Cooperation of GroEL/GroES and DnaK/DnaJ heat shock proteins in preventing protein misfolding in Escherichia coli.

Abstract

Newly synthesized proteins aggregate extensively in Escherichia coil rpoH mutants, which are deficient in the heat shock proteins (hsp (13,14), suggesting that one or more hsp allows proteins to achieve their normal soluble conformation. Protein aggregation in rpoH mutants was dependent on protein synthesis, implying that the correct folding and/or assembly of newly synthesized polypeptides was impaired (13). Similarly, proteins of rpoH mutant cells labeled at permissive temperature remained largely soluble after transfer to elevated temperature (unpublished observation). Two families of hsp, hsp60 and hsp70 (60-and 70-kDa hsp) have been implicated in protein folding and assembly. The E. coli hsp60, GroEL, binds newly formed polypeptides (9) and acts in concert with GroES to promote proper assembly of a variety of proteins, including phage structural proteins (5-7). The hsp70 analogue of E. coli, DnaK, along with DnaJ and GrpE, is required for replication of phage A and of plasmids P1 and F (reviewed in ref. 15). DnaK and DnaJ stimulate replication of A by dissociating the AP protein from a Aori complex (15) and of P1 by converting P1 RepA dimers to the active monomeric form (8). DnaK also activates in vitro the E. coli replication initiator protein DnaA (16). In eukaryotes, hsp70 and cognate proteins are associated with nascent polypeptide chains (10) and, along with hsp60, are required for proper folding of proteins imported into mitochondria (11,12).In this manuscript, we report the effects of expressing four hsp-GroEL, GroES, DnaK, and DnaJ-in rpoH mutants. We find that, at high concentrations, either GroEL/GroES or DnaK/DnaJ prevents aggregation of newly synthesized proteins, while at physiological concentrations the expression of all four hsp is required. MATERIALS AND METHODSStrains and Plaids. The E. coli strains used are listed in Table 1. fl585 and f1587 are thr+ derivatives of (419 to which the groEL100 or groES30 alleles were introduced by P1 transduction via their linkage with the TnWO marker. Plasmid pKJ, described as pJM2 by McCarty and Walker (22), contains the dnaK operon placed under control of the lac promoter. Plasmids pK (having only the dnaK gene) and pJ (having only the dnaJ gene) are pKJ derivatives obtained by deletion of an appropriate restriction fragment. pSL expresses the groE operon from Plac. It was constructed by inserting the groE operon-containing EcoRI restriction fragment, trimmed upstream with BAL-31 nuclease, into Sma I/EcoRI-digested pUC19. pS and pL are derivatives of this plasmid having the groEL and groES genes, respectively, inactivated by deletion of appropriate restriction fragments.The lacIq-containing plasmid was constructed by insertion of an EcoRI restriction fragment with the lacIq gene into pACYC184.Media and Growth Conditions. Strains were grown in LB medium at 300C to mid-logarithmic phase. The cells were then incubated at 420C or at 300C for 1 hr. For plasmidcontaining cells, ampicillin (100 pg/ml) was added to the medium. Cells having rpoH null alle...

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL

“…Activity is expressed as the percentage recovery of the input material. severe aggregation in RpoH mutants, affecting the heat shock transcription factor 32, has been rescued by strong overexpression of GroEL͞GroES (27).…”

Structural Basis for Accommodation of Nonsteroidal Ligands in the Androgen Receptor

“…Purification of AR LBD bound to agonist ligands is well documented and involves removal of contamination chaperones, dnak and groEL, through cation exchange chromatography (21). The AR LBD is not retained by cation exchange chromatography in the presence of antagonist compounds and co-elutes with groEL after anion exchange chromatography, suggesting that antagonist-bound AR LBD is tightly associated with groEL, possibly as a result of partial receptor unfolding (25)(26)(27)(28). Thus, mutations to the AR that confer resistance to antiandrogens offer a viable method to obtain crystallographic evidence for antagonist binding conformations to the AR and provide insight as to how they would alter the conformation in the WT AR to stabilize association with heat shock proteins (3,29).…”

Mutations in the C-terminal fragment of DnaK affecting peptide binding.

“…As with peptide substrates in vitro, CTF would dissociate slowly from these complexes, thus inhibiting the folding or activity of bound substrates and/or blocking their accessibility to functional endogenous DnaK. The phenotypes of CTF induction, filamentation, and protein aggregation are in fact reminiscent of E. coli strains defective in chaperone function (32)(33)(34) (Fig. 2).…”