TPR Domains Drive the Functional Phase Separation of HOP and its Regulation by Hsp90 and Hsp70

HOP is a cochaperone that facilitates client transfer between two major chaperones, Hsp90 and Hsp70. Emerging evidence, however, suggests that HOP plays additional roles in coordinating complex proteostasis networks. We demonstrate here that stress-dependent HOP foci are biomolecular condensates formed by liquid-liquid phase separation. Purified HOP forms protein droplets that closely resemble the foci observed in cells. Our biophysical analyses show that the phase separation of HOP is driven by electrostatic interactions between its tandem TPR domains, with a critical role of its TPR2A domain. Of note, Hsp90 and Hsp70 regulate the extent of HOP phase separation, with Hsp70 driving HOP droplet formation and Hsp90 reversing it. Finally, we find that the Y354E phosphomimetic variant of HOP impairs phase separation and sensitizes cells to acute misfolding stress, suggesting a key role of HOP condensation in mitigating protein misfolding stress. Our work thus identifies a new mechanism by which HOP phase separation mitigates protein misfolding stress in eukaryotic cells and is regulated by Hsp70 and Hsp90.