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Abstract

Cells have evolved a complex network of mechanisms to maintain protein homeostasis. Among these, protein sequestration executed by small heat shock proteins (sHsps) serves as a strategy to prevent deleterious aggregation by capturing misfolded proteins into complexes that remain amenable to disaggregation by ATP-dependent chaperones. The size and morphology of these complexes are determined by the sHsp involved, the substrate, and the aggregation conditions. Some sHsps form only small, soluble assemblies (holdase activity), while others additionally generate large, microscopically visible inclusions (aggregase activity). The precise mechanisms governing the architecture of these complexes remain incompletely understood. In this study, I investigated the structure and function of the Saccharomyces cerevisiae sHsp Hsp42, which exhibits both holdase and aggregase activities. Hsp42 is distinguished from other sHsps by an extended intrinsically disordered N-terminal region, which comprises a prion-like domain (PrLD) and a classical intrinsically disordered domain (IDD), defined by their amino acid composition. To dissect the structural organization of Hsp42, I employed a combination of biophysical, microscopic, and computational approaches. My data reveal that Hsp42 assembles into a range of oligomeric states, from dimers to decamers, with octamers being the predominant species. This oligomerization is dynamic and responsive to environmental triggers such as temperature and pH. Additionally, Hsp42 undergoes extremely rapid subunit exchange, a feature critical for its chaperone function. Structural modeling predicts that Hsp42 forms planar ring-like octamers made of folded domains, flanked by disordered regions extending outward. This novel arrangement of ACDs in sHsp was never reported in other sHsps. This model was partially validated by cross-linking mass spectrometry, which identified proximity regions within the oligomer, and by limited proteolysis coupled to mass spectrometry, which identified exposed and protected regions. I further demonstrate that Hsp42 forms substrate-dependent complexes of varying size. Cross�linking mass spectrometry identified multiple substrate-binding regions within Hsp42. Importantly, my findings confirm that Hsp42 and bound substrate are not passively released from these complexes and that complex dissolution requires the coordinated action of the Hsp70/Hsp40/Hsp100 disaggregation machinery. Finally, I dissected the contributions of PrLD, IDD, and other domains and conserved motifs of Hsp42 to substrate sequestration and recovery. Using a series of deletion and point mutants, I show that distinct domains of Hsp42 mediate substrate interaction and complex formation, with the PrLD playing a central role – its deletion markedly reduced chaperone activity. In contrast, other domains are required for efficient substrate handover to the disaggregase system. The IDD was found to be essential for forming large Hsp42-substrate complexes and to confer temperature-dependent aggregase activity. Moreover, the IDD appears to influence complex architecture in a manner that facilitates access by Hsp70, while blocking access by standalone Hsp100 disaggregases