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.     

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.     

Role of molecular chaperones in inclusion body formation

Protein misfolding and aggregation are linked to several degenerative diseases and are responsible for the formation of bacterial inclusion bodies. Roles of molecular chaperones in promoting protein deposition have been speculated but not proven in vivo. We have investigated the involvement of individual chaperones in inclusion body formation by producing the misfolding-prone but partially soluble VP1LAC protein in chaperone null bacterial strains. Unexpectedly, the absence of a functional GroEL significantly reduced aggregation and favoured the incidence of the soluble protein form, from 4 to 35% of the total VP1LAC protein. On the other hand, no regular inclusion bodies were then formed but more abundant small aggregates up to 0.05 microm(3). Contrarily, in a DnaK(-) background, the amount of inclusion body protein was 2.5-fold higher than in the wild-type strain and the average volume of the inclusion bodies increased from 0.25 to 0.38 microm(3). Also in the absence of DnaK, the minor fraction of soluble protein appears as highly proteolytically stable, suggesting an inverse connection between proteolysis and aggregation managed by this chaperone. In summary, GroEL and DnaK appear as major antagonist controllers of inclusion body formation by promoting and preventing, respectively, the aggregation of misfolded polypeptides. GroEL might have, in addition, a key role in driving the protein transit from the soluble to the insoluble cell fraction and also in the opposite direction. Although chaperones ClpB, ClpA, IbpA and IbpB also participate in these processes, the impact of the respective null mutations on bacterial inclusion body formation is much more moderate.     

Low temperature of GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of deltatigdeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C deltatigdeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of deltatigdeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/= 60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Low temperature or GroEL/ES overproduction permits growth of Escherichia coli cells lacking trigger factor and DnaK

Escherichia coli trigger factor (TF) and DnaK cooperate in the folding of newly synthesized proteins. The combined deletion of the TF-encoding tig gene and the dnaK gene causes protein aggregation and synthetic lethality at 30 degrees C. Here we show that the synthetic lethality of DeltatigDeltadnaK52 cells is abrogated either by growth below 30 degrees C or by overproduction of GroEL/GroES. At 23 degrees C DeltatigDeltadnaK52 cells were viable and showed only minor protein aggregation. Overproduction of GroEL/GroES, but not of other chaperones, restored growth of DeltatigDeltadnaK52 cells at 30 degrees C and suppressed protein aggregation including proteins >/=60 kDa, which normally require TF and DnaK for folding. GroEL/GroES thus influences the folding of proteins previously identified as DnaK/TF substrates.     

Expression of a highly toxic protein, Bax, in Escherichia coli by attachment of a leader peptide derived from the GroES cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384-11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

Proteome-wide analysis of chaperonin-dependent protein folding in Escherichia coli

The E. coli chaperonin GroEL and its cofactor GroES promote protein folding by sequestering nonnative polypeptides in a cage-like structure. Here we define the contribution of this system to protein folding across the entire E. coli proteome. Approximately 250 different proteins interact with GroEL, but most of these can utilize either GroEL or the upstream chaperones trigger factor (TF) and DnaK for folding. Obligate GroEL-dependence is limited to only approximately 85 substrates, including 13 essential proteins, and occupying more than 75% of GroEL capacity. These proteins appear to populate kinetically trapped intermediates during folding; they are stabilized by TF/DnaK against aggregation but reach native state only upon transfer to GroEL/GroES. Interestingly, substantially enriched among the GroEL substrates are proteins with (betaalpha)8 TIM-barrel domains. We suggest that the chaperonin system may have facilitated the evolution of this fold into a versatile platform for the implementation of numerous enzymatic functions.     

DnaK functions as a central hub in the E. coli chaperone network

Cellular chaperone networks prevent potentially toxic protein aggregation and ensure proteome integrity. Here, we used Escherichia coli as a model to understand the organization of these networks, focusing on the cooperation of the DnaK system with the upstream chaperone Trigger factor (TF) and the downstream GroEL. Quantitative proteomics revealed that DnaK interacts with at least ~700 mostly cytosolic proteins, including ~180 relatively aggregation-prone proteins that utilize DnaK extensively during and after initial folding. Upon deletion of TF, DnaK interacts increasingly with ribosomal and other small, basic proteins, while its association with large multidomain proteins is reduced. DnaK also functions prominently in stabilizing proteins for subsequent folding by GroEL. These proteins accumulate on DnaK upon GroEL depletion and are then degraded, thus defining DnaK as a central organizer of the chaperone network. Combined loss of DnaK and TF causes proteostasis collapse with disruption of GroEL function, defective ribosomal biogenesis, and extensive aggregation of large proteins.     

Monitoring protein conformation along the pathway of chaperonin-assisted folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.     

The chaperonin GroEL and other heat-shock proteins, besides DnaK, participate in ribosome biogenesis in Escherichia coli

It has been shown that in Escherichia coli the chaperone DnaK is necessary for the late stages of 50S and 30S ribosomal subunit assembly in vivo. Here we focus on the roles of other HSPs (heat-shock proteins), including the chaperonin GroEL, in addition to DnaK, in ribosome biogenesis at high temperature. GroEL is shown to be required for the very late 45S-->50S step in the biogenesis of the large ribosome subunit, but not for 30S assembly. Interestingly, overproduction of GroES/GroEL can partially compensate for a lack of DnaK/DnaJ at 44 degrees C.     

Cell-free system derived from heat-shocked Escherichia coli: Synthesis of enzyme protein possessing higher specific activity

heat-shocked S30 extract (HS-S30 extract) was prepared from cells of Escherichia coli strain Q13 exposed to elevated temperatures (from 37°C to 42°C) for 30 min. In a cell-free system with HS-S30 extract, the synthesized CAT protein had higher specific activity than that synthesized by a cell-free system with S30 extract prepared from Q13 cells incubated at 37°C. SDS-PAGE analysis showed that the heat-shock proteins, GroEL and DnaK, which are known to be molecular chaperones, were significantly increased in the HS-S30 extract. The addition of GroEL or DnaK to the S30 extract system increased the specific activity of the synthesized CAT protein. Heat-shock induction thus offers an effective method of modifying E. coli cell extracts.     

DnaK/DnaJ chaperone system reactivates endogenous E. coli thermostable FBP aldolase in vivo and in vitro; the effect is enhanced by GroE heat shock proteins

Thermally aggregated, endogenous proteins in Escherichia coli cells form the S fraction, which is separable by sucrose density gradient centrifugation. To date, relatively little is known about the mechanisms of elimination of the heat-aggregated proteins from E. coli cells and the composition of the S fraction. We have identified several proteins of the S fraction using 2D-gel electrophoresis and microsequencing. A thermostable II class fructose-1,6-bisphosphate aldolase (Fda protein) appeared to be one of numerous proteins of the S fraction. Fda was purified from E. coli overproducer strain and used as a model substrate for investigation of the role of Hsps in prevention and repair of thermal denaturation of proteins both in vivo and in vitro. We found that the heat inactivation of Fda was reversible and that its reactivation in vivo and in vitro required mainly the assistance of the DnaK/DnaJ chaperone system. The dnaK756 and dnaJ259 mutations had a negative effect on the reactivation of thermally inactivated Fda. Moreover, we showed that the reactivation process in vitro was enhanced when GroEL/GroES were added together with DnaK/DnaJ. GroEL/GroES alone were inefficient in the resolubilization or reactivation of the heat-aggregated Fda. It is supposed that the denaturation of the thermostable Fda in vivo results rather from a temporary and transient deficit of Hsps than from the direct heat effect.     

Activation and expression of proteins during synchronous germination of aerial spores of Streptomyces granaticolor

Synchronously germinating aerial spores of Streptomyces granaticolor were used to study protein activation and expression during the transition from dormant to metabolically active vegetative forms. The first phase of protein activation is associated with the solubility of proteins. Three major chaperones, DnaK, Trigger factor, and GroEL, were identified in spores. Enhancement in rate of protein synthesis during germination was accompanied by the association of TF and DnaK with ribosomes. During germination, the chaperones TF, GroEL, and DnaK undergo reversible phosphorylation. GroEL was phosphorylated on both Ser and Thr, whereas phosphorylation of DnaK and TF was detected on Thr only. A proteomic approach was used to gain more information on protein expression during germination on two types of media differing in the ability of cells to produce antibiotic granaticin. To obtain an overview of the metabolic activity of germinating spores, glycolytic enzymes, enzymes of citric acid cycle, metabolism of amino acids and nucleic acids, and components of the protein synthesis system were identified and analyzed using the proteomic database. The results were deposited on the SWICZ proteomic server and are accessible on http://proteom.biomed.cas.cz.     

Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli

During production in recombinant Escherichia coli, the human basic fibroblast growth factor (hFGF-2) partly aggregates into stable cytoplasmic inclusion bodies. These inclusion bodies additionally contain significant amounts of the heat-shock chaperone DnaK, and putative DnaK substrates such as the elongation factor Tu (ET-Tu) and the metabolic enzymes dihydrolipoamide dehydrogenase (LpdA), tryptophanase (TnaA), and d-tagatose-1,6-bisphosphate aldolase (GatY). Guanidinium hydrochloride induced disaggregation studies carried out in vitro on artificial aggregates generated through thermal aggregation of purified hFGF-2 revealed identical disaggregation profiles as hFGF-2 inclusion bodies indicating that the heterogenic composition of inclusion bodies did not influence the strength of interactions of hFGF-2 in aggregates formed in vivo as inclusion bodies compared to those generated in vitro from native and pure hFGF-2 through thermal aggregation. Compared to unfolding of native hFGF-2, higher concentrations of denaturant were required to dissolve hFGF-2 aggregates showing that more energy is required for disruption of interactions in both types of protein aggregates compared to the unfolding of the native protein. In vivo dissolution of hFGF-2 inclusion bodies was studied through coexpression of chaperones of the DnaK and GroEL family and ClpB and combinations thereof. None of the chaperone combinations was able to completely prevent the initial formation of inclusion bodies, but upon prolonged incubation mediated disaggregation of otherwise stable inclusion bodies. The GroEL system was particularly efficient in inclusion body dissolution but did not lead to a corresponding increase in soluble hFGF-2 rather was promoting the proteolysis of the recombinant growth factor. Coproduction of the disaggregating DnaK system and ClpB in conjunction with small amounts of the chaperonins GroELS was most efficient in disaggregation with concomitant formation of soluble hFGF-2. Thus, fine-balanced coproduction of chaperone combinations can play an important role in the production of soluble recombinant proteins with a high aggregation propensity not through prevention of aggregation but predominantly through their disaggregating properties.     

Expression of a Highly Toxic Protein, Bax, in Escherichia coli by Attachment of a Leader Peptide Derived from the GroES Cochaperone

Expression of the human apoptosis modulator protein Bax in Escherichia coli is highly toxic, resulting in cell lysis at very low concentrations (Asoh, S., et al., J. Biol. Chem. 273, 11384–11391, 1998). Attempts to express a truncated form of murine Bax in the periplasm by using an expression vector that attached the OmpA signal sequence to the protein failed to alleviate this toxicity. In contrast, attachment of a peptide based on a portion of the E. coli cochaperone GroES reduced Bax's toxicity significantly and allowed good expression. The peptide, which was attached to the N-terminus, included the amino acid sequence of the mobile loop of GroES that has been demonstrated to interact with the chaperonin, GroEL. Under normal growth conditions, expression of this construct was still toxic, but generated a small amount of detectable recombinant Bax. However, when cells were grown in the presence of 2% ethanol, which stimulated overproduction of the molecular chaperones GroEL and DnaK, toxicity was reduced and good overexpression occurred. Two-dimensional gel electrophoresis analysis showed that approximately 15-fold more GroES-loop-Bax was produced under these conditions than under standard conditions and that GroEL and DnaK were elevated approximately 3-fold.     

DnaK plays a pivotal role in Tat targeting of CueO and functions beside SlyD as a general Tat signal binding chaperone

The Tat (twin-arginine translocation) system from Escherichia coli transports folded proteins with N-terminal twin-arginine signal peptides across the cytoplasmic membrane. The influence of general chaperones on Tat substrate targeting has not been clarified so far. Here we show that the chaperones SlyD and DnaK bind to a broad range of different Tat signal sequences in vitro and in vivo. Initially, SlyD and GroEL were purified from DnaK-deficient extracts by their affinity to various Tat signal sequences. Of these, only SlyD bound Tat signal sequences also in the presence of DnaK. SlyD and DnaK also co-purified with Tat substrate precursors, demonstrating the binding to Tat signal sequences in vivo. Deletion of dnaK completely abolished Tat-dependent translocation of CueO, but not of DmsA, YcdB, or HiPIP, indicating that DnaK has an essential role specifically for CueO. DnaK was not required for stability of the CueO precursor and thus served in some essential step after folding. A CueO signal sequence fusion to HiPIP was Tat-dependently transported without the need of DnaK, indicating that the mature domain of CueO is responsible for the DnaK dependence. The overall results suggest that SlyD and DnaK are in the set of chaperones that can serve as general Tat signal-binding proteins. DnaK has additional functions that are indispensable for the targeting of CueO.     

DnaK, DnaJ and GrpE form a cellular chaperone machinery capable of repairing heat-induced protein damage.

Members of the conserved Hsp70 chaperone family are assumed to constitute a main cellular system for the prevention and the amelioration of stress-induced protein damage, though little direct evidence exists for this function. We investigated the roles of the DnaK (Hsp70), DnaJ and GrpE chaperones of Escherichia coli in prevention and repair of thermally induced protein damage using firefly luciferase as a test substrate. In vivo, luciferase was rapidly inactivated at 42 degrees C, but was efficiently reactivated to 50% of its initial activity during subsequent incubation at 30 degrees C. DnaK, DnaJ and GrpE did not prevent luciferase inactivation, but were essential for its reactivation. In vitro, reactivation of heat-inactivated luciferase to 80% of its initial activity required the combined activity of DnaK, DnaJ and GrpE as well as ATP, but not GroEL and GroES. DnaJ associated with denatured luciferase, targeted DnaK to the substrate and co-operated with DnaK to prevent luciferase aggregation at 42 degrees C, an activity that was required for subsequent reactivation. The protein repair function of DnaK, GrpE and, in particular, DnaJ is likely to be part of the role of these proteins in regulation of the heat shock response.     

Identification of a redox-regulated chaperone network

We have identified and reconstituted a multicomponent redox-chaperone network that appears to be designed to protect proteins against stress-induced unfolding and to refold proteins when conditions return to normal. The central player is Hsp33, a redox-regulated molecular chaperone. Hsp33, which is activated by disulfide bond formation and subsequent dimerization, works as an efficient chaperone holdase that binds to unfolding protein intermediates and maintains them in a folding competent conformation. Reduction of Hsp33 is catalyzed by the glutaredoxin and thioredoxin systems in vivo, and leads to the formation of highly active, reduced Hsp33 dimers. Reduction of Hsp33 is necessary but not sufficient for substrate protein release. Substrate dissociation from Hsp33 is linked to the presence of the DnaK/DnaJ/GrpE foldase system, which alone, or in concert with the GroEL/GroES system, then supports the refolding of the substrate proteins. Upon substrate release, reduced Hsp33 dimers dissociate into inactive monomers. This regulated substrate transfer ultimately links substrate release and Hsp33 inactivation to the presence of available DnaK/DnaJ/GrpE, and, therefore, to the return of cells to non-stress conditions.