STI1 domain engages transient helices to mediate Dsk2 phase separation and proteasome condensation

Ubiquitin-binding shuttle proteins are important components of stress-induced biomolecular condensates in cells. Yeast Dsk2 scaffolds proteasome-containing condensates via multivalent interactions with proteasomes and polyubiquitinated substrates under stress conditions. Here, we identify the chaperone-binding STI1 domain as the main driver of Dsk2 self-association and phase separation. Using nuclear magnetic resonance (NMR) spectroscopy and computational simulations, we find that the STI1 domain interacts with three transient amphipathic helices within the intrinsically disordered regions of Dsk2. Removal of either the STI1 domain or these helices significantly reduces Dsk2's propensity to form condensates. In vivo, perturbing STI1-helix interactions, specifically removal of the transient helices, reduces the formation of azide stress-induced Dsk2/proteasome condensates, in line with our in vitro results. Modeling of Dsk2 STI1-helix interactions reveals a binding mode reminiscent of chaperone STI1/DP2 domains interacting with client helices. Our findings support a model whereby STI1-helix interactions important for Dsk2 condensate formation can be replaced by STI1-client interactions for downstream chaperone or other protein quality control outcomes.