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The oligomeric AAA+ chaperones ClpB/Hsp104 mediate the reactivation of aggregated proteins, an activity that is crucial for the survival of cells during severe stress. Hsp104 is also essential for the propagation of yeast prions by severing prion fibres. Protein disaggregation depends on the cooperation of ClpB/Hsp104 with a cognate Hsp70 chaperone system. While Hsp70 chaperones are also involved in prion propagation, their precise role is much less well defined compared with its function in aggregate solubilization. Therefore, it remained unclear whether both ClpB/Hsp104 activities are based on common or different mechanisms. Novel data show that ClpB/Hsp104 uses a motor threading activity to remodel both protein aggregates and prion fibrils. Moreover, transfer of both types of substrates to the ClpB/Hsp104 processing pore site requires initial substrate interaction of Hsp70. Together these data emphasize the similarity of thermotolerance and prion propagation pathways and point to a shared mechanistic principle of Hsp70-ClpB/Hsp104-mediated solubilization of amorphous and ordered aggregates.

General and regulated proteolysis in bacteria is crucial for cellular homeostasis and relies on high substrate specificity of the executing AAA+ proteases. Here we summarize the various strategies that tightly control substrate degradation from both sides: the generation of accessible degrons and their specific recognition by AAA+ proteases and cognate adaptor proteins.

A novel class of antibiotic acyldepsipeptides (designated ADEPs) exerts its unique antibacterial activity by targeting the peptidase caseinolytic protease P (ClpP). ClpP forms proteolytic complexes with heat shock proteins (Hsp100) that select and process substrate proteins for ClpP-mediated degradation. Here, we analyse the molecular mechanism of ADEP action and demonstrate that ADEPs abrogate ClpP interaction with cooperating Hsp100 adenosine triphosphatases (ATPases). Consequently, ADEP treated bacteria are affected in ClpP-dependent general and regulatory proteolysis. At the same time, ADEPs also activate ClpP by converting it from a tightly regulated peptidase, which can only degrade short peptides, into a proteolytic machinery that recognizes and degrades unfolded polypeptides. In vivo nascent polypeptide chains represent the putative primary target of ADEP-activated ClpP, providing a rationale for the antibacterial activity of the ADEPs. Thus, ADEPs cause a complete functional reprogramming of the Clp-protease complex.

The oligomeric AAA+ chaperone Hsp104 is essential for thermotolerance development and prion propagation in yeast. Thermotolerance relies on the ability of Hsp104 to cooperate with the Hsp70 chaperone system in the reactivation of heat-aggregated proteins. Prion propagation requires the Hsp104-dependent fragmentation of prion fibrils to create infectious seeds. It remained elusive whether both processes rely on common or different activities of Hsp104. Specifically, protein reactivation has been suggested to require a substrate threading activity of Hsp104 whereas fibril fragmentation may be mediated by a crowbar activity. Here we engineered an Hsp104 variant, HAP, which cooperates with the bacterial peptidase ClpP to form a novel proteolytic system. HAP threads aggregated model substrates as well as the yeast prion Sup35 through its central pore into associated ClpP. HAP variants that harbour a reduced threading activity were affected in both protein disaggregation and prion propagation, demonstrating that substrate threading represents the common mechanism for the processing of both substrate classes.

ClpC of Bacillus subtilis is an ATP-dependent HSP100Clp protein involved in general stress survival. A complex of ClpC with the protease ClpP and the adaptor protein MecA also controls competence development by regulated proteolysis of the transcription factor ComK. We investigated the in vitro chaperone activity of ClpC and found that the presence of MecA was crucial for the major chaperone activities of ClpC. In particular, MecA enabled ClpC to solubilize and refold aggregated proteins. Finally, in the presence of ClpP, MecA allowed the ClpC-dependent degradation of unfolded or heat-aggregated proteins. This study demonstrates that adaptor proteins like MecA through interaction with their cognate ClpC proteins can have a dual role in the protein quality-control network by rescuing, or together with ClpP, by degrading, aggregated proteins. MecA can thereby coordinate substrate targeting with ClpC activation, adding another layer to the regulation of HSP100/Clp protein activity.