The in vivo and in vitro characterization of DnaK from Agrobacterium tumefaciens RUOR

Molecular chaperones of the heat shock protein 70 family (Hsp70; also called DnaK in prokaryotes) play an important role in the folding and functioning of cellular protein machinery. The dnaK gene from the plant pathogen Agrobacterium tumefaciens RUOR was amplified using the polymerase chain reaction and the DnaK protein (Agt DnaK) was over-produced as a His-tagged protein in Escherichia coli. The Agt DnaK amino acid sequence was 96% identical to the A. tumefaciens C58 DnaK sequence and 65% identical to the E. coli DnaK sequence. Agt DnaK was shown to be able to functionally replace E. coli DnaK in vivo using complementation assays with an E. coli dnaK756 mutant strain and a dnaK52 deletion strain. Over-production and purification of Agt DnaK was successful, and allowed for further characterization of the protein. Kinetic analysis of the basal ATPase activity of purified Agt DnaK revealed a Vmax of 1.3 nmol phosphate released per minute per milligram DnaK, and a Km of 62 microM ATP. Thus, this is the first study to provide both in vivo and in vitro evidence that Agt DnaK has the properties of a molecular chaperone of the Hsp70 family.     

Interaction between heat shock protein DnaK and recombinant staphylococcal protein A.

When a protein derived from the immunoglobulin G (IgG)-binding domains of staphylococcal protein A was expressed in Escherichia coli and recovered from cell extract by IgG affinity chromatography, the 69-kilodalton heat shock protein DnaK was found to be copurified. DnaK could be selectively eluted from the IgG column by ATP or by lowering the pH to 4.7. Protein A could subsequently be eluted by lowering the pH to 3.2. Thus, this procedure allows a one-step purification of both DnaK and protein A from cell extract. In vitro experiments with pure DnaK and protein A revealed that DnaK did not interfere with the IgG-binding properties of protein A but associated with its unfolded C-terminal in a salt-resistant manner. In addition, a specific interaction between DnaK and denaturated casein was found.     

Characterization of the dnaK multigene family in the Cyanobacterium Synechococcus sp. strain PCC7942

The cyanobacterium Synechococcus sp. strain PCC7942 has three dnaK homologues (dnaK1, dnaK2, and dnaK3), and a gene disruption experiment was carried out for each dnaK gene by inserting an antibiotic resistance marker. Our findings revealed that DnaK1 was not essential for normal growth, whereas DnaK2 and DnaK3 were essential. We also examined the effect of heat shock on the levels of these three DnaK and GroEL proteins and found a varied response to heat shock, with levels depending on each protein. The DnaK2 and GroEL proteins exhibited a typical heat shock response, that is, their synthesis increased upon temperature upshift. In contrast, the synthesis of DnaK1 and DnaK3 did not respond to heat shock; in fact, the level of DnaK1 protein decreased. We also analyzed the effect of overproduction of each DnaK protein in Escherichia coli cells using an inducible expression system. Overproduction of DnaK1 or DnaK2 resulted in defects in cell septation and formation of cell filaments. On the other hand, overproduction of DnaK3 did not result in filamentous cells; rather a swollen and twisted cell morphology was observed. When expressed in an E. coli dnaK756 mutant, dnaK2 could suppress the growth deficiency at the nonpermissive temperature, while dnaK1 and dnaK3 could not suppress this phenotype. On the contrary, overproduction of DnaK1 or DnaK3 resulted in growth inhibition at the permissive temperature. These results suggest that different types of Hsp70 in the same cellular compartment have specific functions in the cell.     

The heat-shock-regulated grpE gene of Escherichia coli is required for bacterial growth at all temperatures but is dispensable in certain mutant backgrounds.

Previous work has established that the grpE+ gene product is a heat shock protein that is essential for bacteriophage lambda growth at all temperatures and for Escherichia coli growth at temperatures above 43 degrees C. Here it is shown that the grpE+ gene product is essential for bacterial viability at all temperatures. The strategy required constructing a grpE deletion derivative carrying a selectable chloramphenicol drug resistance marker provided by an omega insertion and showing that this deletion construct can be crossed into the bacterial chromosome if and only if a functional grpE+ gene is present elsewhere in the same cell. As a control, the same omega insertion could be placed immediately downstream of the grpE+ coding sequence without any observable effects on host growth. This result demonstrates that the inability to construct a grpE-deleted E. coli strain is not simply due to a lethal polar effect on neighboring gene expression. Unexpectedly, it was found that the grpE deletion derivative could be crossed into the bacterial chromosome in a strain that was defective in DnaK function. Further analysis showed that it was not the lack of DnaK function per se that allowed E. coli to tolerate a deletion in the grpE+ gene. Rather, it was the presence of unknown extragenic suppressors of a dnaK mutation that somehow compensated for the deficiency in both DnaK and GrpE function.     

Hsc62, Hsc56, and GrpE,the third Hsp70 chaperone system of Escherichia coli

Hsc62 is the third Hsp70 homolog of Escherichia coli, which we found previously. Hsc62 is structurally and biochemically similar to DnaK, but hscC gene encoding Hsc62 did not compensate for the defects in the dnaK-null mutant of E. coli MC4100 strain. We cloned the ybeV gene and purified the gene product named Hsc56, a 55,687-Da protein with a J-domain like sequence. Hsc56 stimulated the ATPase activity of only Hsc62 but not those of the other Hsp70 homologs, DnaK and Hsc66. Hsc56 contains the -His-Pro-Glu- sequence corresponding to the His-Pro-Asp motif in DnaJ, which is indispensable for DnaJ to interact with DnaK. Conversion of -His-Pro-Glu- to -Ala-Ala-Ala- abolished the ability of Hsc56 to stimulate the ATPase activity of Hsc62. GrpE, a nucleotide exchange factor for DnaK, also stimulated the ATPase activity of Hsc62 in the presence of Hsc56. Hsc62-Hsc56-GrpE is probably a new Hsp70 chaperone system of E. coli.     

CbpA, a DnaJ homolog, is a DnaK co-chaperone, and its activity is modulated by CbpM

The DnaK chaperone system, consisting of DnaK, DnaJ, and GrpE, remodels and refolds proteins during both normal growth and stress conditions. CbpA, one of several DnaJ analogs in Escherichia coli, is known to function as a multicopy suppressor for dnaJ mutations and to bind nonspecifically to DNA and preferentially to curved DNA. We found that CbpA functions as a DnaJ-like co-chaperone in vitro. CbpA acted in an ATP-dependent reaction with DnaK and GrpE to remodel inactive dimers of plasmid P1 RepA into monomers active in P1 DNA binding. Additionally, CbpA participated with DnaK in an ATP-dependent reaction to prevent aggregation of denatured rhodanese. The cbpA gene is in an operon with an open reading frame, yccD, which encodes a protein that has some homology to DafA of Thermus thermophilus. DafA is a protein required for the assembly of ring-like particles that contain trimers each of T. thermophilus DnaK, DnaJ, and DafA. The E. coli YccD was isolated because of its potential functional relationship to CbpA. Purified YccD specifically inhibited both the co-chaperone activity and the DNA binding activity of CbpA, suggesting that YccD modulates the activity of CbpA. We named the product of the yccD gene CbpM for "CbpA modulator."     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

Formation in vitro of complexes between an abnormal fusion protein and the heat shock proteins from Escherichia coli and yeast mitochondria.

Heat shock proteins (HSPs) of the Hsp70 and GroEL families associate with a variety of cell proteins in vivo. However, the formation of such complexes has not been systematically studied. A 31-kDa fusion protein (CRAG), which contains 12 residues of cro repressor, truncated protein A, and 14 residues of beta-galactosidase, when expressed in Escherichia coli, was found in complexes with DnaK, GrpE, protease La, and GroEL. When an E. coli extract not containing CRAG was applied to an affinity column containing CRAG, DnaK, GroEL, and GrpE were selectively bound. These HSPs did not bind to a normal protein A column. DnaK, GrpE, and the fraction of GroEL could be eluted from the CRAG column with ATP but not with a nonhydrolyzable ATP analog. The ATP-dependent release of DnaK and GroEL also required Mg2+, but GrpE dissociated with ATP alone. The binding and release of DnaK and GroEL were independent events, but the binding of GrpE required DnaK. Inactivation of DnaJ, GrpE, and GroES did not affect the association or dissociation of DnaK or GroEL from CRAG. The DnaK and GrpE proteins could be eluted with 10(-6) M ATP, but 10(-4) M was required for GroEL release. This approach allows a one-step purification of these proteins from E. coli and also the isolation of the DnaK and GroEL homologs from yeast mitochondria. Competition experiments with oligopeptide fragments of CRAG showed that DnaK and GroEL interact with different sites on CRAG and that the cro-derived domain of CRAG contains the DnaK-binding site.     

Precursor of mitochondrial aspartate aminotransferase synthesized in Escherichia coli is complexed with heat-shock protein DnaK.

On expression of the cDNA encoding the precursor of chicken mitochondrial aspartate aminotransferase (pmAspAT) in Escherichia coli, the bulk of pmAspAT was found to be associated with the 70-kDa heat-shock protein DnaK which is closely related to mitochondrial 70-kDa heat-shock protein (HSP70). Purification protocols for the DnaK/pmAspAT complex and its individual components were elaborated. The complex dissociated on treatment with MgATP or at pH 5.5. Like the mature enzyme, pmAspAT is a dimer (2 x 47 kDa) and exhibits about a third of its enzyme activity. In the DnaK/pmAspAT complex, one DnaK molecule is bound to each subunit of pmAspAT; this tetramer may further aggregate to an octamer. The complex is catalytically almost as active as free pmAspAT. It could be reconstituted from isolated DnaK and pmAspAT. No complex was formed with mAspAT. Apparently, DnaK binds to the solvent-exposed presequence of folded pmAspAT without significantly changing the structure and functional properties of its mature moiety.     

Molecular characterization of a plant mitochondrial chaperone GrpE

Escherichia coli DnaK (Hsp70) cooperates with DnaJ and GrpE in its essential role as a molecular chaperone. Function of mitochondrial Hsp70 (mHsp70) in protein folding and organellar import in eukaryotes is critically dependent on GrpE. We cloned two genes from tobacco (Nicotiana tabacum) BY2 cells based on peptide sequences from a purified protein. The predicted amino acid sequences of both clones resembled that of GrpE from E. coli and its homologues from eukaryotes, and a cDNA clone from Arabidopsis thaliana. One gene (Type 1) encoded a deduced protein that was identical to the purified protein while the other (Type 2) encoded a deduced protein that has 80% sequence identity to Type 1. Both tobacco and Arabidopsis thaliana GrpE homologues bound to DnaK and ATP inhibited this binding. The tobacco GrpE homologue contained a typical N-terminal mitochondrial target presequence of 64 residues and the presequence directed the green fluorescent protein to tobacco mitochondria. The tobacco GrpE homologue also associated with mHsp70 when reintroduced into BY2 protoplasts, and this association was disrupted by ATP. A three-dimensional structure for the tobacco GrpE homologue was modeled based on the X-ray structure of E. coli GrpE complexed with DnaK. The modeled structure has the same overall structure as E. coli GrpE. We propose that the tobacco GrpE homologue interacts with mHsp70 in a manner analogous to E. coli GrpE with DnaK and designate it as tobacco mitochondrial GrpE (NtmGrpE).     

Escherichia coli heat shock protein DnaK: production and consequences in terms of monitoring cooking

Through use of commercially available DnaK proteins and anti-DnaK monoclonal antibodies, a competitive enzyme-linked immunosorbent assay was developed to quantify this heat shock protein in Escherichia coli ATCC 25922 subjected to various heating regimens. For a given process lethality (F(70)(10) of 1, 3, and 5 min), the intracellular concentration of DnaK in E. coli varied with the heating temperature (50 or 55 degrees C). In fact, the highest DnaK concentrations were found after treatments at the lower temperature (50 degrees C) applied for a longer time. Residual DnaK after heating was found to be necessary for cell recovery, and additional DnaK was produced during the recovery process. Overall, higher intracellular concentrations of DnaK tended to enhance cell resistance to a subsequent lethal stress. Indeed, E. coli cells that had undergone a sublethal heat shock (105 min at 55 degrees C, F(70)(10) = 3 min) accompanied by a 12-h recovery (containing 76,786 +/- 25,230 molecules/cell) resisted better than exponentially growing cells (38,500 +/- 6,056 molecules/cell) when later heated to 60 degrees C for 50 min (F(70)(10) = 5 min). Results reported here suggest that using stress protein to determine cell adaptation and survival, rather than cell counts alone, may lead to more efficient heat treatment.     

A novel plastid-targeted J-domain protein in Arabidopsis thaliana

Arabidopsis cDNAs encoding ATJ11, the smallest known J-domain protein, have been isolated and characterized. The precursor protein of 161 amino acid residues was synthesized in vitro and imported by isolated pea chloroplasts where it was localized to the stroma and cleaved to a mature protein of 125 amino acid residues. The mature protein consists of an 80 amino acid J-domain, and N- and C-terminal extensions of 24 and 21 amino acid residues, respectively, which show no similarity to regions in other DnaJ-related proteins. ATJ11 produced in Escherichia coli stimulated the weak ATPase activity of E. coli DnaK, but was unable to stimulate refolding of firefly luciferase by DnaK, and inhibited refolding by DnaK, DnaJ and GrpE. ATJ11 is encoded by a single-copy gene on chromosome 4, and is expressed in all plant organs examined. A paralogue of ATJ11, showing 72% identity, is encoded in a 4.5 Mb duplication of chromosome 4 on chromosome 2. These proteins represent a novel class of J-domain proteins.     

Phylogenetic analysis of the third hsp70 homolog in Escherichia coli; a novel member of the Hsc66 subfamily and its possible co-chaperone.

Novel members of the highly conserved protein family, Hsp70, have been found in the complete sequences of several genomes. To elucidate a phylogenetic relationship among Hsp70 proteins of Escherichia coli, we searched all open reading frames derived from 13 complete genomes for Hsp70/actin-related proteins by the single-linkage clustering method. Phylogenetic analysis of this superfamily revealed that E. coli possesses at least three Hsp70 homologs (DnaK, Hsc66 and Hsc62). We found that Hsc62, which is the product of hscC, is a new member of the Hsc66 subfamily, and is specific to E. coli. The analysis also suggested that YegD of E. coli is closely related to the actin family, which consists of the actin, FtsA and MreB subfamilies. A further database search revealed that two dnaJ homologs, ybeS and ybeV, were located on the opposite strand near hscC. Consequently, E. coli seems to have three gene clusters composed of DnaK and DnaJ homologs.     

Escherichia coli DnaK protein possesses a 5''-nucleotidase activity that is inhibited by AppppA.

AppppA and the DnaK protein have both been hypothesized to function in regulating the heat shock response of Escherichia coli. The proposals are that AppppA serves as a signal (alarmone) to turn on the heat shock response, whereas the DnaK protein is necessary to turn off the heat shock response. A simple model would be that the DnaK protein turns off the response by degrading AppppA. We disproved this model by demonstrating that the DnaK protein possesses a 5'-nucleotidase activity capable of degrading many cellular nucleotides but not AppppA. Although AppppA was not a substrate, it did inhibit the 5'-nucleotidase activity of the DnaK protein. This inhibition may be specific and have biological function since the mutant DnaK756 protein, which is defective in turning off the heat shock response, is partially desensitized to AppppA inhibition. These findings led us to consider other possible mechanisms for AppppA and the DnaK protein in heat shock regulation.     

Functional similarities and differences of an archaeal Hsp70(DnaK) stress protein compared with its homologue from the bacterium Escherichia coli

Archaea are prokaryotes but some of their chaperoning systems resemble those of eukaryotes. Also, not all archaea possess the stress protein Hsp70(DnaK), in contrast with bacteria and eukaryotes, which possess it without any known exception. Further, the primary structure of the archaeal DnaK resembles more the bacterial than the eukaryotic homologues. The work reported here addresses two questions: Is the archaeal Hsp70 protein a chaperone, like its homologues in the other two phylogenetic domains? And, if so, is the chaperoning mechanism of bacterial or eukaryotic type? The data have shown that the DnaK protein of the archaeon Methanosarcina mazei functions efficiently as a chaperone in luciferase renaturation in vitro, and that it requires DnaJ, and the other bacterial-type chaperone, GrpE, to perform its function. The M. mazei DnaK chaperone activity was enhanced by interaction with the bacterial co-chaperone DnaJ, but not by the eukaryotic homologue HDJ-2. Both the bacterial GrpE and DnaJ stimulated the ATPase activity of the M. mazei DnaK. The M. mazei DnaK-dependent chaperoning pathway in vitro is similar to that of the bacterium Escherichia coli used for comparison. However, in vivo analyses indicate that there are also significant differences. The M. mazei dnaJ and grpE genes rescued E.coli mutants lacking these genes, but E.coli dnaK mutants were not complemented by the M. mazei dnaK gene. Thus, while the data from in vitro tests demonstrate functional similarities between the M. mazei and E.coli DnaK proteins, in vivo results indicate that, intracellularly, the chaperones from the two species differ.     

Molecular cloning of mtp70, a mitochondrial member of the hsp70 family.

We have isolated a gene from the protozoan parasite Trypanosoma cruzi that encodes a previously unidentified member of the 70-kilodalton heat shock protein (hsp70) family. Among all the eucaryotic hsp70 proteins described to date, this trypanosome protein, mtp70, is uniquely related in sequence and structure to the hsp70 of Escherichia coli, DnaK, which functions in the initiation of DNA replication. This relationship to DnaK is especially relevant in view of the intracellular location of the protein. Within the trypanosome, mtp70 is located in the mitochondrion, where it associates with kinetoplast DNA (kDNA), the unusual mitochondrial DNA that distinguishes this order of protozoa. Moreover, mtp70 is located in the specific region of the kinetoplast in which kDNA replication occurs. In view of the known functions of DnaK, the localization of mtp70 to the site of kDNA replication suggests that mtp70 may participate in eucaryotic mitochondrial DNA replication in a manner analogous to that of DnaK in E. coli.     

Construction and analysis of hybrid Escherichia coli-Bacillus subtilis dnaK genes

The highly conserved DnaK chaperones consist of an N-terminal ATPase domain, a central substrate-binding domain, and a C-terminal domain whose function is not known. Since Bacillus subtilis dnaK was not able to complement an Escherichia coli dnaK null mutant, we performed domain element swap experiments to identify the regions responsible for this finding. It turned out that the B. subtilis DnaK protein needed approximately normal amounts of the cochaperone DnaJ to be functional in E. coli. The ATPase domain and the substrate-binding domain form a species-specific functional unit, while the C-terminal domains, although less conserved, are exchangeable. Deletion of the C-terminal domain in E. coli DnaK affected neither complementation of growth at high temperatures nor propagation of phage lambda but abolished degradation of sigma32.     

Interaction of the DnaK and DnaJ chaperone system with a native substrate,P1 RepA

DnaK, the Hsp70 chaperone of Escherichia coli interacts with protein substrates in an ATP-dependent manner, in conjunction with DnaJ and GrpE co-chaperones, to carry out protein folding, protein remodeling, and assembly and disassembly of multisubunit protein complexes. To understand how DnaJ targets specific proteins for recognition by the DnaK chaperone system, we investigated the interaction of DnaJ and DnaK with a known natural substrate, bacteriophage P1 RepA protein. By characterizing RepA deletion derivatives, we found that DnaJ interacts with a region of RepA located between amino acids 180 and 200 of the 286-amino acid protein. A peptide corresponding to amino acids 180-195 inhibited the interaction of RepA and DnaJ. Two site-directed RepA mutants with alanine substitutions in this region were about 4-fold less efficiently activated for oriP1 DNA binding by DnaJ and DnaK than wild type RepA. We also identified by deletion analysis a site in RepA, in the region of amino acids 35-49, which interacts with DnaK. An alanine substitution mutant in amino acids 36-39 was constructed and found defective in activation by DnaJ and DnaK. Taken together the results suggest that DnaJ and DnaK interact with separate sites on RepA.     

Identification and characterization of GroEL and DnaK homologues in Thiobacillus ferrooxidans

The major heat shock proteins from Thiobacillus ferrooxidans were identified as DnaK and GroEL equivalents by Western blotting and analysis of the N-terminal amino acid sequence of spots isolated from dried 2-D polyacrylamide electrophoresis gels. The T. ferrooxidans chaperonins showed 70% and 80% identity with the Escherichia coli GroEL and DnaK, respectively. By using electrophoresis with a transverse pore gradient of cross-linked polyacrylamide and nondenaturing conditions followed by Western blotting, we found that the GroEL proteins from both bacteria formed a 14-mer, whereas E. coli DnaK protein existed partially as a dimer and the T. ferrooxidans DnaK-equivalent showed only a monomeric nature under our experimental conditions.