The Lon protease temporally restricts polar cell differentiation events during the Caulobacter cell cycle

Abstract

The highly conserved protease Lon has important regulatory and protein quality control functions in cells from the three domains of life. Despite many years of research on Lon, only a few specific protein substrates are known in most organisms. Here, we used a quantitative proteomics approach to identify novel substrates of Lon in the dimorphic bacterium Caulobacter crescentus. We focused our study on proteins involved in polar cell differentiation and investigated the developmental regulator StaR and the flagella hook length regulator FliK as specific Lon substrates in detail. We show that Lon recognizes these proteins at their C-termini, and that Lon-dependent degradation ensures their temporally restricted accumulation in the cell cycle phase when their function is needed. Disruption of this precise temporal regulation of StaR and FliK levels in a Δlon mutant contributes to defects in stalk biogenesis and motility, respectively, revealing a critical role of Lon in coordinating developmental processes with cell cycle progression. Our work underscores the importance of Lon in the regulation of complex temporally controlled processes by adjusting the concentrations of critical regulatory proteins. Furthermore, this study includes the first characterization of FliK in C. crescentus and uncovers a dual role of the C-terminal amino acids of FliK in protein function and degradation.

The heat shock protein LarA activates the Lon protease at the onset of proteotoxic stress

“…Among the enriched proteins were several previously validated Lon substrates, including DnaA, StaR and FliK, as well as proteins that were in a previous study classified as putative Lon substrates (Fig. 1B) 20, 29 . CCNA_03707 (hereafter referred to as LarA for L on a ctivity r egulator A ) (Fig.…”

“…6A-B). Specifically, we tested the effect of LarA on the previously identified native C. crescentus Lon substrates CcrM and DnaA, as well as FliK-C, the C-terminal portion of FliK that contains the Londependent degron 20, 29, 35 . Additionally, we assessed LarA-dependent effects on the degradation of the flagellar assembly protein FliX, a protein that was strongly enriched in the Lon trap (Fig.…”

“…WT cells harbouring either an empty vector (VC) or the plasmid carrying Pxyl-3xFLAG-larA were grown for two hours with xylose to induce F-larA overexpression. Lon, the confirmed substrates SciP and FlgE (green label) as well as the ten most significantly changed proteins are indicated (previously reported putative substrates 29 are labeled in purple). Mean values from three biological replicates are shown (see Dataset S2).…”

“…S6A), which is consistent with our in vitro data. Prompted by our finding that the levels of FliK, a critical regulator of flagella hook length 29 , were strongly downregulated in F-larA -overexpressing cells, we also tested the effect of F-larA overexpression on soft agar motility and observed a notable swimming defect. In contrast, no motility defect was observed when the F-LarA Δ5 mutant was overproduced (Fig.…”

“…In vitro degradation assays were performed as described previously 29 Afterwards, the solutions were mixed and measured immediately in a Spark microplate reader (Tecan) as follows: After shaking briefly (5 s, double orbital, 1 mm amplitude), absorption at 900 nm and 1000 nm (10 flashes, 50 ms settle time) for path length correction was measured once, followed by 300 cycles of measurements of absorption at 340 nm (25 flashes, 500 ms settle time) and shaking (as before). The analysis of the raw measurements were carried out in RStudio (https://www.rstudio.com) using R version 4.2.0 (markdown notebook provided at github.com/Matthionine/DetermineATPaseRate).…”