{"doi":"10.1101/2023.09.27.559790","title":"Short N-terminal disordered regions and the proline-rich domain are major regulators of phase transitions for full-length UBQLN1, UBQLN2 and UBQLN4","abstract":"Abstract Highly 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, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts. Highlights 1) Full-length UBQLN1, UBQLN2 and UBQLN4 all exhibit LCST phase transitions but to different degrees. 2) The N-terminal region (N-terminal to the UBL) substantially regulates UBQLN phase separation. 3) Removal of the disease-associated proline-rich (Pxx) region in UBQLN2 removes the strong temperature dependence of UBQLN2 phase separation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":400967,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9632,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1070258,"name":"Anitha Rajendran","orcid":null,"position":1,"is_corresponding":false},{"id":691792,"name":"Sarasi K. K. Galagedera","orcid":null,"position":2,"is_corresponding":false},{"id":1070259,"name":"William Haws","orcid":null,"position":3,"is_corresponding":false},{"id":576329,"name":"Carlos A. Castañeda","orcid":"0000-0001-9634-0867","position":4,"is_corresponding":false},{"id":813337,"name":"Thuy P. Dao","orcid":"0000-0002-2168-7679","position":0,"is_corresponding":true}],"reference_count":48,"raw_metadata":null,"created_at":"2026-07-19T01:20:08.195423Z","pmid":"37808720","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}