Ubiquitin Modulates Liquid-Liquid Phase Separation of UBQLN2 via Disruption of Multivalent Interactions

Abstract

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.

Ubiquilin 2: Shuttling Clients Out of Phase?

“…Dao et al (2018) now establish that UBQLN2 forms ubiquitin-labile liquids, which may enable UBQLN2 to specifically extract ubiquitylated clients from stress granules for degradation. …”

“…However, the mechanisms underlying the complete functional repertoire of UBQLNs are not understood. In this issue of Molecular Cell , Castañeda and coworkers determine that UBQLN2 undergoes LLPS and accumulates in SGs (Dao et al, 2018). Ubiquitin or polyubiquitin abolishes UBQLN2 LLPS and may enable UBQLN2 to extract ubiquitylated clients from SGs for degradation (Dao et al, 2018).…”

“…Dao et al establish that UBQLN2 is diffusely localized in the cytoplasm, but accumulates in SGs under a variety of stresses (Dao et al, 2018). Importantly, purified UBQLN2 undergoes reversible LLPS in vitro under relatively physiological conditions, which likely enables UBQLN2 recruitment to SGs in vivo (Dao et al, 2018).…”

“…Importantly, purified UBQLN2 undergoes reversible LLPS in vitro under relatively physiological conditions, which likely enables UBQLN2 recruitment to SGs in vivo (Dao et al, 2018). To map the domain requirements for UBQLN2 LLPS, several UBQLN2 deletion constructs were assessed for propensity to form liquid drops.…”

“…PrLDs also drive functional LLPS and deleterious aggregation of several ALS-linked RBPs such as TDP-43 and FUS (Figure 1A) (March et al, 2016). Dao et al show that UBQLN2 450-624, which includes a portion of the PrLD, STI1-II, Pxx, and UBA domains, is the minimal region necessary for LLPS in vitro and accumulation in SGs in vivo (Dao et al, 2018). NMR revealed this region to be largely disordered.…”