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The Hsp104 protein from Saccharomyces cerevisiae is a member of the Hsp100/Clp family of molecular chaperones. It mediates the solubilization of aggregated proteins in an ATP-dependent process assisted by the Hsp70/40 system. Although the principal function of Hsp104 is well established, the mechanistic details of this catalyzed disaggregation are poorly understood. In this work, we have investigated the interaction of Hsp104 with reduced, carboxymethylated ␣-lactalbumin (RCMLa), a permanently unfolded model substrate. Our results demonstrate that the affinity of Hsp104 toward polypeptides is regulated by nucleotides. In the presence of ATP or adenosine-5-O-(3-thiotriphosphate), the chaperone formed complexes with RCMLa, whereas no binding was observed in the presence of ADP. In particular, the occupation of the N-terminally located nucleotide-binding domain with ATP seems to be crucial for substrate interaction. When ATP binding to this domain was impaired by mutation, Hsp104 lost its ability to interact with RCMLa. Our results also indicate that upon association with a polypeptide, a conformational change occurs within Hsp104 that strongly reduces the dynamics of nucleotide exchange and commits the bound polypeptide to ATP hydrolysis.Molecular chaperones are important constituents of the cellular protein quality control system (1, 2). In response to severe growth conditions, the synthesis of many chaperones is up-regulated to cope with potentially harmful consequences of cellular stress such as protein unfolding and aggregation. The Hsp100/Clp chaperone family, a subclass of the AAA ϩ 3 proteins, is involved in the unfolding and subsequent degradation of misfolded or damaged polypeptides (3, 4) as well as in the resolubilization of protein aggregates (5-7). The high degree of sequence homology suggests that, despite their diverse functions, Hsp100/Clp proteins employ a common, ATP-dependent mechanism to exert their biological roles, for which the disruption of non-covalent interactions appears to be central (8).Among the Hsp100/Clp chaperones involved in cellular protein disaggregation, Hsp104 of Saccharomyces cerevisiae and its bacterial homologue ClpB are best understood on the molecular level. Like other members of the Hsp100/Clp family, both proteins assemble into ringshaped hexamers. Several studies have shown that disaggregation is a complex process requiring the assistance of DnaK/DnaJ/GrpE in bacteria and Ssa1/Ydj1,Sis1 in the yeast system (5, 6). Two models have been proposed to describe the action of Hsp104/ClpB in substrate resolubilization. According to the "molecular crowbar" hypothesis, the flexible linker arms located at the perimeter of the Hsp104/ClpB oligomer serve as binding sites for aggregated polypeptides. In response to nucleotide binding and hydrolysis, a conformational change in these arms could provide the mechanical force necessary to disrupt protein aggregates (9 -11). More recent findings favor an unfolding/threading mechanism similar to that observed for the protease-associated Hs...

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