Phase separation drives NLRP3 inflammasome activation independently of Golgi localization

The NLRP3 inflammasome plays a pivotal role in sterile inflammation and various diseases, yet the mechanisms underlying its activation remain elusive. Previous studies have implicated both NLRP3 Golgi localization and palmitoylation-mediated phase separation in its activation; however, mutations in palmitoylation sites or the FISNA domain’s intrinsically disordered region (IDR) concurrently disrupt both processes, hindering delineation of their individual contributions to inflammasome activation. Here, we show that NLRP3 exhibits phase separation and Golgi localization simultaneously during inflammasome activation. The polybasic region and FISNA IDR each mediate both processes, whereas lipid-binding motif deletion abolishes Golgi localization without compromising phase separation or inflammasome activity. Conversely, the C-terminal IDR is essential for phase separation and inflammasome activity but dispensable for Golgi localization. By mechanistically uncoupling these processes, our findings establish that phase separation drives NLRP3 activation independently of Golgi localization, thus redefining the spatial and biophysical paradigm of inflammasome assembly.