ClpB cooperates with DnaK, DnaJ, and GrpE in suppressing protein aggregation. A novel multi-chaperone system from Escherichia coli.

ClpB is a heat-shock protein from Escherichia coli with an unknown function. We studied a possible molecular chaperone activity of ClpB in vitro. Firefly luciferase was denatured in urea and then diluted into the refolding buffer (in the presence of 5 mM ATP and 0.1 mg/ml bovine serum albumin). Spontaneous reactivation of luciferase was very weak (less than 0.02% of the native activity) because of extensive aggregation. Conventional chaperone systems (GroEL/GroES and DnaK/DnaJ/GrpE) or ClpB alone did not reactivate luciferase under those conditions. However, ClpB together with DnaK/DnaJ/GrpE greatly enhanced the luciferase activity regain (up to 57% of native activity) by suppressing luciferase aggregation. This coordinated function of ClpB and DnaK/DnaJ/GrpE required ATP hydrolysis, although the ClpB ATPase was not activated by native or denatured luciferase. When the chaperones were added to the luciferase refolding solutions after 5-25 min of refolding, ClpB and DnaK/DnaJ/GrpE recovered the luciferase activity from preformed aggregates. Thus, we have identified a novel multi-chaperone system from E. coli, which is analogous to the Hsp104/Ssa1/Ydj1 system from yeast. ClpB is the only known bacterial Hsp100 protein capable of cooperating with other heat-shock proteins in suppressing and reversing protein aggregation.     

ClpB/Hsp100 proteins and heat stress tolerance in plants

High-temperature stress can disrupt cellular proteostasis, resulting in the accumulation of insoluble protein aggregates. For survival under stressful conditions, it is important for cells to maintain a pool of native soluble proteins by preventing and/or dissociating these aggregates. Chaperones such as GroEL/GroES (Hsp60/Hsp10) and DnaK/DnaJ/GrpE (Hsp70/Hsp40/nucleotide exchange factor) help cells minimize protein aggregation. Protein disaggregation is accomplished by chaperones belonging to the Caseinolytic Protease (Clp) family of proteins. ClpB/Hsp100 proteins are strikingly ubiquitous and are found in bacteria, yeast and multi-cellular plants. The expression of these proteins is regulated by heat stress (HS) and developmental cues. Bacteria and yeast contain one and two forms of ClpB proteins, respectively. Plants possess multiple forms of these proteins that are localized to different cellular compartments (i.e. cytoplasm/nucleus, chloroplast or mitochondria). Overwhelming evidence suggests that ClpB/Hsp100 proteins play decisive roles in cell adaptation to HS. Mutant bacteria and yeast cells lacking active ClpB/Hsp100 proteins are critically sensitive to high-temperature stress. Likewise, Arabidopsis, maize and rice mutants lacking cytoplasmic ClpB proteins are very sensitive to heat. In this study, we present the structural and functional attributes of plant ClpB forms.     

Cooperative action of Escherichia coli ClpB protein and DnaK chaperone in the activation of a replication initiation protein

The Escherichia coli molecular chaperone protein ClpB is a member of the highly conserved Hsp100/Clp protein family. Previous studies have shown that the ClpB protein is needed for bacterial thermotolerance. Purified ClpB protein has been shown to reactivate chemically and heat-denatured proteins. In this work we demonstrate that the combined action of ClpB and the DnaK, DnaJ, and GrpE chaperones leads to the activation of DNA replication of the broad-host-range plasmid RK2. In contrast, ClpB is not needed for the activation of the oriC-dependent replication of E. coli. Using purified protein components we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the plasmid RK2 replication initiation protein TrfA by converting inactive dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1, the corresponding protein system from yeast mitochondria, cannot activate the TrfA replication protein. Our results demonstrate for the first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein monomerization and in the activation of a DNA replication factor.     

Successive and synergistic action of the Hsp70 and Hsp100 chaperones in protein disaggregation

Proteins belonging to the B-subtype of the Hsp100/Clp chaperone family execute a crucial role in cellular thermotolerance. They cooperate with the Hsp70 chaperones in reactivation of thermally aggregated protein substrates. We investigated the initial events of the disaggregation reaction in real time using denatured, aggregated green fluorescent protein (GFP) as a substrate. Bacterial Hsp70 (DnaK), its co-chaperones (DnaJ and GrpE), and Hsp100 (ClpB) were incubated with aggregated GFP, and the increase in GFP fluorescence was monitored. Incubation of aggregated GFP with DnaK/DnaJ/GrpE but not with ClpB resulted in the rapid initiation of the disaggregation reaction. Under the same conditions a complex between DnaK, DnaJ, and GFP, but not ClpB, was formed as demonstrated by sedimentation analysis and light scattering experiments. Chaperone-dependent disaggregation of chemically denatured aggregated luciferase showed that, similar to GFP disaggregation, incubation with Hsp70 results in the rapid start of the reactivation reaction. For both aggregated GFP and luciferase, incubation with Hsp70 chaperones changes the initial rate but not the overall efficiency or rate of the refolding reaction. Our results clearly demonstrate that the interaction of DnaK and its co-chaperones with aggregated substrate is the rate-limiting reaction at the initial steps of disaggregation.     

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.     

Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells

The molecular chaperone protein Hsp78, a member of the Clp/Hsp100 family localized in the mitochondria of Saccharomyces cerevisiae, is required for maintenance of mitochondrial functions under heat stress. To characterize the biochemical mechanisms of Hsp78 function, Hsp78 was purified to homogeneity and its role in the reactivation of chemically and heat-denatured substrate protein was analyzed in vitro. Hsp78 alone was not able to mediate reactivation of firefly luciferase. Rather, efficient refolding was dependent on the simultaneous presence of Hsp78 and the mitochondrial Hsp70 machinery, composed of Ssc1p/Mdj1p/Mge1p. Bacterial DnaK/DnaJ/GrpE, which cooperates with the Hsp78 homolog, ClpB in Escherichia coli, could not substitute for the mitochondrial Hsp70 system. However, efficient Hsp78-dependent refolding of luciferase was observed if DnaK was replaced by Ssc1p in these experiments, suggesting a specific functional interaction of both chaperone proteins. These findings establish the cooperation of Hsp78 with the Hsp70 machinery in the refolding of heat-inactivated proteins and demonstrate a conserved mode of action of ClpB homologs.     

Tuning of DnaK chaperone action by nonnative protein sensor DnaJ and thermosensor GrpE

DnaK, an Hsp70 molecular chaperone, processes its substrates in an ATP-driven cycle, which is controlled by the co-chaperones DnaJ and GrpE. The kinetic analysis of substrate binding and release has as yet been limited to fluorescence-labeled peptides. Here, we report a comprehensive kinetic analysis of the chaperone action with protein substrates. The kinetic partitioning of the (ATP x DnaK) x substrate complexes between dissociation and conversion into stable (ADP x DnaK) x substrate complexes is determined by DnaJ. In the case of substrates that allow the formation of ternary (ATP x DnaK) x substrate x DnaJ complexes, the cis-effect of DnaJ markedly accelerates ATP hydrolysis. This triage mechanism efficiently selects from the (ATP x DnaK) x substrate complexes those to be processed in the chaperone cycle; at 45 degrees C, the fraction of protein complexes fed into the cycle is 20 times higher than that of peptide complexes. The thermosensor effect of the ADP/ATP exchange factor GrpE retards the release of substrate from the cycle at higher temperatures; the fraction of total DnaK in stable (ADP x DnaK) x substrate complexes is 2 times higher at 45 degrees C than at 25 degrees C. Monitoring the cellular situation by DnaJ as nonnative protein sensor and GrpE as thermosensor thus directly adapts the operational mode of the DnaK system to heat shock conditions.     

The importance of having thermosensor control in the DnaK chaperone system

In addition to the sigma(32)-mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADP-liganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40 degrees C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.     

Role of Escherichia coli molecular chaperones in the protection of bacterial cells against irreversible aggregation induced by heat shock

All living organisms respond to environmental stresses, such as heat or ethanol by increasing the synthesis of a specific group of proteins termed heat shock proteins (Hsps) or stress proteins. Major Hsps are molecular chaperones and proteases. Molecular chaperones facilitate the proper folding of polypeptides, protect other proteins from inactivation, and reactivate aggregated proteins. Heat shock proteases eliminate proteins irreversibly damaged by stress. This review describes the role of heat shock proteins of the model bacterial cell, E. coli in the protection of other proteins against aggregation and in the mechanism of removal of protein aggregates from the cell. This mechanism remains unclear and it is believed to involve substrate renaturation and proteolysis by molecular chaperones and heat shock proteases. Recently, many studies have been focused on the disaggregation and reactivation of proteins by a bi-chaperone system consisting of DnaK/DnaJ/GrpE and ClpB, an ATPase from the AAA superfamily of proteins.     

Chloroplastic isoforms of DnaJ and GrpE in pea

The folding and assembly of proteins in cells often requires the assistance of molecular chaperones such as the Hsp70 and Hsp60 heat shock proteins. Hsp70 chaperones cooperate with DnaJ and GrpE homologues to ensure a productive folding cycle. In this study we describe the gene for the first chloroplast localized DnaJ homologue and present evidence that the gene product is at least partially associated with the inner envelope membrane. Immunoblot analysis also provides evidence for the presence of a GrpE homologue in plastids.     

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.     

Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation

Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.     

Heat shock protein-mediated disassembly of nucleoprotein structures is required for the initiation of bacteriophage lambda DNA replication

Three Escherichia coli heat shock proteins, DnaJ, DnaK, and GrpE, are required for replication of the bacteriophage lambda chromosome in vivo. We show that the GrpE heat shock protein is not required for initiation of lambda DNA replication in vitro when the concentration of DnaK is sufficiently high. GrpE does, however, greatly potentiate the action of DnaK in the initiation process when the DnaK concentration is reduced to a subsaturating level. We demonstrate in the accompanying articles (Alfano, C. and McMacken, R. (1989) J. Biol. Chem. 264, 10699-10708; Dodson, M., McMacken, R., and Echols, H. (1989) J. Biol. Chem. 264, 10719-10725) that DnaJ and DnaK bind to prepriming nucleoprotein structures that are assembled at the lambda replication origin (ori lambda). Binding of DnaJ and DnaK completes the ordered assembly of an ori lambda initiation complex that also contains the lambda O and P initiators and the E. coli DnaB helicase. With the addition of ATP, the DnaJ and DnaK heat shock proteins mediate the partial disassembly of the initiation complex, and the P and DnaJ proteins are largely removed from the template. Concomitantly, on supercoiled ori lambda plasmid templates, the intrinsic helicase activity of DnaB is activated and DnaB initiates localized unwinding of the DNA duplex, thereby preparing the template for priming and DNA chain elongation. We infer from our results that DnaK and DnaJ function in normal E. coli metabolism to promote ATP-dependent protein unfolding and disassembly reactions. We also provide evidence that neither the lambda O and P initiators nor the E. coli DnaJ and DnaK heat shock proteins play a direct role in the propagation of lambda replication forks in vitro.     

A small heat shock protein cooperates with heat shock protein 70 systems to reactivate a heat-denatured protein

Small heat shock proteins (sHsps) are a diverse group of heat-induced proteins that are conserved in prokaryotes and eukaryotes and are especially abundant in plants. Recent in vitro data indicate that sHsps act as molecular chaperones to prevent thermal aggregation of proteins by binding non-native intermediates, which can then be refolded in an ATP-dependent fashion by other chaperones. We used heat-denatured firefly luciferase (Luc) bound to pea (Pisum sativum) Hsp18.1 as a model to define the minimum chaperone system required for refolding of a sHsp-bound substrate. Heat-denatured Luc bound to Hsp18.1 was effectively refolded either with Hsc/Hsp70 from diverse eukaryotes plus the DnaJ homologs Hdj1 and Ydj1 (maximum = 97% Luc reactivation with k(ob) = 1.0 x 10(-2)/min), or with prokaryotic Escherichia coli DnaK plus DnaJ and GrpE (100% Luc reactivation, k(ob) = 11.3 x 10(-2)/min). Furthermore, we show that Hsp18.1 is more effective in preventing Luc thermal aggregation than the Hsc70 or DnaK systems, and that Hsp18.1 enhances the yields of refolded Luc even when other chaperones are present during heat inactivation. These findings integrate the aggregation-preventive activity of sHsps with the protein-folding activity of the Hsp70 system and define an in vitro system for further investigation of the mechanism of sHsp action.