Chaperones with aggregase activity promote and organize the aggregation of misfolded proteins and their deposition at specific intracellular sites. This activity represents a novel cytoprotective strategy of protein quality control systems; however, little is known about its mechanism. In yeast, the small heat shock protein Hsp42 orchestrates the stress-induced sequestration of misfolded proteins into cytosolic aggregates (CytoQ). In this study, we show that Hsp42 harbors a prion-like domain (PrLD) and a canonical intrinsically disordered domain (IDD) that act coordinately to promote and control protein aggregation. Hsp42 PrLD is essential for CytoQ formation and is bifunctional, mediating self-association as well as binding to misfolded proteins. Hsp42 IDD confines chaperone and aggregase activity and affects CytoQ numbers and stability in vivo. Hsp42 PrLD and IDD are both crucial for cellular fitness during heat stress, demonstrating the need for sequestering misfolded proteins in a regulated manner.

Hsp100 and Hsp70 chaperones in bacteria, yeast, and plants cooperate to reactivate aggregated proteins. Disaggregation relies on Hsp70 function and on ATP-dependent threading of aggregated polypeptides through the pore of the Hsp100 AAA + hexamer. In yeast, both chaperones also promote propagation of prions by fibril fragmentation, but their functional interplay is controversial. Here, we demonstrate that Hsp70 chaperones were essential for species-specific targeting of their Hsp100 partner chaperones ClpB and Hsp104, respectively, to heat-induced protein aggregates in vivo. Hsp70 inactivation in yeast also abrogated Hsp104 targeting to almost all prions tested and reduced fibril mobility, which indicates that fibril fragmentation by Hsp104 requires Hsp70. The Sup35 prion was unique in allowing Hsp70-independent association of Hsp104 via its N-terminal domain, which, however, was nonproductive. Hsp104 overproduction even outcompeted Hsp70 for Sup35 prion binding, which explains why this condition prevented Sup35 fragmentation and caused prion curing. Our findings indicate a conserved mechanism of Hsp70–Hsp100 cooperation at the surface of protein aggregates and prion fibrils.

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae , aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors.