Thermotolerance requires refolding of aggregated proteins by substrate translocation through the central pore of ClpB

Cell survival under severe thermal stress requires the activity of the CIpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on CIpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a CIpB variant, BAP, which associates with the CIpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into CIpP for degradation. CIpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/CIpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.