Proteolysis is crucial in cells to maintain a functional proteome. It is required for removal of damaged and unfolded proteins during protein quality control, and serves as a mechanism to regulate protein levels through regulated proteolysis. The latter targets specific proteins under certain conditions to rapidly adjust the levels of these proteins. Thereby, activity of the targeted proteins is reduced or even eliminated. Many cellular processes like cell cycle progression, differentiation and stress response/adaptation depend on proteolytic removal of regulatory proteins via proteolysis. One protease with crucial roles in both protein quality control and regulated proteolysis is called Lon and is found in many species across all domains of life.

Extensive research has revealed many aspects of proteolysis by Lon and identified several Lon substrates. The dimorphic bacterium Caulobacter crescentus is a model organisms to study proteolysis and Lon as the two cell types, a flagellated swarmer cell and a stalked sessile cell, and the transition between them heavily depends on proteolysis. However, certain details, like recognition of substrates and its regulation are largely unknown. Here I focus on regulated proteolysis by Lon in C. crescentus, specifically on novel substrates, their recognition, and the regulation of Lon.

Study I: Using quantitative proteomics of wildtype and mutant C. crescentus strains we identified potential substrates of Lon. Out of these, we focused on the stalk biogenesis regulator StaR and the flagellar hook length determination factor FliK. Both proteins are developmental regulators, whose protein levels oscillate during the cell cycle. Our experiments showed that turnover by Lon is required to maintain these oscillations and disruption thereof results in deregulation of the stalk and the flagellum.

Study II: We used proteolytically inactive Lon to co-purify interactors of Lon and identify them by mass spectrometry. Thereby, we found an uncharacterized heat shock protein that regulates the activity of Lon, and due to our findings named it LarA (Lon activity regulator A). We showed that LarA interacts with Lon at an allosteric site and modulates the activity of Lon via its C-terminal amino acids. In most cases LarA exhibits a stimulating effect on the degradation of the substrates, indicating that LarA regulates substrate specificity and guarantees efficient degradation of the affected substrates. The same residues involved in the modulating interaction also serve as a degron for degradation of LarA by Lon to shut off LarA-mediated modulation if not needed anymore.

Study III: SigT is a driver of gene expression of the general stress response in C. crescentus. It was detected in both previous studies, which indicates that it is degraded by Lon. We showed that it is a substrate of Lon in vitro. Based on steady-state levels of SigT during and after sucrose-induced stress, we could show that Lon-mediated degradation is important during the recovery. In addition, LarA-mediated regulation of Lon stimulates turnover of SigT, indicating that SigT levels are fine-tuned by LarA under certain conditions.

In summary, we identified novel regulatory roles of Lon on differentiation and stress response in C. crescentus and discovered LarA as a novel modulator of Lon activity. The results of these studies once more emphasize the importance of Lon as a regulator of various cellular processes. 

The Lon protease is widely conserved in both prokaryotic and eukaryotic organisms and fulfills important regulatory functions. Nevertheless, the number of identified Lon substrates is limited in most organisms, and the precise role of Lon in regulating these proteins is poorly understood. Here, we describe the α-proteobacterial general stress response sigma factor σ^T as a novel Lon substrate in Caulobacter crescentus. Based on previously published quantitative proteomics data, we find σ^T to be a promising putative Lon substrate and confirm a direct role of Lon in degrading σ^T. We show that Lon contributes to the downregulation of σ^T abundance under optimal conditions and during recovery from sucrose-induced osmotic stress. Furthermore, the presence of the Lon activity regulator LarA enhances Lon-mediated degradation of σ^T in vitro and reduces σ^T levels in vivo indicating a role of LarA in modulating Lon-mediated degradation of σ^T. Together, our results highlight the importance of Lon during the recovery phase following stress exposure by adjusting the concentrations of critical regulators of stress responses.

The Lon protease is a highly conserved protein degradation machine that has critical regulatory and protein quality control functions in cells from the three domains of life. Here, we report the discovery of a α-proteobacterial heat shock protein, LarA, that functions as a dedicated Lon regulator. We show that LarA accumulates at the onset of proteotoxic stress and allosterically activates Lon-catalysed degradation of a large group of substrates through a five amino acid sequence at its C-terminus. Further, we find that high levels of LarA cause growth inhibition in a Lon-dependent manner and that Lon-mediated degradation of LarA itself ensures low LarA levels in the absence of stress. We suggest that the temporal LarA-dependent activation of Lon helps to meet an increased proteolysis demand in response to protein unfolding stress. Our study defines a regulatory interaction of a conserved protease with a heat shock protein, serving as a paradigm of how protease activity can be tuned under changing environmental conditions.