress. : Mühlhofer et al. show that under mild and severe heat stress, proteins lost due to increased aggregation and degradation are replenished by increased protein synthesis to ensure a constant pool of proteins, together with the molecular chaperones. The heat shock response is activated in modules by transcriptional regulation. Keywords: heat shock response, protein aggregation, chaperones, transcriptome, translatome, proteome, S. cerevisiae, ribosome profiling, mass spectrometry

Summary The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.