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Polyubiquitin effects on phase transitions of shuttle protein UBQLN2

ALS‐linked ubiquitin‐binding shuttle protein UBQLN2 mediates crosstalk between proteasomal degradation and autophagy pathways, which are thought to be controlled by interactions involving K48‐linked and K63‐linked polyubiquitin chains, respectively. We previously showed that UBQLN2 is recruited to stress granules in cells and undergoes liquid‐liquid phase separation (LLPS) in vitro. Interactions with ubiquitin or multivalent K48‐linked chains inhibit UBQLN2 LLPS. Here, we show that other multivalent chains of longer lengths, including K63‐ and M1‐linked chains and a designed tetrameric ubiquitin construct that mimics a multi‐monoubiquitinated substrate, significantly enhanced UBQLN2 LLPS over a wide range of Ub:UBQLN2 ratios. With nuclear magnetic resonance (NMR) spectroscopy and complementary biophysical techniques, we demonstrated that these opposing effects stem from differences in polyUb chain conformations, but not in affinities between polyUb chains and UBQLN2. Using microscopy and turbidity assay experiments, we obtained multi‐component phase diagrams to demonstrate that compact K11 and K48‐linked Ub4 inhibit UBQLN2 LLPS whereas extended K63 and M1‐linked Ub4 stabilize UBQLN2 LLPS. Increasing chain flexibility and accessibility to the ubiquitin binding surface enables a switch between homotypically‐driven to partially heterotypically‐driven UBQLN2 phase separation. These observations provide mechanistic insights into how UBQLN2 could differentiate between proteasomal degradation and autophagy pathways via LLPS.