Single-molecule analyses of the dynamics of heat shock protein 104 (Hsp104) and protein aggregates.

Hsp104 solubilizes protein aggregates in cooperation with Hsp70/40. Although the framework of the disaggregase function has been elucidated, the actual process of aggregate solubilization by Hsp104-Hsp70/40 remains poorly understood. Here we developed several methods to investigate the functions of Hsp104 and Hsp70/40 from Saccharomyces cerevisiae, at single-molecule levels. The single-molecule methods, which provide the size distribution of the aggregates, revealed that Hsp70/40 prevented the formation of large aggregates from small aggregates and that the solubilization of the small aggregates required both Hsp104 and Hsp70/40. We directly visualized the individual association-dissociation dynamics of Hsp104 on immobilized aggregates and found that the lifetimes of the Hsp104-aggregate complex are divided into two groups: short (∼4 s) and long (∼30 s). Hsp70/40 stimulated the association of Hsp104 with aggregates and increased the duration of this association. The single-molecule data provide novel insights into the functional mechanism of the Hsp104 disaggregation machine.