From the Biochimie Génétique, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75005 Paris, France
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ABSTRACT |
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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 the Escherichia 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 -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
-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.
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INTRODUCTION |
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EF-Tu1 is responsible
for binding and transporting the appropriate codon-specified
aminoacyl-tRNA to the aminoacyl (A) site of the ribosome (1, 2). In
this role, EF-Tu interacts with GTP, aminoacyl-tRNA, ribosomes, and a
second protein factor, EF-Ts, which mediates GDP/GTP exchange on EF-Tu.
Peptide bonds are formed by transfer of the nascent peptide from its
tRNA at the peptidyl site to the -amino group of the newly bound
aminoacyl-tRNA at the aminoacyl site, and elongation factor EF-G allows
translocation of ribosomes on the mRNA (1, 2). There is a high
level of EF-Tu in Escherichia coli cells, comprising 5-10%
total cell protein, in vast molar excess over the other essential
protein components of the translation machinery (3). Several sets of
results suggest that EF-Tu and its eukaryotic counterpart EF1
may
have other functions in addition to the conventional role that they
play in polypeptide elongation. EF-Tu is an essential host-donated subunit of the replicative complex of Q
phage (4), and it may
interact with the transcriptional apparatus as a positive regulator of
RNA synthesis (5). EF-Tu is associated in part with the plasma membrane
(6) and is methylated in response to nutrient deprivation (7). EF-1
binds to actin filaments and microtubules both in vitro and
in vivo (8) and influences the assembly and stability of
cytoskeletal polymers (9) in a manner that is reminiscent of the
ability of chaperones to control the state of aggregation of multimeric
proteins (10-12).
Molecular chaperones form a class of polypeptide-binding proteins that are implicated in protein folding, protein targeting to membranes, protein renaturation or degradation after stress, and the control of protein-protein interactions. They can distinguish native proteins from their non-native forms, owing to the specificity of their peptide binding site, and they catalyze protein folding and renaturation in vitro (reviewed in Refs. 10-12). The major classes of bacterial chaperones comprise DnaK/Hsp70 (and its assistants DnaJ and GrpE), GroEL/Hsp60 (and its assistant GroES), HtpG/Hsp90, and the small heat shock proteins (10-12). In the present study, we show that elongation factor EF-Tu, in a manner similar to that of molecular chaperones, increases the refolding of unfolded proteins, protects proteins against thermal denaturation, and forms complexes with unfolded proteins. We propose that, in addition to its role in translational elongation, this overabundant protein might help in protein folding and renaturation in the cytoplasm.
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EXPERIMENTAL PROCEDURES |
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Materials--
Citrate synthase (from porcine heart),
-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 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 mM dithiothreitol, 10% glycerol; elution with a linear 0-0.35 M NaCl gradient in the same buffer) followed by covalent chromatography on thiol-Sepharose (column buffer: 20 mM potassium 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 mM dithiothreitol, 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
-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 M
urea, 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 mM
potassium acetate, pH 8.0. The enzymatic activity of citrate synthase
was measured as described (18).
-Glucosidase was denatured at a
concentration of 1.5 µM in 8 M urea, 0.1 M 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
-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 mM potassium 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 were 3H-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 -glucosidase
renaturation buffers supplemented with 200 µM
MgCl2. 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
-glucosidase
renaturation buffers supplemented with 200 µM
MgCl2 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
-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).
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RESULTS |
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EF-Tu Increases the Amount of Correctly Folded Citrate Synthase and
-Glucosidase--
We first investigated whether EF-Tu acts as
molecular chaperones in the folding of proteins. Citrate synthase and
-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 8 M 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 µM
citrate 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.
1A). The dependence of citrate
synthase reactivation on the concentration of EF-Tu is shown in Fig.
1B. 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.
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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.
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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. 4A). 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.
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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 -glucosidase. As
shown in Fig. 5, EF-Tu·GDP stimulated
renaturation of citrate synthase and
-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.
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DISCUSSION |
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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
-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 and 25-27). 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 proteins in 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 relative thermo 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-1 (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 1
(46).
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ACKNOWLEDGEMENT |
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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.
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FOOTNOTES |
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* This work was supported in part by CNRS Grant 96N88/0006 "Physique et Chimie du Vivant" (to G. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported successively by a Leonardo de Vinci grant and by
Ministerio de Ciência e Tecnologia of Portugal Grant
PRAXIS/BD/13898/97.
§ Present address: Dept. de Biologie, Faculté des Sciences, Université Mohamed-1, Oujda, Morocco.
¶ To whom correspondence should be addressed. Tel.: 33 01 44 27 50 98; Fax: 33 01 44 27 35 80; E-mail: richarme{at}ccr.jussieu.fr.
1
The abbreviations used are: EF-Tu, elongation
factor Tu; R-CMLA, reduced carboxymethyl -lactalbumin; BPTI, bovine
pancreatic trypsin inhibitor; Hsp, heat shock protein.
2 T. D. Caldas, A. El Yaagoubi, and G. Richarme submitted for publication.
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REFERENCES |
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