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Summary Heat shock protein 90 (Hsp90) is a molecular chaperone regulating the activity of diverse client proteins together with a plethora of different co-chaperones. Whether these functionally cooperate has remained enigmatic. We analyze all double mutants of 11 Saccharomyces cerevisiae Hsp90 co-chaperones in vivo concerning effects on cell physiology and the activation of specific client proteins. We find that client activation is supported by a genetic network with weak epistasis between most co-chaperones and a few modules with strong genetic interactions. These include an epistatic module regulating protein translation and dedicated epistatic networks for specific clients. For kinases, the bridging of Hsp70 and Hsp90 by Sti1/Hop is essential for activation, whereas for steroid hormone receptors, an epistatic module regulating their dwell time on Hsp90 is crucial, highlighting the specific needs of different clients. Thus, the Hsp90 system is characterized by plastic co-chaperone networks fine-tuning the conformational processing in a client-specific manner.

The heat shock protein 90 (HSP90) chaperone machinery is a key regulator of proteostasis under both physiological and stress conditions in eukaryotic cells. As HSP90 has several hundred protein substrates (or 'clients'), it is involved in many cellular processes beyond protein folding, which include DNA repair, development, the immune response and neurodegenerative disease. A large number of co-chaperones interact with HSP90 and regulate the ATPase-associated conformational changes of the HSP90 dimer that occur during the processing of clients. Recent progress has allowed the interactions of clients with HSP90 and its co-chaperones to be defined. Owing to the importance of HSP90 in the regulation of many cellular proteins, it has become a promising drug target for the treatment of several diseases, which include cancer and diseases associated with protein misfolding.

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.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the maturation of a plethora of substrates (“clients”), including protein kinases, transcription factors, and E3 ubiquitin ligases, positioning Hsp90 as a central regulator of cellular proteostasis. Hsp90 undergoes large conformational changes during its ATPase cycle. The processing of clients by cytosolic Hsp90 is assisted by a cohort of cochaperones that affect client recruitment, Hsp90 ATPase function or conformational rearrangements in Hsp90. Because of the importance of Hsp90 in regulating central cellular pathways, strategies for the pharmacological inhibition of the Hsp90 machinery in diseases such as cancer and neurodegeneration are being developed. In this review, we summarize recent structural and mechanistic progress in defining the function of organelle-specific and cytosolic Hsp90, including the impact of individual cochaperones on the maturation of specific clients and complexes with clients as well as ways of exploiting Hsp90 as a drug target.