Degradation of Lon in Caulobacter crescentus

Abstract

Protein degradation is an essential process in all organisms. This process is irreversible and energetically costly; therefore, protein destruction must be tightly controlled. While environmental stresses often lead to upregulation of proteases at the transcriptional level, little is known about posttranslational control of these critical machines. In this study, we show that in Caulobacter crescentus levels of the Lon protease are controlled through proteolysis. Lon turnover requires active Lon and ClpAP proteases. We show that specific determinants dictate Lon stability with a key carboxy-terminal histidine residue driving recognition. Expression of stabilized Lon variants results in toxic levels of protease that deplete normal Lon substrates, such as the replication initiator DnaA, to lethally low levels. Taken together, results of this work demonstrate a feedback mechanism in which ClpAP and Lon collaborate to tune Lon proteolytic capacity for the cell.IMPORTANCE Proteases are essential, but unrestrained activity can also kill cells by degrading essential proteins. The quality-control protease Lon must degrade many misfolded and native substrates. We show that Lon is itself controlled through proteolysis and that bypassing this control results in toxic consequences for the cell.

Keywords: AAA+; Caulobacter crescentus; Lon; degradation; protease.

FIG 1

Lon degradation in vivo requires Lon catalytic activity. In vivo degradation of Lon, CtrA, DnaA, and ClpP following translation shutoff in wt, Δlon xylX::lon, and Δlon xylX::lon S674A strains under inducing conditions. Quantification of biological triplicates is shown below, with error bars representing standard deviations; **, P < 0.005.

FIG 2

The C terminus of Lon is necessary for degradation. (A) Steady-state protein levels of Lon, DnaA, and ClpP in wt or Δlon strains alone or with Lon, amino terminus M2-FLAG epitope Lon (M2lon), or carboxy terminus M2-FLAG epitope Lon (lonM2). Three conditions were tested; − and + were samples grown overnight under noninducing conditions and then back diluted for outgrowth in either noninducing (−) or inducing (+) medium. ON samples were grown under inducing conditions overnight and back diluted under inducing conditions for outgrowth. In all cases, the outgrowth was done for 6 h. Samples were normalized to starting OD₆₀₀ in lysis buffer prior to Western blot analysis. Cropped images are of Western blots probing for Lon, DnaA, and ClpP. (B) In vivo stability of Lon following translation inhibition in wt, Δlon xylX::lon, Δlon xylX::M2lon, and Δlon xylX::lonM2 strains. Levels of DnaA are shown as a control for Lon proteolytic activity and ClpP as a loading control.

FIG 3

Overexpression of a nondegradable Lon results in toxicity and aberrant morphology. (A) Induction of lonM2 from the xylX promoter results in cell death in wt and Δlon strains. Cells were grown to exponential phase in 0.2% glucose, normalized by OD₆₀₀, serially diluted 10-fold, and spotted onto medium supplemented with 0.2% glucose (− induction) or 0.2% xylose (+ induction). Images of plates were taken on day 2 of growth. (B) Induction of lonM2 from the xylX promoter results in aberrant morphologies of wt and Δlon xylX::M2lon or xylX::lonM2 strains. Representative images of indicated strains grown in exponential phase in the presence of 0.2% xylose or 0.2% glucose for 6 h prior to imaging. (C) Cell curvatures (n, >100) were quantified using MicrobeJ and graphed with Prism. (D) Induction of lonM2 from the xylX promoter shifts cells toward G₁. Strains grown were overnight in the presence of 0.2% xylose or 0.2% glucose and then treated with rifampin for 3 hours. Cells were fixed and stained with Sytox green before measuring their DNA content by flow cytometry. Because of the rifampin treatment, cells which have initiated replication will complete replication, resulting in two distinct peaks of fluorescence and representing either 1 or 2 chromosomes per cell. The wt cells are represented by purple, and wt strains containing M2lon or lonM2 are in yellow.

FIG 4

A C-terminal histidine on Lon is necessary for its proteolysis. (A) Structural alignment of the C terminus of Lon. The highly conserved C-terminal histidine residue is indicated by an arrow. (B) Steady-state protein levels of Lon DnaA and ClpP in Δlon xylX::lon (lon native, lon H799K, and lon H799D) strains. Samples were normalized to starting OD₆₀₀ values in lysis buffer prior to Western blot analysis. Cropped images are of Western blots probing for Lon, DnaA, and ClpP. (C) In vivo stability of Lon following translation inhibition in Δlon xylX::lon (wild-type lon, lon H799K, and lon H799D) strains. Levels of DnaA are shown as a control for Lon proteolytic activity and ClpP as a loading control.

FIG 5

In vivo degradation of Lon is dependent on ClpA. (A) Steady-state protein levels in wt and clpA deletion strains. Representative image of Western blot using anti-Lon. Anti-ClpP was used as a loading control. Protein quantifications of biological triplicates with error bars are represented as standard deviations; *, P < 0.05. (B) Protein stability of Lon in wt and ΔclpA strains. Strains were grown to exponential phase at 30°C prior to inhibition of protein synthesis by the addition of chloramphenicol. Samples were withdrawn at the indicated time points and normalized to starting OD₆₀₀ values in lysis buffer. Lysates were used for Western blot analysis and probed with antibodies to Lon and ClpP. Cropped representative images of biological triplicates with quantifications of biological triplicates are shown with error bars representing standard deviations; ***, P < 0.0005.

FIG 6

Lon stability is dependent on ClpAP and Lon activity. Lon degradation is dependent on ClpAP and Lon activity, but these proteases are insufficient on their own in vitro. This finding suggests a need for a factor (Y) that enhances the degradation of Lon dependent on a C-terminal histidine residue by either ClpAP or Lon itself. An inhibitor (X) of Y is degraded by either Lon or ClpAP, thereby linking the in vivo observations with our in vitro biochemistry.