Short disordered N-termini & amp; proline-rich domain are major regulators of UBQLN1/2/4 phase separation

Biophys J. 2023 Nov 30:S0006-3495(23)04117-6. doi: 10.1016/j.bpj.2023.11.3401. Online ahead of print.ABSTRACTHighly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, includin...

Source: Biophysical Journal - December 2, 2023 Category: Physics Authors: Thuy P Dao Anitha Rajendran Sarasi K K Galagedera William Haws Carlos A Casta ñeda Source Type: research