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.Molecular chaperones comprise a large group of highly conserved proteins that aid protein folding, transit across biological membranes, and quality control as well as assembly and disassembly of protein complexes (1-3). Molecular chaperones act by repeated binding and release of aggregation-sensitive polypeptide segments normally found buried in the final folded structure of a substrate. Although predominantly found during de novo protein synthesis or after a denaturing stress leading to protein misfolding or aggregation, such aggregation-prone segments can also be transiently exposed in native multiprotein complexes. In this case molecular chaperone binding coordinates subtle conformational changes affecting biological functions (4, 5).In the bacterium Escherichia coli, intracellular protein folding is mainly orchestrated by the chaperones Trigger Factor (TF), 2 DnaK/Hsp70, and GroEL/Hsp60. The chaperone TF binds near the ribosomal polypeptide exit with a 1:1 stoichiometry, forming a protective "cradle" for emerging nascent chains. Because of its advantageous localization, TF may interact cotranslationally with most nascent polypeptides (6, 7). TF cycles on and off the ribosome, and both ribosome-bound or released polypeptide chains can stay post-translationally bound to ribosome-free TF for a prolonged period ...

Chaperone-assisted Excisive Recombination, a Solitary Role for DnaJ (Hsp40) Chaperone in Lysogeny Escape

“…Such an observation is in sharp contrast with the role performed by DnaJ during protein export where presecretory substrate release from DnaJ to the degradation machinery required both DnaK and GrpE (51). As a bona fide chaperone, DnaJ plays a major role in mini-F plasmid replication by stimulating RepE binding to the origin in vitro (47).…”

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

“…For example, E. coli HscA specifically assists in the maturation of iron-sulfur cluster assembly protein IscA (55) and has a natural C-terminal deletion. The chaperones SecB and DnaJ, which are functionally linked to the C-terminal role of DnaK, play related roles in the protection of misfolded states from aggregation and degradation as well in the maintenance of the unfolded state for delivery to the secretory pathway (38,39,56,57).…”

SecB-like chaperone controls a toxin–antitoxin stress-responsive system in Mycobacterium tuberculosis

“…As shown in Fig. 4A, the antitoxin was efficiently detected in whole-cell fractions only when the Rv1957 chaperone was coexpressed, indicating that Rv1957 protects HigA from degradation by a yet unknown protease(s) (28,34). A direct in vivo interaction between Rv1957 and HigA was confirmed by pulldown experiments performed in E. coli (Fig.…”

“…Deletion of secB in E. coli confers a SecB-dependent coldsensitive growth phenotype at temperatures below 23°C. This is due to a strong export defect for many proteins (27,28). To examine whether Rv1957 could replace SecB in vivo, the Rv1957 gene was cloned on a low-copy plasmid under the control of a lac-inducible promoter and tested for its ability to complement the secB mutant phenotype in E. coli (Fig.…”

“…Purified Rv1957 was then compared with SecB for its ability to prevent aggregation of denatured proOmpC, a model outer membrane protein known as a SecB substrate in E. coli (28). Rv1957 efficiently prevented proOmpC aggregation in a manner comparable to that exhibited by the native E. coli SecB (Fig.…”