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Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones.

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.

SUBMITTER: Sakr S

PROVIDER: S-EPMC2906341 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones.

Sakr Samer S Cirinesi Anne-Marie AM Ullers Ronald S RS Schwager Françoise F Georgopoulos Costa C Genevaux Pierre P

The Journal of biological chemistry 20100526 30

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 sec ...[more]

PMID: 20504766

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Project description:Correct targeting of membrane proteins is essential for membrane integrity, cell physiology, and viability. Cotranslational targeting depends on the universally conserved signal recognition particle (SRP), which is a ribonucleoprotein complex comprised of the protein component Ffh and the 4.5S RNA in Escherichia coli About 25?years ago it was reported that Ffh is an unstable protein, but the underlying mechanism has never been explored. Here, we show that Lon is the primary protease responsible for adjusting the cellular Ffh level. When overproduced, Ffh is particularly prone to degradation during transition from exponential to stationary growth and the cellular Ffh amount is lowest in stationary phase. The Ffh protein consists of two domains, the NG domain, responsible for GTP hydrolysis and docking to the membrane receptor FtsY, and the RNA-binding M domain. We find that the NG domain alone is stable, whereas the isolated M domain is degraded. Consistent with the importance of Lon in this process, the M domain confers synthetic lethality to the lon mutant. The Ffh homolog from the model plant Arabidopsis thaliana, which forms a protein-protein complex rather than a protein-RNA complex, is stable, suggesting that the RNA-binding ability residing in the M domain of E. coli Ffh is important for proteolysis. Our results support a model in which excess Ffh not bound to 4.5S RNA is subjected to proteolysis until an appropriate Ffh concentration is reached. The differential proteolysis adjusts Ffh levels to the cellular demand and maintains cotranslational protein transport and membrane integrity.IMPORTANCE Since one-third of all bacterial proteins reside outside the cytoplasm, protein targeting to the appropriate address is an essential process. Cotranslational targeting to the membrane relies on the signal recognition particle (SRP), which is a protein-RNA complex in bacteria. We report that the protein component Ffh is a substrate of the Lon protease. Regulated proteolysis of Ffh provides a simple mechanism to adjust the concentration of the essential protein to the cellular demand. This is important because elevated or depleted SRP levels negatively impact protein targeting and bacterial fitness.

Project description:Evolutionary trajectories and mutational landscapes of drug-resistant bacteria are influenced by cell-intrinsic and extrinsic factors. In this study, I demonstrated that loss of the Lon protease altered susceptibility of Escherichia coli to trimethoprim and that these effects were strongly contingent on the drug concentration and genetic background. Lon, an AAA+ ATPase, is a bacterial master regulator protease involved in cytokinesis, suppression of transposition events, and clearance of misfolded proteins. I show that Lon deficiency enhances intrinsic drug tolerance at sub-MIC levels of trimethoprim. As a result, loss of Lon, though disadvantageous under drug-free conditions, has a selective advantage at low concentrations of trimethoprim. At high drug concentrations, however, Lon deficiency is detrimental for E. coli I show that the former is explained by suppression of drug efflux by Lon, while the latter can be attributed to SulA-dependent hyperfilamentation. On the other hand, deletion of lon in a trimethoprim-resistant mutant E. coli strain (harboring the Trp30Gly dihydrofolate reductase [DHFR] allele) directly potentiates resistance by enhancing the in vivo stability of mutant DHFR. Using extensive mutational analysis at 3 hot spots of resistance, I show that many resistance-conferring mutations render DHFR prone to proteolysis. This trade-off between gaining resistance and losing in vivo stability limits the number of mutations in DHFR that can confer trimethoprim resistance. Loss of Lon expands the mutational capacity for acquisition of trimethoprim resistance. This paper identifies the multipronged action of Lon in trimethoprim resistance in E. coli and provides mechanistic insight into how genetic backgrounds and drug concentrations may alter the potential for antimicrobial resistance evolution.IMPORTANCE Understanding the evolutionary dynamics of antimicrobial resistance is vital to curb its emergence and spread. Being fundamentally similar to natural selection, the fitness of resistant mutants is a key parameter to consider in the evolutionary dynamics of antimicrobial resistance (AMR). Various intrinsic and extrinsic factors modulate the fitness of resistant bacteria. This study demonstrated that Lon, a bacterial master regulator protease, influences drug tolerance and resistance. Lon is a key regulator of several fundamental processes in bacteria, including cytokinesis. I demonstrated that Lon deficiency produces highly contingent phenotypes in E. coli challenged with trimethoprim and can expand the mutational repertoire available to E. coli to evolve resistance. This multipronged influence of Lon on drug resistance provides an illustrative instance of how master regulators shape the response of bacteria to antibiotics.

Dataset Information

Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones.

Publications

Lon protease quality control of presecretory proteins in Escherichia coli and its dependence on the SecB and DnaJ (Hsp40) chaperones.

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