Teshirogi, Y.; Mihara, R.; Saito, Y.; Rhee, H.-W.; Terada, T.; Tate, S.-i.; Kyota, Y.

Stress granules (SGs) are dynamic, membrane-less assemblies that form in the cytoplasm in response to cellular stress. The ordered recruitment of proteins into SGs is fundamental to condensate composition and function, yet the molecular determinants of this ordered client recruitment remain incompletely understood. Using proximity photo-crosslinking proteomics, we identified heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) as a TIA1-proximal protein preferentially enriched in SGs under arsenite stress. Knockdown of hnRNPA2B1 preferentially delayed TIA1 enrichment in G3BP1-marked SGs at 20 min without affecting G3BP1 or the overall SG-positive cell fraction, and this phenotype showed directional rescue upon re-expression. In vitro droplet reconstitution assays with purified proteins revealed that hnRNPA2B1 and RNA cooperatively increased TIA1 incorporation capacity into G3BP1 condensates, an effect not attributable to changes in droplet size. Kinetic fitting identified hnRNPA2B1 + RNA as uniquely increasing the plateau amplitude of TIA1 recruitment (Cohen's d = 1.62 versus RNA-alone condition). Coarse-grained simulations support an inside-out assembly model in which hnRNPA2B1 stabilizes the condensate core through homotypic interactions while RNA-bound TIA1 accumulates at the periphery. Together, these findings identify hnRNPA2B1 as a capacity-determining modulator of early TIA1 recruitment and provide a framework for understanding ordered protein assembly within stress granules.