SmiA is a hybrid priming/scaffolding adaptor for the LonA protease in Bacillus subtilis

Regulatory proteolysis targets properly folded clients via a combination of cis-encoded degron sequences and trans-expressed specificity factors called adaptors. SmiA of Bacillus subtilis was identified as the first adaptor protein for the Lon family of proteases, but the mechanism of SmiAdependent proteolysis is unknown. Here, we develop a fluorescence-based assay to measure the kinetics of SmiAdependent degradation of its client SwrA and show that SmiA-SwrA interaction and the SwrA degron were both necessary, but not sufficient, for proteolysis. Consistent with a scaffolding adaptor mechanism, we found that stoichiometric excess of SmiA caused substrate-independent inhibition of LonA-dependent turnover. Furthermore, SmiA was strictly required even when SwrA levels were high suggesting that a local increase in substrate concentration mediated by the scaffold was not sufficient for proteolysis. Moreover, SmiA function could not be substituted by thermal denaturation of the substrate, consistent with a priming adaptor mechanism. Taken together, we conclude that SmiA functions via a mechanism that is a hybrid between scaffolding and priming models.

Automated summary

In this study, a fluorescence-based assay was developed to investigate the degradation kinetics of SmiA-dependent on its client protein SwrA. The results showed that the interaction between SmiA and SwrA, as well as the SwrA degron, are necessary but not sufficient for proteolysis. Additionally, excess SmiA was found to inhibit LonA-dependent turnover, and the presence of SmiA was required even when SwrA levels were high.