Role of the MecA adaptor protein in regulation of the AAA + chaperone ClpC of Bacillus subtilis
ClpC, a member of the AAA+ protein superfamily from Bacillus subtilis, is forming with ClpP a proteolytic system, that is part of the protein quality control system and involved in general proteolysis of misfolded and aggregated proteins. In addition ClpCP together with the adaptor MecA is necessary for the regulated proteolysis of the transcription factor ComK in competence development of B. subtilis. The ClpCP mediated regulatory proteolysis controls also stress response and sporulation in B. subtilis. In this work the in vitro chaperone activity of ClpC was investigated. It was discovered that the presence of the adaptor protein MecA is essential for the chaperone activity of ClpC, because it targets substrate to ClpC and activates ClpC by assisting the oligomerisation of ClpC. In particular MecA enabled ClpC to disaggregate and refold previously heat aggregated Luciferase and Malate Dehydrogenase. In the presence of ClpP, MecA enabled the subsequent degradation of unfolded or previously heat-aggregated proteins by ClpCP while native proteins were not degraded. In addition it was demonstrated that the MecA paralogue YpbH, which is not involved in the regulatory proteolysis in B. subtilis, displayed comparable chaperone activities. Therefore MecA and YpbH may have a general and complementary function in protein quality control. These and other experiments suggested that MecA can coordinate substrate targeting with ClpC activation and that the ATPase induction of ClpC by MecA was necessary but not sufficient for this activation. The question why MecA is necessary for the general activation of ClpC was addressed in more detail. It could be demonstrated that in the presence of ATP MecA assists the assembly of an active higher oligomer of ClpC via formation of a ClpC-MecA heterodimer. This higher oligomeric complex is a prerequisite for all the activities of AAA+ proteins and consists presumably of a hexamer of ClpC interacting with up to six MecA molecules. The N-terminal and the Linker domain of the first AAA+ domain of ClpC were identified as MecA interaction sites and structural determinants necessary for this process. Controlling the ability of an AAA+ protein to form an active ring is an important functional aspect by which the activity of this protein family can be specifically regulated by an adaptor protein.
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