Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains

Ubiquitin‐binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48‐ and K63‐linked polyubiquitin chains, respectively. UBQLN2 comprises self‐associating regions that drive its homotypic liquid–liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11‐Ub4 and K48‐Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63‐Ub4, M1‐Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin‐binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self‐interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin‐binding shuttles and adaptors.SynopsisDifferent polyubiquitin chains on substrate proteins can bind to UBQLN2 and signal distinct cellular events. Here, we found that polyubiquitin chains can enhance or inhibit liquid–liquid phase separation of UBQLN2 due to differences in chain linkage and solution conformation.•Ubiquitin or short polyubiquitin chains bind to phase separation‐driving stickers on UBQLN2 and inhibit its phase separation, whereas longer chains provide the multivalency needed to enhance UBQLN2 phase separation.•Phase separation of UBQLN2 is promoted over a wide range of Ub:UBQLN2 ratios in the presence of extended M1‐ and K63‐linked Ub4 chains but not compact K11‐ and K48‐linked Ub4 chains.•Chain conformation and accessibility of the Ub‐interacting surface is a driving factor of UBQLN2/polyUb co‐phase separation.