Under stress, certain eukaryotic proteins and RNA assemble to form membraneless organelles known as stress granules. The most well-studied stress granule components are RNA-binding proteins that undergo liquid-liquid phase separation (LLPS) into protein-rich droplets mediated by intrinsically disordered low-complexity domains (LCDs). Here we show that stress granules include proteasomal shuttle factor UBQLN2, an LCD-containing protein structurally and functionally distinct from RNA-binding proteins. In vitro, UBQLN2 exhibits LLPS at physiological conditions. Deletion studies correlate oligomerization with UBQLN2's ability to phase-separate and form stress-induced cytoplasmic puncta in cells. Using nuclear magnetic resonance (NMR) spectroscopy, we mapped weak, multivalent interactions that promote UBQLN2 oligomerization and LLPS. Ubiquitin or polyubiquitin binding, obligatory for UBQLN2's biological functions, eliminates UBQLN2 LLPS, thus serving as a switch between droplet and disperse phases. We postulate that UBQLN2 LLPS enables its recruitment to stress granules, where its interactions with ubiquitinated substrates reverse LLPS to enable shuttling of clients out of stress granules.

Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.

Liquid‐liquid phase separation (LLPS) is hypothesized to be the dominant mechanism that underlies the formation of membraneless organelles, including stress granules formed under cellular stress containing sequestered RNA and proteins. Our lab has recently shown that Ubiquilin‐2 (UBQLN2), a protein involved in protein quality control through proteasomal degradation and autophagy pathways, is recruited to stress granules in vivo and undergoes LLPS in vitro under physiological conditions. Using NMR spectroscopy, we identified both intrinsically‐disordered and folded regions that are involved in UBQLN2 self‐association that also mediate UBQLN2 LLPS. One of these regions includes the proline‐rich (Pxx) segment, where known disease‐linked mutations have been shown to cause 1–2% of familial ALS or ALS/dementia cases. Using size‐exclusion chromatography, NMR spectroscopy, and microscopy, we demonstrated that Pxx mutations modulate UBQLN2 self‐association and phase separation in vitro. Pxx mutations at P497, P506 and P525 affected droplet morphology and dynamics to significantly different extents. Our data suggested that increased hydrophobicity of the amino acid promoted UBQLN2 LLPS and lowered the phase transition temperature at which UBQLN2 LLPS is first observed. To systematically investigate the molecular basis for how amino acid composition affects UBQLN2 phase separation, we generated all 19 possible amino acid replacements at residues P497, P506, P525, and V538. Our data indicate that the phase behavior of UBQLN2 is significantly modulated by mutations at positions 497 and 506, but weakly modulated by mutations at positions 525 and 538. These results suggest that positions 497 and 506 are ‘stickers’ whereas positions 525 and 538 are ‘spacers’ using the language of associative polymers. Consistent with our data above, hydrophobic amino acid substitutions promoted UBQLN2 LLPS. Our experiments suggest that UBQLN2 may be used as a model system to understand the sequence determinants of phase separation. Hydrophobic mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2‐containing biomolecular condensates in the cell, promoting disease states.Support or Funding InformationC.A.C. graciously acknowledges funding from the ALS Association via grants 17‐IIP‐369 and 18‐IIP‐400, as well as from the National Science Foundation (CAREER award 1750462).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Liquid-liquid phase separation (LLPS) underlies the assembly of biomolecular condensates such as stress granules. Stress granule persistence or disrupted stress granule dynamics is hypothesized to lead to the characteristic protein inclusions that are a hallmark of ALS (amyotrophic lateral sclerosis) and other neurological disorders. We recently demonstrated that Ubiquilin-2 (UBQLN2), an ALS-linked protein critical for maintaining protein quality control, is recruited to stress granules in cells and undergoes LLPS in vitro under physiological conditions. Mutations in the intrinsically-disordered, proline-rich (Pxx) region of UBQLN2 cause ALS and ALS/dementia and are linked to protein inclusions that form in degenerated motor neurons. The molecular mechanisms for how these Pxx mutations cause disease are unknown or poorly understood. We hypothesized that Pxx mutations disrupt UBQLN2 LLPS. Using spectrophotometric assays, light and fluorescence microscopy, we show that a subset of these mutations at positions T487, P497 or P506 significantly increase UBQLN2 LLPS propensity and/or alter material properties of UBQLN2 protein droplets in vitro. Importantly, these UBQLN2 mutants still undergo LLPS reversibly, and are all eliminated by ubiquitin binding. Biophysical characterization reveal that these single point mutations do not alter UBQLN2 structure, but likely promote UBQLN2 self-association and oligomerization, a prerequisite for LLPS. Our preliminary results suggested that increased hydrophobicity of the amino acid promotes UBQLN2 LLPS. We speculate that increased hydrophobicity promotes UBQLN2 oligomerization and self-assembly, which in turn, promotes UBQLN2 phase separation. Overall, our experiments suggest that disease-linked mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2-containing biomolecular condensates in the cell, promoting disease states.