Leptomycin B

N4BP1 (NEDD4-binding partner 1) is a key checkpoint for proper inflammatory responses; however, its cellular localization, biologic nature, and functions in other cellular processes remain largely unknown. In this study, we demonstrate that N4BP1 is a nucleocytoplasmic shuttling protein. Treatment with leptomycin B induces nuclear accumulation of N4BP1, and the region responsible for its nuclear distribution maps to amino acids 151 to 338. Further analysis identified a nuclear localization signal (NLS) spanning residues 279 to 299. Deletion or mutation of this NLS abolishes N4BP1 nuclear import, while fusing the NLS to GFP is sufficient to drive GFP into the nucleus. Notably, we found that N4BP1 forms protein aggregates in both the cytoplasm and nucleus. These aggregates lack ubiquitin-modified proteins but instead colocalize with NEDD8-modified proteins. Consistently, N4BP1 aggregates contain cullin-1 and cullin-2. The CoCUN domain is essential for recognizing neddylated proteins and mediating N4BP1 aggregate formation. N4BP1 aggregates exhibit liquid-liquid phase separation, as evidenced by their sensitivity to 1,6-hexanediol (an liquid-liquid phase separation inhibitor). Smaller N4BP1 aggregates can fuse into larger one and reassemble after 1,6-hexanediol-induced disruption. Furthermore, heat shock promotes N4BP1 aggregation, which confers cellular protection under stress conditions. Taken together, our findings reveal that N4BP1 is a nucleocytoplasmic shuttling protein. N4BP1 forms protein aggregates that contain neddylated proteins such as cullin-1 and cullin-2. This study uncovers the previously unrecognized role of N4BP1 in organizing neddylated protein aggregates and highlights its functional significance in stress adaption.