Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Samer Sakr,Anne-Marie Cirinesi,Ronald S Ullers,Françoise Schwager,Costa Georgopoulos,Pierre Genevaux

doi:10.1074/jbc.m110.133058

Abstract

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.

Highlights

Polypeptide segments normally found buried in the final folded structure of a substrate
The presence of ribosomebound Trigger Factor (TF) generally retards protein export [29, 30]. In agreement with this observation, the protein export defect, the cold-sensitive (Cs) phenotype, and the increased protein aggregation observed in the absence of SecB are fully suppressed by null mutations in the tig gene, encoding TF. In this case more SecA and ribosomes are found associated with the inner membrane, suggesting that TF directly or indirectly interferes with the process of co-translational targeting to the Sec translocon, driving presecretory proteins toward a post-translational mode of export that relies on the cytosolic chaperone SecB [30, 31]
We have dissected the roles of the chaperone SecB and the ATP-dependent protease Lon in this process, finding that they represent a dedicated system for the quality control of presecretory proteins in E. coli

Summary

Introduction

Polypeptide segments normally found buried in the final folded structure of a substrate. The Deleterious Effect of Lon Is Specific and Proteasedependent—To test whether the detrimental role of Lon in the absence of the SecB chaperone is due to its protease activity and not to substrate sequestration, plasmid pSE-LonSA, carrying the catalytic site mutation Ser-679 to Ala that abolishes Lon protease activity [55], was engineered and tested for its effect on growth of the ⌬secB mutant at low temperature.

Results

Conclusion