J-domain proteins cooperate with Hsp70 to drive multiphase separation of RNA-binding-deficient TDP-43

RNA-free TDP-43, resulting from mutations or post-translational modifications in its RNA-binding domain, forms multiphase condensates with Hsp70 chaperones enriched in the core. The presence of this structure in the nucleus is thought to be associated with disease states. However, the mechanisms underlying its formation remain poorly understood. In particular, it is unclear whether J-domain proteins (JDPs), critical co-chaperones of Hsp70, are incorporated into multiphase condensates, and if so, how they contribute to the phase separation process. Using yeast as a model organism with a relatively small JDP family, we identified Sis1, but not Ydj1, as an important factor in TDP-43 multiphase separation. RNA-binding-deficient TDP-43 initially forms uniform condensates enriched with the JDP Sis1 but not with Hsp70. Subsequent recruitment of Hsp70 transforms these uniform structures into multiphase condensates, with both Sis1 and Hsp70 enriched in the core. This transition requires a functional J-domain, which stimulates Hsp70 ATPase activity. These findings reveal a key role for JDPs in TDP-43 multiphase separation and highlight JDP specificity in this process.