Phase separation of polyubiquitinated proteins in UBQLN2 condensates controls substrate fate

Abstract

Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2s phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code. SignificanceCovalent attachment of polyubiquitin chains to eukaryotic proteins is a common protein quality control signal. Ubiquitination often marks proteins for degradation by the proteasome, but can also drive non-degradative outcomes. Proteins, including UBQLN2, that bind both polyubiquitin and the proteasome can either enhance or inhibit degradation. The ALS-related UBQLN2 is recruited to membraneless organelles, including stress granules, and undergoes phase separation in vitro, but the effects of phase separation on substrate fate are unknown. Herein we show that UBQLN2 phase separation is modulated by polyubiquitinated substrates in a linkage-dependent fashion. We show that two functional outcomes, degradation and deubiquitination, are differentially affected by phase separation. Our results suggest that phase separation of substrates and UBQLN2 could control protein fates.