Abstract Heat shock triggers formation of intracellular protein aggregates and induction of a molecular disaggregation system. Although this system (Hsp100/Hsp70/Hsp40 in most cellular life) can disperse aggregates of model misfolded proteins, its activity on these model substrates is puzzlingly weak, and its endogenous heat-induced substrates have largely eluded biochemical study. Recent work has revealed that several cases of apparent heat-induced aggregation instead reflect evolved, adaptive biomolecular condensation. In budding yeast Saccharomyces cerevisiae , the resulting condensates depend on molecular chaperones for timely dispersal in vivo , hinting that condensates may be major endogenous substrates of the disaggregation system. Here, we show that the yeast disaggregation system disperses heat-induced biomolecular condensates of poly(A)-binding protein (Pab1) orders of magnitude more rapidly than aggregates of the most commonly used model substrate, firefly luciferase. Pab1 condensate dispersal also differs from aggregate dispersal in its molecular requirements, showing no dependence on small heat-shock proteins and a strict requirement for type II Hsp40. Unlike luciferase, Pab1 is not fully threaded (and thus not fully unfolded) by the disaggregase Hsp104 during dispersal, which we show can contribute to the extreme differences in dispersal efficiency. The Hsp70-related disaggregase Hsp110 shows some Pab1 dispersal activity, a potentially important link to animal systems, which lack cytosolic Hsp104. Finally, we show that the long-observed dependence of the disaggregation system on excess Hsp70 stems from the precise mechanism of the disaggregation system, which depends on the presence of multiple, closely spaced Hsp70s for Hsp104 recruitment and activation. Our results establish heat-induced biomolecular condensates of Pab1 as a direct endogenous substrate of the disaggregation machinery which differs markedly from previously studied foreign substrates, opening a crucial new window into the native mechanistic behavior and biological roles of this ancient system.

Chaperones directly and efficiently disperse stress-triggered biomolecular condensates