ClpB from Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp superfamily of AAA⁺ proteins and is composed of two ATP-binding AAA⁺ modules, the middle domain which forms a coiled-coil structure and the N-teminal and C-terminal domains. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the ClpB80, which does not contain the N-terminal domain. The physiological function of the different isoforms of ClpB has not been explained yet. We examined the ability of the two different-sized ClpB homologues to confer thermotolerance and remove heat-aggregated proteins from Escherichia coli cells (the S fraction). For this purpose we constructed series of plasmids allowing production of the ClpB95/ClpB80 pair, ClpB95 alone, or ClpB80 alone. We found that in the ΔclpB mutant cells expressing ClpB95/ClpB80, the S fraction disappeared 20 min after transfer of the culture from 45° C to 37° C. In contrast to this, in the clpB mutant overexpressing ClpB95 or ClpB80 alone elimination of the S fraction was retarded- nearly 50% of the aggregated proteins remained stable 20 min after heat shock. These results indicate that cooperation of both isoforms of ClpB is required to protect Escherichia coli from thermal killing and to efficiently remove protein aggregates after heat shock.

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