STI1 domain dynamically engages transient helices in disordered regions to drive self-association and phase separation of yeast ubiquilin Dsk2

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 ubiquitinated substrates under azide-induced mitochondrial stress or extended growth conditions. However, the molecular mechanisms underlying how these shuttle proteins work are unknown. Here, we identify that the middle chaperone-binding STI1 domain is the main driver of Dsk2 self-association and phase separation in vitro . On a molecular level, 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 the propensity for Dsk2 to phase separate. In vivo , removal of the STI1 domain in Dsk2 has the opposite effect, resulting in an increase of proteasome-containing condensates due to an accumulation of polyubiquitinated substrates. Modeling of STI1-helix interactions reveals a binding mode that is reminiscent of interactions between chaperone STI1/DP2 domains and client proteins containing amphipathic or transmembrane 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.