Lon protease degrades transfer-messenger RNA-tagged proteins

Abstract

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

FIG. 1.

Endogenous protein tagging assays performed with selected secondary candidates and wild-type tmRNA or variant tmRNA^H6. Purified His₆-tagged proteins were analyzed by SDS-PAGE and Western blotting, using HRP-conjugated anti-His₆ antibody. (A) The ssrA::Tn mutant had higher levels of tmRNA^H6-tagged proteins than the wild-type strain due to the absence of endogenous tmRNA (lanes 1 and 3). As expected, wild-type tmRNA-tagged proteins were not detected in this assay (lane 2). The smpB::Tn mutant is completely defective in the tagging function of trans translation (lane 4). (B) Compared to the wild type, tolR::Tn, tolQ::Tn, and tolA::Tn mutants showed slightly decreased levels of tmRNA^H6-tagged proteins (lanes 1, 3, and 6), while the lon::Tn mutant (lon-1) accumulated higher levels of tmRNA^H6-tagged proteins (lane 5). (C) Similar endogenous tagging profiles were obtained for lon-1, lon-2, and lon-3 mutants and an otherwise isogenic, previously characterized lon::Tet mutant (from AP401). wt, wild type.

FIG. 2.

GFP-SsrA exhibits increased stability in the lon-1 mutant. Expression of GFP or GFP-SsrA was induced using 0.01% arabinose. After removal of the inducer, protein levels were chased in medium containing spectinomycin. In vivo levels of GFP or GFP-SsrA were determined by SDS-PAGE and Western blot analysis using HRP-conjugated anti-GFP antibody. Parallel assays were performed with clpA and clpX mutants for comparative analysis. wt, wild type.

FIG. 3.

trans-translation reporter protein λ-CI-N is tagged but not efficiently degraded in the lon-1 mutant. Expression of the λ-CI-N protein from a nonstop mRNA activates trans translation, generating tmRNA-tagged λ-CI-N. Reporter expression was induced using 1 mM IPTG. After removal of the inducer, protein levels were chased in medium containing spectinomycin. In vivo levels of λ-CI-N were determined by Tris-Tricine-PAGE and Western blot analysis using anti-FLAG M2 (λ-CI-N has an internal FLAG epitope) and anti-mouse IgG-HRP antibodies. Parallel assays were performed with clpA and clpX mutants for comparative analysis. wt, wild type.

FIG. 4.

Lon preferentially degrades tmRNA-tagged λ-CI-N protein in vitro. In vitro proteolysis assays were carried out at 37°C in a minimal activity buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 1 mM dithiothreitol. Complete reaction mixtures contained 1 μM Lon, 10 μM substrate, and an ATP regeneration system. Time point samples were taken at the indicated times and analyzed by Tris-Tricine-PAGE followed by Coomassie blue staining.

FIG. 5.

Lon preferentially degrades tmRNA-tagged λ-CI-N protein in vitro. A quantitative analysis of in vitro reactions performed with Lon and tagged and untagged forms of λ-CI-N is shown. Coomassie blue-stained λ-CI-N species were quantified using an imaging densitometer.

FIG. 6.

Lon degrades GFP-SsrA but not GFP in vitro. Reactions were carried out with GFP-SsrA or GFP as described in the legend to Fig. 4. The levels of GFP-SsrA and GFP at various time points were determined by microplate fluorimetry.