Proteasome condensate formation is driven by multivalent interactions with shuttle factors and K48-linked ubiquitin chains

Abstract

Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome (Rpn1, Rpn10, and Rpn13) are critical under different condensate inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes. SignificanceStress conditions can cause the relocalization of proteasomes to condensates in yeast as well as mammalian cells. Our work shows that the formation of proteasome condensates in yeast depends on long K48-linked ubiquitin chains, the proteasome binding shuttle factors Rad23 and Dsk2 and proteasome intrinsic ubiquitin receptors. Here, different receptors are critical for different condensate inducers. These results indicate distinct condensates can form with specific functionality. Our identification of key factors involved in the process is crucial for understanding the function of proteasome relocalization to condensates. We propose that cellular accumulation of substrates with long ubiquitin chains results in the formation of condensates comprising those ubiquitinated substrates, proteasomes, and proteasome shuttle factors, where the ubiquitin chains serve as the scaffold for condensate formation.