Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides

A large fraction of the newly translated polypeptides emerging from the ribosome require certain proteins, the so-called molecular chaperones, to assist in their folding. In Escherichia coli, three major chaperone systems are considered to contribute to the folding of newly synthesized cytosolic polypeptides. Trigger factor (TF), a ribosome-tethered chaperone, and DnaK are known to exhibit overlapping co-translational roles, whereas the cage-shaped GroEL, with the aid of the co-chaperonin, GroES, and ATP, is believed to be implicated in folding only after the polypeptides are released from the ribosome. However, the recent finding that GroEL-GroES overproduction permits the growth of E. coli cells lacking both TF and DnaK raised questions regarding the separate roles of these chaperones. Here, we report the puromycin-sensitive association of GroEL-GroES with translating ribosomes in vivo. Further experiments in vitro, using a reconstituted cell-free translation system, clearly demonstrate that GroEL associates with the translation complex and accomplishes proper folding by encapsulating the newly translated polypeptides in the central cavity formed by GroES. Therefore, we propose that GroEL is a versatile chaperone, which participates in the folding pathway co-translationally and also achieves correct folding post-translationally.     

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.     

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.     

Hsp33 Controls Elongation Factor-Tu Stability and Allows Escherichia coli Growth in the Absence of the Major DnaK and Trigger Factor Chaperones

Intracellular de novo protein folding is assisted by cellular networks of molecular chaperones. In Escherichia coli, cooperation between the chaperones trigger factor (TF) and DnaK is central to this process. Accordingly, the simultaneous deletion of both chaperone-encoding genes leads to severe growth and protein folding defects. Herein, we took advantage of such defective phenotypes to further elucidate the interactions of chaperone networks in vivo. We show that disruption of the TF/DnaK chaperone pathway is efficiently rescued by overexpression of the redox-regulated chaperone Hsp33. Consistent with this observation, the deletion of hslO, the Hsp33 structural gene, is no longer tolerated in the absence of the TF/DnaK pathway. However, in contrast with other chaperones like GroEL or SecB, suppression by Hsp33 was not attributed to its potential overlapping general chaperone function(s). Instead, we show that overexpressed Hsp33 specifically binds to elongation factor-Tu (EF-Tu) and targets it for degradation by the protease Lon. This synergistic action of Hsp33 and Lon was responsible for the rescue of bacterial growth in the absence of TF and DnaK, by presumably restoring the coupling between translation and the downstream folding capacity of the cell. In support of this hypothesis, we show that overexpression of the stress-responsive toxin HipA, which inhibits EF-Tu, also rescues bacterial growth and protein folding in the absence of TF and DnaK. The relevance for such a convergence of networks of chaperones and proteases acting directly on EF-Tu to modulate the intracellular rate of protein synthesis in response to protein aggregation is discussed.     

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.     

Chemical chaperones regulate molecular chaperones in vitro and in cells under combined salt and heat stresses

Salt and heat stresses, which are often combined in nature, induce complementing defense mechanisms. Organisms adapt to high external salinity by accumulating small organic compounds known as osmolytes, which equilibrate cellular osmotic pressure. Osmolytes can also act as "chemical chaperones" by increasing the stability of native proteins and assisting refolding of unfolded polypeptides. Adaptation to heat stress depends on the expression of heat-shock proteins, many of which are molecular chaperones, that prevent protein aggregation, disassemble protein aggregates, and assist protein refolding. We show here that Escherichia coli cells preadapted to high salinity contain increased levels of glycine betaine that prevent protein aggregation under thermal stress. After heat shock, the aggregated proteins, which escaped protection, were disaggregated in salt-adapted cells as efficiently as in low salt. Here we address the effects of four common osmolytes on chaperone activity in vitro. Systematic dose responses of glycine betaine, glycerol, proline, and trehalose revealed a regulatory effect on the folding activities of individual and combinations of chaperones GroEL, DnaK, and ClpB. With the exception of trehalose, low physiological concentrations of proline, glycerol, and especially glycine betaine activated the molecular chaperones, likely by assisting local folding in chaperone-bound polypeptides and stabilizing the native end product of the reaction. High osmolyte concentrations, especially trehalose, strongly inhibited DnaK-dependent chaperone networks, such as DnaK+GroEL and DnaK+ClpB, likely because high viscosity affects dynamic interactions between chaperones and folding substrates and stabilizes protein aggregates. Thus, during combined salt and heat stresses, cells can specifically control protein stability and chaperone-mediated disaggregation and refolding by modulating the intracellular levels of different osmolytes.     

Ribosome-based protein folding systems are structurally divergent but functionally universal across biological kingdoms

In bacteria, Trigger factor (TF) is the first chaperone that interacts with nascent polypeptides as soon as they emerge from the exit tunnel of the ribosome. TF binds to the ribosomal protein L23 located next to the tunnel exit of the large subunit, with which it forms a cradle-like space embracing the polypeptide exit region. It cooperates with the DnaK Hsp70 chaperone system to ensure correct folding of a number of newly translated cytosolic proteins in Escherichia coli. Whereas TF is exclusively found in prokaryotes and chloroplasts, Saccharomyces cerevisiae, a eukaryotic microorganism, has a three-member Hsp70-J protein complex, Ssb-Ssz-Zuo, which could act as a ribosome-associated folding facilitator. In the work reported in this volume of Molecular Microbiology, Rauch et al. (2005, Mol Microbiol, doi:10.1111/j.1365-2958.2005.04690.x) examined the functional similarity of the ribosome-associated chaperones in prokaryotes and eukaryotes. In spite of the fact that TF and the Hsp70-based triad are structurally unrelated, TF can bind to the yeast ribosome via Rpl25 (the L23 counterpart) and can substitute for some, but not all, of the functions assigned to Ssb-Ssz-Zuo in yeast. The functional conservation of the ribosome-associated chaperones without structural similarity is remarkable and suggests that during evolution nature has employed a common design but divergent components to facilitate folding of polypeptides as they emerge from the ribosomal exit, a fundamental process required for the efficient expression of genetic information.     

Chaperone networking facilitates protein targeting to the bacterial cytoplasmic membrane

Nascent polypeptides emerging from the ribosome are assisted by a pool of molecular chaperones and targeting factors, which enable them to efficiently partition as cytosolic, integral membrane or exported proteins. Extensive genetic and biochemical analyses have significantly expanded our knowledge of chaperone tasking throughout this process. In bacteria, it is known that the folding of newly-synthesized cytosolic proteins is mainly orchestrated by three highly conserved molecular chaperones, namely Trigger Factor (TF), DnaK (HSP70) and GroEL (HSP60). Yet, it has been reported that these major chaperones are strongly involved in protein translocation pathways as well. This review describes such essential molecular chaperone functions, with emphasis on both the biogenesis of inner membrane proteins and the post-translational targeting of presecretory proteins to the Sec and the twin-arginine translocation (Tat) pathways. Critical interplay between TF, DnaK, GroEL and other molecular chaperones and targeting factors, including SecB, SecA, the signal recognition particle (SRP) and the redox enzyme maturation proteins (REMPs) is also discussed. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Trigger Factor and DnaK possess overlapping substrate pools and binding specificities

Ribosome-associated Trigger Factor (TF) and the DnaK chaperone system assist the folding of newly synthesized proteins in Escherichia coli. Here, we show that DnaK and TF share a common substrate pool in vivo. In TF-deficient cells, deltatig, depleted for DnaK and DnaJ the amount of aggregated proteins increases with increasing temperature, amounting to 10% of total soluble protein (approximately 340 protein species) at 37 degrees C. A similar population of proteins aggregated in DnaK depleted tig+ cells, albeit to a much lower extent. Ninety-four aggregated proteins isolated from DnaK- and DnaJ-depleted deltatig cells were identified by mass spectrometry and found to include essential cytosolic proteins. Four potential in vivo substrates were screened for chaperone binding sites using peptide libraries. Although TF and DnaK recognize different binding motifs, 77% of TF binding peptides also associated with DnaK. In the case of the nascent polypeptides TF and DnaK competed for binding, however, with competitive advantage for TF. In vivo, the loss of TF is compensated by the induction of the heat shock response and thus enhanced levels of DnaK. In summary, our results demonstrate that the co-operation of the two mechanistically distinct chaperones in protein folding is based on their overlap in substrate specificities.     

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.     

Trigger factor lacking the PPIase domain can enhance the folding of eukaryotic multi-domain proteins in Escherichia coli

Recombinant expression of eukaryotic proteins in bacteria often results in misfolding and aggregation. The ribosome-binding Trigger factor (TF) is the first molecular chaperone that interacts with nascent polypeptide chains in bacteria. Here we show that mutant TF lacking the PPIase domain (TFNC) is more efficient than wild-type TF in enhancing the folding yield of multi-domain proteins such as firefly luciferase. We find that TFNC has a shorter residence time on nascent chains, thus facilitating co-translational folding. By delaying folding relative to translation, the PPIase domain may increase the propensity of misfolding for certain eukaryotic proteins that rely on a mechanism of co-translational, domain-wise folding.     

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.     

Severe oxidative stress causes inactivation of DnaK and activation of the redox-regulated chaperone Hsp33

DnaK/DnaJ/GrpE constitutes the primary chaperone machinery in E. coli that functions to protect proteins against heat-induced protein aggregation. Surprisingly, upon exposure of cells to reactive oxygen species at elevated temperature, proteins are no longer protected by the DnaK system. Instead, they bind now to the redox-regulated chaperone Hsp33, which is activated by the same conditions that inactivate DnaK. The inactivation of DnaK seems to be induced by the dramatic decrease in intracellular ATP levels that occurs upon exposure of cells to reactive oxygen species. This appears to render DnaK's N-terminal ATPase domain nucleotide depleted and thermolabile. DnaK's N terminus reversibly unfolds in vivo, and DnaK loses its ability to protect proteins against stress-induced aggregation. Now, the ATP-independent chaperone holdase Hsp33 binds to a large number of cellular proteins and prevents their irreversible aggregation. Upon return to nonstress conditions, Hsp33 becomes inactivated while DnaK reactivates and resumes its task to support protein folding.     

The thioredoxin homolog YbbN functions as a chaperone rather than as an oxidoreductase

Escherichia coli contains two thioredoxins, Trx1 and Trx2, and a thioredoxin-like protein, YbbN, which presents a strong homology in its N-terminal part with thioredoxins, and possesses a 20 kDa C-terminal part of unknown function. We reported previously that YbbN displays both protein oxido-reductase and chaperone properties in vitro. In this study, we show that an ybbN-deficient strain displays an increased sensitivity to thermal stress but not to oxidative stress, a normal redox state of its cellular proteins but a decreased expression of several cytoplasmic proteins, including EF-Tu, DnaK, GroEL, trigger factor and several Krebs cycle enzymes, suggesting that the chaperone properties of YbbN are more important in vivo than its redox properties. YbbN specifically interacts with DnaK and GroEL, as shown by reverse purification. It increases 4-fold the rate of protein renaturation in vitro by the DnaK chaperone machine, suggesting that it cooperates with DnaK for the optimal expression of several cytoplasmic proteins.     

GroEL chaperone binding to beetle luciferases and the implications for refolding when co-expressed

The folding of many proteins including luciferase in vivo requires the assistance of molecular chaperone proteins. To understand how a chaperone targets luciferase, we took three luciferases that give different bioluminescence with the same luciferin substrate and with differences in homology. The three luciferase genes, firefly luciferase (FF-Luc) (from Pyrocoelia miyako), and red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), were expressed in Escherichia coli to produce fusion proteins with predicted molecular masses. Subsequently, we observed that DnaK and GroEL were co-purified along with recombinant luciferase. Although the amount of co-purified DnaK was almost the same compared to FF-Luc, GroEL was 25 and 32 times higher in GR-Luc and RE-Luc respectively. Furthermore, co-expression of GroEL/GroES along with luciferase substantially refolded RE-Luc and GR-Luc compared to FF-Luc.     

Improvement of productivity of active form of glutamate racemase in Escherichia coli by coexpression of folding accessory proteins

Since two classes of folding accessory proteins, molecular chaperones and foldases, prevent the misfolding of newly synthesized polypeptides in the cell, their coexpression could be expected to improve the productivity of soluble and active recombinant proteins. Escherichia coli cytoplasmic glutamate racemase (GluR), which has five cysteine thiol groups and no disulfide bond, was selected as a model enzyme and overexpressed in E. coli. The effects of coexpressing a series of folding accessory proteins (DnaK, DnaJ, GrpE, GroEL/ES, trigger factor (TF), DsbA, DsbB, DsbC, DsbD, and thioredoxin (Trx)) on the productivity of active GluR in E. coli were examined. A relatively large amount of active GluR produced by mild induction with 10 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Active GluR productivity was further increased 2.2–2.3-fold by coexpression of GroEL/ES, Trx, or DsbB–DsbD (DsbBD), while it was decreased by coexpression of DnaK–DnaJ–GrpE and TF. These results demonstrate that coexpression of appropriate folding accessory proteins could significantly improve the productivity of active form of proteins in E. coli.     

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

Size-dependent disaggregation of stable protein aggregates by the DnaK chaperone machinery

Classic in vitro studies show that the Hsp70 chaperone system from Escherichia coli (DnaK-DnaJ-GrpE, the DnaK system) can bind to proteins, prevent aggregation, and promote the correct refolding of chaperone-bound polypeptides into native proteins. However, little is known about how the DnaK system handles proteins that have already aggregated. In this study, glucose-6-phosphate dehydrogenase was used as a model system to generate stable populations of protein aggregates comprising controlled ranges of particle sizes. The DnaK system recognized the glucose-6-phosphate dehydrogenase aggregates as authentic substrates and specifically solubilized and refolded the protein into a native enzyme. The efficiency of disaggregation by the DnaK system was high with small aggregates, but the efficiency decreased as the size of the aggregates increased. High folding efficiency was restored by either excess DnaK or substoichiometric amounts of the chaperone ClpB. We suggest a mechanism whereby the DnaK system can readily solubilize small aggregates and refold them into active proteins. With large aggregates, however, the binding sites for the DnaK system had to be dynamically exposed with excess DnaK or the catalytic action of ClpB and ATP. Disaggregation by the DnaK machinery in the cell can solubilize early aggregates that formed accidentally during chaperone-assisted protein folding or that escaped the protection of "holding" chaperones during stress.     

In vivo analysis of the overlapping functions of DnaK and trigger factor

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.