Elongation factor Tu (EF-Tu) is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We report herewith that theEscherichia coli EF-Tu interacts with unfolded and denatured proteins as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-Tu promotes the functional folding of citrate synthase and α-glucosidase after urea denaturation. It prevents the aggregation of citrate synthase under heat shock conditions, and it forms stable complexes with several unfolded proteins such as reduced carboxymethyl α-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. The EF-Tu·GDP complex is much more active than EF-Tu·GTP in stimulating protein renaturation. These chaperone-like functions of EF-Tu occur at concentrations that are at least 20-fold lower than the cellular concentration of this factor. These results suggest that EF-Tu, in addition to its function in translation elongation, might be implicated in protein folding and protection from stress.

# EXPERIMENTAL PROCEDURES

### Materials

Citrate synthase (from porcine heart), α-glucosidase (from yeast), BPTI, R-CMLA, bovine serum albumin, ovalbumin, lysozyme, kirromycin, and all other chemicals were from Sigma and were reagent grade. DEAE-Sephacel and thiol-Sepharose resins were from Amersham Pharmacia Biotech, and hydroxylapatite (Bio-Gel HTP) was from Bio-Rad.

### Purification of EF-Tu and DnaK

EF-Tu was purified by covalent chromatography on thiol-Sepharose.2
                              
                                 2
                                 T. D. Caldas, A. El Yaagoubi, and G. Richarme submitted for publication.
                               Crude extracts from the E. coli K12 strain C600 (leuB6 thi-1thr-1 supE44) were prepared by a lysozyme-EDTA method (13). EF-Tu was purified by DEAE-Sephacel chromatography (column buffer: 20 mm Tris, pH 8.0, 0.2 mm EDTA, 1 mmdithiothreitol, 10% glycerol; elution with a linear 0–0.35m NaCl gradient in the same buffer) followed by covalent chromatography on thiol-Sepharose (column buffer: 20 mmpotassium phosphate, 1 mm EDTA, pH 7.0; elution with a gradient of 0–10 mm cysteine in the same buffer) and hydroxylapatite chromatography (column buffer: 20 mm Tris, pH 7.4, 1 mm dithiothreitol, 10% glycerol, elution with a linear 0–0.1 m gradient of potassium phosphate in the same buffer). The purified protein was dialyzed for 3 h against 20 mm Tris, pH 8.0, 50 mm KCl, 1 mmdithiothreitol, 10% glycerol and concentrated by ultrafiltration. Its 280/260-nm absorbancy was 1.25, indicating that it contains 0.9 equivalent of bound GDP (2). EF-Tu was more than 98% pure as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Unless otherwise indicated, we used this preparation of EF-Tu. DnaK was prepared as described previously (14-16) from an overproducing strain of E. coli-bearing plasmid pLNA2 derived from plasmid pDM38 (17) (a gift from Dr. O. Fayet, Microbiologie et Génétique Microbienne CNRS, Toulouse, France).

### Refolding of Citrate Synthase and α-Glucosidase

Denaturation and renaturation reactions were carried out at 20 °C. For both proteins, renaturation was initiated by pouring the renaturation solvent on the unfolded protein under vortex agitation in Eppendorf polyethylene tubes. Citrate synthase was denatured at a concentration of 10 μm in 8 murea, 50 mm Tris-HCl, 2 mm EDTA, 20 mm dithiothreitol, pH 8.0, for 50 min. Renaturation was initiated by a 100-fold dilution in 40 mm Hepes, 50 mm KCl, 10 mm(NH4)2SO4, 2 mmpotassium acetate, pH 8.0. The enzymatic activity of citrate synthase was measured as described (18). α-Glucosidase was denatured at a concentration of 1.5 μm in 8 m urea, 0.1m potassium phosphate, 2 mm EDTA, 20 mm dithiothreitol, pH 7.0, for 15 min. Renaturation was initiated by a 30-fold dilution in 40 mm Hepes-KOH, pH 7.8, at 20 °C. The enzymatic activity of α-glucosidase was measured as described (18).

### Thermal Aggregation of Citrate Synthase

The native enzyme (80 μm) was diluted 100-fold in 40 mm Hepes, 50 mm KCl, 10 mm(NH4)2SO4, 2 mmpotassium acetate, pH 8.0, at 43 °C in the absence of added proteins or in the presence of DnaK or EF-Tu supplemented with 10 μm GDP (10 μm GDP was added in order to increase the thermal stability of EF-Tu (19) and did not modify citrate synthase aggregation). Citrate synthase aggregation was monitored by measuring the absorbance at 650 nm as described in (18).

### Size Exclusion Chromatography

For binding assays of R-CMLA and unfolded BPTI to EF-Tu and DnaK, gel permeation columns (Bio-Gel P-200 from Bio-Rad for studies with R-CMLA or Sephadex G-75 from Amersham for studies with BPTI, 300 μl bed volume each) were equilibrated with column buffer containing 50 mm Tris-HCl (pH 8.2 for studies with R-CMLA and pH 7.4 for studies with BPTI), 50 mm KCl, 1 mm dithiothreitol, 100 μg/ml bovine serum albumin. Reaction mixtures containing EF-Tu or DnaK and radiolabeled unfolded BPTI or radiolabeled R-CMLA at indicated concentrations were incubated for 20 min at 23 °C in column buffer without serum albumin and applied to the column at room temperature. Fractions were collected at a flow rate of 1 drop/fraction/30 s and counted for radioactivity. DnaK was incubated for 3 h at 37 °C before use. Unfolded BPTI was prepared as described previously from native BPTI (20). Unfolded BPTI, native BPTI, and R-CMLA were3H-labeled by reductive methylation (21).

### Preparation of EF-Tu·GDP and EF-Tu·GTP

EF-Tu·GDP was formed by incubating EF-Tu (1–10 μm) in the presence of 20 μm GDP in citrate synthase or α-glucosidase renaturation buffers supplemented with 200 μmMgCl2. Because EF-Tu binds GDP much more tightly than GTP and since GDP always contaminates GTP preparations, EF-Tu·GTP was formed by incubating EF-Tu (1–10 μm) in the presence of 20 μm GTP, 4 mm phosphoenolpyruvate, and 10 μg of pyruvate kinase in citrate synthase or α-glucosidase renaturation buffers supplemented with 200 μmMgCl2 so as to recycle GDP into GTP (22). GDP, phosphoenolpyruvate or pyruvate kinase had no effect on substrate-protein renaturation. GTP had no effect on α-glucosidase renaturation but stimulated 1.7-fold citrate synthase renaturation, probably as an allosteric ligand of citrate synthase (23).

### EF-Tu GTPase Assay

The assay was performed as described (24) in the presence of 10 μm kirromycin, except that GDP was separated from GTP by chromatography on polyethyleneimine-cellulose (20). 14C-Labeled GTP was from Amersham and was used at 1 Ci/mmol. The specific activities of EF-Tu in the absence and in the presence of kirromycin were similar to those reported by Wolf et al. (24).

# RESULTS

### EF-Tu Increases the Amount of Correctly Folded Citrate Synthase and α-Glucosidase

We first investigated whether EF-Tu acts as molecular chaperones in the folding of proteins. Citrate synthase and α-glucosidase, whose refolding is facilitated by several chaperones such as GroEL, DnaK, Hsp90, and small Hsps (18, 25-27) were chosen as substrates for this reaction. They were unfolded in the presence of 8m urea and allowed to refold upon dilution of the denaturant in the absence or in the presence of EF-Tu (protein folding in the presence of DnaK was studied in parallel). Under our experimental conditions, the refolding yield of 0.1 μmcitrate synthase was increased from 8% in the absence of added proteins to 30% in the presence of 3 μm EF-Tu and 33% in the presence of 3 μm DnaK (Fig.1
                               
                              A). The dependence of citrate synthase reactivation on the concentration of EF-Tu is shown in Fig.1 
                              B. The maximal recovery of citrate synthase activity attains 32% in the presence of 10 μm EF-Tu, and half-maximal reactivation occurs at 1 μm EF-Tu, a concentration similar to the concentration of DnaK (1 μm) required for half-maximal reactivation of citrate synthase in similar conditions (not shown and Ref. 27). The EF-Tu concentration required for half-maximal reactivation of citrate synthase is somewhat higher than the concentration of citrate synthase but is impressively lower than the concentrations of EF-Tu in the cytoplasm (around 100 μm (28)). As reported previously (27), other proteins such as ovalbumin and lysozyme were unable to stimulate citrate renaturation, whereas serum albumin could stimulate it to some extent.

As shown in Fig. 2
                              , the refolding of 0.05 μm α-glucosidase was increased from 8% in the absence of added protein to 24% in the presence of EF-Tu, and half-maximal reactivation of α-glucosidase occurs at 0.4 μm EF-Tu. In similar conditions, the refolding of α-glucosidase was 27% in the presence of 2 μm DnaK, and half-maximal reactivation of α-glucosidase occurred at 0.2 μm DnaK (data not shown). These results suggest that, like molecular chaperones, EF-Tu interacts with unfolded proteins, and increases their productive folding.

### EF-Tu Protects Citrate Synthase from Irreversible Aggregation during Thermal Stress

We investigated the function of EF-Tu under heat shock conditions. As reported previously (18, 25, 27), citrate synthase loses its native conformation and undergoes aggregation during incubation at 43 °C. The addition of Ef-Tu (2 μm) or DnaK (2 μm) partially reduces citrate synthase (0.8 μm) aggregation, whereas both 5 μm DnaK and 5 μm EF-Tu suppress citrate synthase aggregation (Fig.3
                              ). In contrast, the addition of up to 35 μm bovine serum albumin, ovalbumin, or lysozyme (not shown and Ref. 27) does not protect citrate synthase against thermal aggregation. Thus, EF-Tu is nearly as efficient as DnaK and other chaperones (18, 25) in protecting citrate synthase from thermal denaturation and is much more efficient than other proteins such as bovine serum albumin, ovalbumin, or lysozyme. Furthermore, the concentration of EF-Tu required for an efficient thermal protection of citrate synthase (around 5 μm) is approximately 20-fold lower than its cellular concentration.

### Interaction between EF-Tu and Unfolded Proteins

One characteristic of molecular chaperones is their preferential interaction with unfolded proteins (10-12). R-CMLA, a permanently unfolded protein that maintains an extended conformation without any stable secondary structure in the absence of denaturant, strongly interacts with several chaperones, including DnaK (27, 29). Complex formation between R-CMLA (20,000 Da) and EF-Tu (44,000 Da) was analyzed by gel filtration on a Bio-Gel P-200 column. When R-CMLA (2 μm) is filtered in the presence of EF-Tu (4 μm), 27% of R-CMLA fractionates as a high molecular weight complex as compared with R-CMLA alone (Fig.4
                               
                              A). In the presence of 2 μm EF-Tu this amount is 21%. The interaction between EF-Tu and R-CMLA is not significantly different from that observed between R-CMLA and DnaK; at similar concentrations, DnaK bound 30–50% of R-CMLA (data not shown and Ref. 27). Thus EF-Tu seems to interact strongly with R-CMLA in a manner similar to that of DnaK.

Unfolded BPTI is known to interact with chaperones, including DnaK (27,30). Complex formation between 0.5 μm unfolded BPTI (6000 Da) and 4 μm EF-Tu (44,000 Da) was studied by gel filtration on a Sephadex G-75 column; a significant percentage (18%) of unfolded BPTI fractionates as higher molecular mass material than unfolded BPTI alone (Fig. 4 
                              B). In similar conditions, 4 μm DnaK retained 34% unfolded BPTI (not shown). In contrast, when 0.5 μm native BPTI and 4 μmEF-Tu were loaded on the gel permeation column, native BPTI did not elute as a high molecular mass complex (not shown). When similar experiments were carried out with bovine serum albumin (30 μm) or ovalbumin (30 μm), unfolded BPTI did not elute as a high molecular mass complex (not shown). Thus, EF-Tu, like molecular chaperones, interacts preferentially with unfolded proteins.

### Chaperone Properties of EF-Tu·GDP and EF-Tu·GTP

During the protein elongation cycle, EF-Tu behaves like a GTPase that promotes the binding or aminoacyl-tRNA to codon-programmed ribosomes. The active GTP-bound form of EF-Tu binds aminoacyl-tRNA. When the ternary aminoacyl-tRNA·EF-Tu·GTP complex interacts with ribosomes in the presence of mRNA, the aminoacyl-tRNA is transferred to the ribosomes, and GTP is hydrolyzed, with the formation of the inactive GDP-bound form of EF-Tu. The latter dissociates from the ribosome and is recycled to EF-Tu·GTP by the nucleotide exchange factor EF-Ts (1-3). We measured the ability of EF-Tu·GDP and EF-Tu·GTP to stimulate the renaturation of citrate synthase and α-glucosidase. As shown in Fig. 5
                              , EF-Tu·GDP stimulated renaturation of citrate synthase and α-glucosidase 3-fold and 2.4-fold, respectively, whereas EF-Tu·GTP had almost no effect on this process. Thus the EF-Tu·GDP form, which is inactive for aminoacyl-tRNA binding, behaves like the active form in unfolded protein renaturation.

We also tested the effects of unfolded proteins on the EF-Tu GTPase activity. The EF-Tu GTPase (which is normally stimulated by ribosomes plus aminoacyl-tRNA) can be stimulated by either of these components alone (5-fold and 2-fold, respectively) when assayed in the presence of the antibiotic kirromycin (24, 31). Unfolded R-CMLA did not trigger any stimulation of the kirromycin-dependent GTPase activity of EF-Tu (data not shown). This result is consistent with a preferential interaction of unfolded proteins with EF-Tu·GDP (and with a probable absence of effect of unfolded proteins on GDP dissociation from EF-Tu).

# DISCUSSION

We present biochemical evidence suggesting that EF-Tu has a chaperone-like function in protein folding, protection against thermal denaturation, and interaction with unfolded proteins. EF-Tu increases approximately 3-fold the yield of active citrate synthase and α-glucosidase renaturation, as do molecular chaperones. The stimulation factors of protein renaturation (more than 3-fold) and EF-Tu concentrations required for half-maximal protein renaturation (O.4–1 μm) are not significantly different from those obtained with DnaK, Hsp90, or small Hsps (this study, and Refs. 18 and25-27). Notably, the EF-Tu concentrations used in this study (in the micromolar range) are at least 50-fold lower than its estimated concentration in the bacterial cytoplasm (around 100 μm(28)). At micromolar concentrations, EF-Tu protects citrate synthase from thermal denaturation. These concentrations are similar to those of DnaK (24) and of small Hsp (expressed as monomers) required for a similar protection (18). They are 20-fold lower than the cytoplasmic concentration of EF-Tu. Furthermore, other proteins tested (bovine serum albumin, ovalbumin, and lysozyme) do not protect citrate synthase efficiently (27). EF-Tu forms stable complexes with unfolded proteins. The EF-Tu·R-CMLA and DnaK·R-CMLA complexes (27) display a similar stability. Like DnaK (27, 30), EF-Tu interacts with unfolded BPTI but not with native BPTI and thus appears to discriminate between unfolded and native proteins.

Our results show that EF-Tu can bind polypeptides. The elongation factor possesses an amino acid binding site that binds the aminoacyl moiety of aminoacyl-tRNAs (1-3, 32-34). Interestingly, aminoacyl-tRNAs with hydrophobic amino acid side chains are bound more strongly than those with polar groups (33, 34), suggesting that this site preferentially interacts with hydrophobic amino acids (32, 33), like chaperones (10-12, 20)). Although not proven by our results, a hydrophobic interaction between EF-Tu and unfolded proteins is likely (since unfolded proteins expose hydrophobic surfaces), and the site discussed above could be partly implicated in such an interaction.

The strong interaction shown in this study between EF-Tu and unfolded proteins suggests that EF-Tu might interact with unfolded proteinsin vivo during protein folding or heat shock. During heat shock, the large quantity of EF-Tu molecules present in the cytoplasm and near the cytoplasmic membrane (6) might constitute a reservoir of chaperone-like molecules that serve as chaperone-buffer in preventing the aggregation of non-native proteins until permissive renaturation conditions are restored. EF-Tu takes its name from its relativethermo unstability. However, its stability in the presence of nucleotide cofactors extends up to 50 °C (19), a temperature higher than current heat shock temperatures of 40–45 °C (EF-Tu is bound to GTP or GDP in vivo). Although our results do not indicate whether EF-Tu interacts with proteins during translation and although EF-Tu has not been identified as one of the nascent chain binding proteins of E. coli (these proteins include DnaJ (35), the signal recognition particle (36, 37), and trigger factor (37, 38)), several results suggest that EF-Tu might interact indeed with nascent chains. (i) Two EF-Tu molecules might be involved per translation cycle, both molecules interacting with a single aminoacyl-tRNA (39-40) or one of them interacting with the aminoacyl-tRNA at the ribosomal A-site, the other interacting with the peptidyl-tRNA at the P-site (41); in both cases, there might exist some interaction between one EF-Tu molecule and the nascent peptidyl chain. (ii) The efficiency of EF-Tu-dependent read-through of stop codons depends on the last two amino acids of the nascent peptide at the ribosomal P-site, suggesting that there is an interaction between EF-Tu and the C-terminal amino acids in the nascent chain (42).

EF-Tu·GDP appears more active in binding unfolded proteins than EF-Tu·GTP. There are important conformational differences between both forms, which can explain the preferential affinity of one of them for unfolded proteins (41). Since our manuscript was submitted, EF-Tu has been shown to catalyze rhodanese renaturation (43). In contrast with our results, EF-Tu·GDP is less active than EF·Tu·GTP in catalyzing rhodanese renaturation. This discrepancy could be explained by a quasi irreversible binding of EF-Tu·GDP to rhodanese, which would prevent its renaturation (such a result has been observed for the interaction between chaperones and substrate proteins (12, 44)). Furthermore, the EF-Tu-dependent renaturation of rhodanese is stimulated by EF-Ts and GTP, suggesting that, in the presence of these cofactors, EF-Tu can perform several rounds of protein renaturation.

Finally, several functions of translational elongation factors are reminiscent of molecular chaperones. The microtubule-severing activity of EF-1α (8, 9) is reminiscent of the ability of Hsc73 to disassemble clathrin cages (45), and the slow growth phenotype of yeast mutants deficients in Hsp70 (ssb1 ssb2 mutants) can be suppressed by the increasing copy number of a gene encoding translation elongation factor 1α (46).

We thank Dr. M. Kohiyama for constant support throughout this work, Dr. O. Fayet (Laboratoire de Microbiologie et Génétique Moléculaire, CNRS, Toulouse, France) for the DnaK/DnaJ hyperproducing strain, Dr. A. Parmeggiani (Ecole Polytechnique, Palaiseau, France) and Dr. A. L. Haenni (Institut J. Monod, Université Paris 7, France) for critical reading of the manuscript, and A. Kropfinger for corrections in the English language.