Hsp104 is a yeast chaperone that rescues misfolded proteins from aggregates associated with proteotoxic stress and aging. Hsp104 consists of N-terminal domain, regulatory M-domain and two ATPase domains, assembled into a spiral-shaped hexamer. Protein disaggregation involves polypeptide extraction from an aggregate and its translocation through the central channel. This process relies on Hsp104 cooperation with the Hsp70 chaperone, which also plays important role in regulation of the disaggregase. Although Hsp104 protein-unfolding activity enables cells to survive stress, when uncontrolled, it becomes toxic to the cell. In this work, we investigated the significance of the interaction between Hsp70 and the M-domain of Hsp104 for functioning of the disaggregation system. We identified phenylalanine at position 508 in Hsp104 to be the key site of interaction with Hsp70. Disruption of this site makes Hsp104 unable to bind protein aggregates and to confer tolerance in yeast cells. The use of this Hsp104 variant demonstrates that Hsp70 allows successful initiation of disaggregation only as long as it is able to interact with the disaggregase. As reported previously, this interaction causes release of the M-domain-driven repression of Hsp104. Now we reveal that, apart from this allosteric effect, the interaction between the chaperone partners itself contributes to effective initiation of disaggregation and plays important role in cell protection against Hsp104-induced toxicity. Interaction with Hsp70 shifts Hsp104 substrate specificity from non-aggregated, disordered substrates toward protein aggregates. Accordingly, Hsp70-mediated sequestering of the Hsp104 unfoldase in aggregates makes it less toxic and more productive.
Keywords: molecular chaperones; protein disaggregation; protein misfolding; protein quality control; protein refolding.
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