Rad23B delays Ataxin-3 liquid-to-solid phase transition through heterotypic buffering.

The abnormal expansion of polyglutamine (polyQ) length in Ataxin-3 is associated with Machado-Joseph disease, forming nuclear inclusions in neurons. A truncated variant of Ataxin-3 with two ubiquitin-interacting motifs (UIMs) has recently been shown to undergo liquid-liquid phase separation (LLPS). However, the molecular mechanisms underlying Ataxin-3 aggregation through amyloid formation and phase separation remain unclear. Here we investigated the LLPS properties of Ataxin-3 with three UIMs, the most abundant isoform found inthe brain. Ataxin-3 Q25 forms phase-separated droplets which undergo aging, producing amyloid-like fibrillar structures. PolyQ expanded Q54 forms short-lived droplets that exhibit rapid maturation. Ataxin-3 C-terminal fragment forms unique straight, unbranched amyloid fibrils which display negligible Thioflavin-T binding. Rad23B, the main constituent driving the formation of proteasomal condensates, is a known Ataxin-3 interactor. We found that heterotypic interactions with Rad23B inhibit Ataxin-3 droplet maturation but do not inhibit amyloid formation under dilute conditions, suggesting that Ataxin-3 aggregation via misfolding pathway is distinct from condensation pathway. Finally, we show that Ataxin-3 is incorporated into liquid-like stress granules under arsenite stress, shedding light on its roles in aggregation dynamics and stress responses.