©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Function of Escherichia coli Chaperone DnaJ
PROTEIN-DISULFIDE ISOMERASE (*)

(Received for publication, June 21, 1995)

Axelle de Crouy-Chanel Masamichi Kohiyama Gilbert Richarme (§)

From the Biochimie génétique, Institut Jacques Monod, Université Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes. The DnaJ protein of Escherichia coli and the DnaJ-like proteins of eukaryotes are known as molecular chaperones and specific regulators of DnaK-like proteins and are involved in protein folding and renaturation after stress. In this study we show that DnaJ, like thioredoxin, protein-disulfide isomerase, and DsbA, possesses an active dithiol/disulfide group and catalyzes protein disulfide formation (oxidative renaturation of reduced RNase), reduction (reduction of insulin disulfides), and isomerization (refolding of randomly oxidized RNase). These results suggest that, in addition to its known function as a chaperone, DnaJ might be involved in controlling the redox state of cytoplasmic, membrane, or exported proteins.


INTRODUCTION

Molecular chaperones, protein-disulfide isomerases, and peptidyl prolyl cis-trans isomerases assist protein folding in both prokaryotes and eukaryotes(1, 2, 3) . Protein disulfide isomerases catalyze dithiol/disulfide interchange reactions and promote protein disulfide formation, isomerization, or reduction, depending on the imposed redox potential and on the nature of the polypeptide substrate (4, 5, 6) . The redox state of the bacterial periplasm and of the eukaryotic endoplasmic reticulum is more oxidized than that of the cytoplasm, and protein disulfides are common in these extracytoplasmic compartments(6, 7) . Protein disulfide isomerase in the endoplasmic reticulum and DsbA/DsbC in the Escherichia coli periplasm catalyze disulfide bond formation in extracytoplasmic proteins (4, 7, 8) . Disulfide bonds are rare in cytoplasmic proteins, and this is generally attributed to the reducing environment of the cytoplasm, with a high GSH/GSSG ratio(6) . Meanwhile, recent studies suggest that protein-disulfide isomerases might actively prevent the formation of disulfides in the cytoplasm(9) . The thioredoxin system of E. coli (NADPH, thioredoxin reductase, and thioredoxin), which is known as a hydrogen donor for ribonucleotide and sulfate reduction(5) , might be involved in keeping the cytoplasmic proteins reduced(9) . Thioredoxin reductase mutants allow disulfide bond formation in several cytoplasmic proteins, and thioredoxin exhibits protein disulfide reductase and protein-disulfide isomerase activities in vitro(5, 10, 11) . In this study we show that DnaJ(1, 2, 3) , like thioredoxin, protein-disulfide isomerase, and DsbA, catalyzes protein disulfide formation, reduction, and isomerization.


EXPERIMENTAL PROCEDURES

Purification of DnaK and DnaJ

DnaK and DnaJ were purified as described previously (12, 13) from an overproducing strain of E. coli bearing plasmid pLNA2, derived from plasmid pDM38 (14) (gift from H. Berges and O. Fayet, Microbiologie et Génétique Microbienne CNRS, Toulouse, France).

Assay of Insulin Disulfide Reduction

The reduction of insulin was assayed by measuring the increase in absorbance at 650 nm (7) . The incubation mixture contained in a final volume of 100 µl: O.1 M N(2)-equilibrated potassium phosphate, pH 6.6, 0.3 mM EDTA, 0.13 mM bovine insulin, 0.3 mM dithiothreitol, in the presence of thioredoxin, DnaJ, or DnaK as indicated. Insulin stock solutions were prepared as described in (10) .

RNase Activity

RNase activity was determined by following the hydrolysis of 2 mM cyclic 2`,3`-cytidine monophosphate in 0.1 M Tris, pH 7.4(15) ; 20 µl of RNase was incubated at 23 °C with 100 µl of cyclic CMP. Hydrolysis of cyclic CMP was measured by the increase of absorption at 296 nm. For each experiment, a control without cyclic CMP was made.

Preparation of Reduced and Denatured RNase

Reduced, denatured RNase A was prepared as described in (16) by incubating 20 mg of the native enzyme (RNase A from Sigma) in 1 ml of 0.1 M Tris, pH 8.6, containing O.15 M dithiothreitol and 6 M guanidine hydrochloride. Reduced RNase was separated from dithiothreitol and guanidine hydrochloride using Sephadex G-25 equilibrated with oxygen-free O.O1 M HCl, and the fractions were stored under mineral oil.

Preparation of Scrambled RNase

For the preparation of scrambled RNase(16) , reduced, denatured RNase was made 6 M in guanidine hydrochloride, and the pH was adjusted to 8.6 with solid Tris. The sample was then sparged with oxygen and incubated at room temperature in the dark for 3 days. Free thiol was less than 0.1 mol/mol of RNase, and RNase activity was undectable.

Reactivation of Reduced and Denatured RNase

Reactivation of reduced RNase by oxygen was initiated by diluting the reduced enzyme in air-containing buffer at 23 °C (0.1 M Tris, pH 7.4, 1 mM EDTA) in the presence of thioredoxin, DnaJ, or DnaK as indicated(16) . Samples were withdrawn at intervals and assayed for RNase activity. Reactivation of reduced RNase by redox buffers was initiated by diluting the reduced enzyme in 50 mM Tris, pH 8.2, 1 mM MgCl(2), 0.6 mM dithiothreitol, 0.9 mM oxidized glutathione, at 23 °C, in the presence of DnaJ or DnaK as indicated. Samples were withdrawn at intervals and assayed for RNase activity.

Reactivation of Scrambled RNase

Reactivation of scrambled RNase was initiated by diluting scrambled RNase (final concentration, 30 µM) in 0.1 M Tris, pH 7.4, 1 mM EDTA, 60 µM dithiothreitol at 23 °C in the presence of thioredoxin, DnaJ, or DnaK as indicated(16) . Samples were withdrawn at intervals and assayed for RNase activity.

DnaK ATPase

The stimulation by DnaJ of the DnaK ATPase was carried out in 50 mM Tris, pH 7.4, 100 µM dithiothreitol, 50 mM KCl with 50 µM [^3H]ATP, 1 µM DnaK, and DnaJ concentrations ranging from 0 to 1 µM. ATP and ADP were separated on polyethyleneimine cellulose as described in (17) . The DnaK stimulation factor and DnaJ concentrations required for DnaK stimulation are in accordance with (17) .

Preparation of Reduced and Oxidized DnaJ

40 µl of DnaJ (15 mg/ml) was incubated for 30 min with 10 mM dithiothreitol and isolated by gel filtration through a Bio-Gel P-10 column (2-ml bed volume, Bio-Rad) equilibrated with oxygen-free 0.1 M potassium phosphate, pH 6.8, 1 mM EDTA. SH determination was made immediately by adding 10 µl of each fraction to 100 µl of 1 mM 5,5`-dithiobis(2-nitrobenzoic acid), 5 M guanidine HCl, 50 mM Tris, pH 7.5(18) . The number of SH groups per mol of DnaJ was calculated from the DnaJ concentration and from the absorbance at 412 nm (with an adsorption coefficient of the p-nitrothiophenol anion of 13,600 M cm(18) ). DnaJ concentration (M(r) = 41,000 Da) was calculated from its absorbance at 280 nm, with A = 0.289 calculated from tryptophan and tyrosine content. Oxidized DnaJ was obtained by incubating reduced DnaJ for 72 h in air-containing buffer (Tris, pH 8, 1 mM EDTA).

Materials

Insulin (from bovine pancreas), RNase A, and thioredoxin (from Spirulina sp.) were from Sigma. All other products were reagent grade and were obtained from Sigma. [^3H]ATP was obtained from Amersham Corp. and was used at 1.5 Ci/mmol. [^14C]Iodoacetamide (60 mCi/mmol) was from Amersham Corp.


RESULTS AND DISCUSSION

Reduction of Insulin Disulfide Bonds

The reduction of insulin by protein-disulfide isomerases can be assayed by a rapid spectrophotometric measurement(7) . Insulin contains two polypeptide chains A and B that are linked by disulfide bonds. When these bonds are broken, the free B chain is insoluble and precipitates, leading to an increase in absorbance at 650 nm. Thioredoxin, protein-disulfide isomerase, and DsbA catalyze the dithiothreitol-dependent reduction of insulin(7, 10, 19) . The reduction of insulin (130 µM) by dithiothreitol (0.3 mM) was determined in the presence or absence of DnaJ (Fig. 1). Insulin precipitation can be observed after 30 min in the presence of DnaJ (2 µM), compared with 80 min in the absence of DnaJ. In the same assay, thioredoxin (1 µM) catalyzes insulin reduction, as previously reported(10) . Interestingly, DnaK (2 µM) is inactive in this reaction (Fig. 1). Like DsbA (7) and protein-disulfide isomerase(19) , DnaJ is severalfold less active than thioredoxin in catalyzing the dithiothreitol-dependent reduction of insulin.


Figure 1: DnaJ-catalyzed reduction of insulin by dithiothreitol. The incubation mixtures contained O.1 M N(2)-equilibrated potassium phosphate, pH 6.6, 0.3 mM EDTA, 0.13 mM bovine insulin, and 0.3 mM dithiothreitol alone (circle) or in the presence of 2 µM DnaJ (bullet), 1 µM thioredoxin (times), or 2 µM DnaK (up triangle). The reduction of insulin and its resulting precipitation were monitored by following optical density at 650 nm.



Oxidative Folding of Reduced RNase in the Presence of Air or Redox Buffers

Renaturation of reduced, denatured RNase (which possesses eight -SH groups) involves the oxidation of its thiol groups followed by rearrangement of the disulfides to the native conformation (with 4 S-S bridges)(20) . As previously reported(16) , the spontaneous refolding of reduced, denatured RNase in the presence of air is negligible (Fig. 2A). In contrast, 40 µM thioredoxin or 40 µM DnaJ catalyze the reactivation of reduced, denatured RNase A with an efficiency similar to that previously reported for the E. coli thioredoxin (16) (Fig. 2A). Under the same conditions, DnaK (40 µM) is unable to stimulate the oxidative refolding of reduced RNase A (Fig. 2A).


Figure 2: Oxidative folding of reduced, denatured RNase. A, reactivation of RNase by air. The mixtures (0.1 M Tris, pH 7.4, 1 mM EDTA at 23 °C) containing 30 µM reduced denatured RNase alone (circle) or in the presence of 40 µM DnaJ (bullet), 40 µM thioredoxin (times), or 40 µM DnaK (up triangle) were assayed for RNase activity at the times indicated. B, reactivation of RNase by redox buffer. The mixtures (0.6 mM dithiothreitol, O.9 mM oxidized glutathione, 1 mM MgCl(2), 50 mM Tris, pH 8.2) containing 30 µM reduced, denatured RNase alone (circle) or in the presence of 30 µM DnaJ (bullet) or 30 µM DnaK (up triangle) were assayed for RNase activity at the times indicated.



Appropriate redox buffers are much more efficient than air in reactivating reduced RNase. Furthermore, protein-disulfide isomerases can accelerate the reactivation of reduced RNase in the presence of redox buffers(15) . In the presence of O.9 mM oxidized glutathione and 0.6 mM dithiothreitol (a redox buffer used for the reactivation of reduced RNase by DsbA(15) ), DnaJ stimulates the reactivation of reduced RNase A. The kinetics of RNase A reactivation are severalfold faster in the presence of 40 µM DnaJ than in its absence (Fig. 2B) and are similar to those obtained by others in the presence of 25 µM DsbA(15) . The DnaK protein (40 µM) does not accelerate the reaction.

Refolding of Scrambled RNase

Scrambled RNase A is a fully oxidized form of RNase A obtained by oxidation of reduced RNase A in the presence of guanidine hydrochloride. It consists of a heterogeneous population of molecules containing random disulfide bonds and requires interchange of disulfide bonds to acquire the native conformation. It has been reported that thioredoxin, protein-disulfide isomerase, or DsbA can reactivate scrambled RNase in the presence of dithiothreitol (11, 15, 16) . DnaJ can reactivate scrambled RNase more efficiently than thioredoxin, while DnaK or dithiothreitol alone is unable to do so (Fig. 3).


Figure 3: Refolding of scrambled RNase. The mixtures (0.1 M Tris, pH 7.4, 1 mM EDTA at 23 °C) containing 60 µM dithiothreitol and 30 µM scrambled RNase alone (circle) or in the presence of 30 µM DnaJ (bullet), 5 µM DnaJ (), 12 µM thioredoxin (times), or 5 µM DnaK (up triangle) were assayed for RNase activity at the times indicated.



Copurification of DnaJ with the Protein-disulfide Isomerase Activity

To confirm that the protein-disulfide isomerase activity was inherent to DnaJ, we assayed the fractions obtained at the final step of the purification procedure (heparin-Sepharose affinity chromatography) for activity to stimulate the DnaK ATPase (a known property of DnaJ) (17) and for activity to catalyze insulin precipitation. As shown in Fig. 4A, both enzymatic activities coincide with the DnaJ protein peak. Analysis of the DnaJ preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows purity of more than 95% without visible material corresponding to thioredoxin (12,000 Da), DsbA (21,000 Da), or DsbC (23,000 Da).


Figure 4: A, copurification of the protein-disulfide isomerase-like activity with the activity of DnaK-ATPase stimulation. Purified DnaJ (0.6 mg) was loaded on a heparin-Ultrogel column (IBF France, O.8-ml bed volume) equilibrated in 50 mM Tris, pH 7.4, 100 µM dithiothreitol, 50 mM KCl and eluted with a linear gradient (2 times 5 ml) of 0-0.3 M KCl in the same buffer. The fractions were analyzed for protein content (expressed in mg/ml) (circle), stimulation of the DnaK ATPase (), and stimulation of insulin precipitation (bullet). Stimulation of insulin precipitation is expressed as the ratio between the times required for the onset of insulin precipitation (absorbance, 0.05) in the absence and in the presence of each fraction. Stimulation of insulin precipitation and of the DnaK ATPase were assayed as described under ``Experimental Procedures'' with 10 and 1 µl of each fraction, respectively. Protein determination was made according to Bradford(30) . B, dithiol/disulfide of DnaJ. Gel permeation chromatography of reduced DnaJ. 40 µl of DnaJ (15 mg/ml) (bullet) or 40 µl of buffer (circle) were incubated for 30 min with 10 mM dithiothreitol and isolated by gel filtration through a Bio-Gel P-10 column as described under ``Experimental Procedures.'' The SH concentration of each fraction was determined with 5,5`-dithiobis(nitrobenzoic acid) by measuring the absorbance at 412 mM as described under ``Experimental Procedures.'' C, alkylation of reduced DnaJ by radiolabeled iodoacetamide. Labeling of oxidized (laneA) or reduced DnaJ (laneB) with [^14C]iodoacetamide is shown. 15 µM oxidized DnaJ (prepared as described under ``Experimental Procedures'') was incubated without (laneA) or with 80 µM dithiothreitol (laneB) in 50 mM Tris, pH 7.5 buffer for 10 min at 20 °C. Then [^14C]iodoacetamide (60 mCi/mmol) was added (final concentration, 600 µM), and the incubation was continued for 2 h(31) . The mixture was added to the electrophoresis sample buffer (60 mM Tris, pH 6.8, 10% glycerol, 4% SDS, 5% 2-mercaptoethanol) containing 30 mM unlabeled iodoacetamide and submitted to SDS-polyacrylamide gel electrophoresis (12.5% polyacrylamide). Finally, the gel was processed for autoradiography.



Active Dithiol/Disulfide in DnaJ

Most DnaJ proteins contain a cysteine-rich region, with four repeats of the sequence Cys-X-X-Cys-X-Gly-X-Gly. It has been suggested that the organization of these cysteine regions is similar to that found in zinc finger proteins(21) . However, Cys-X-X-Cys sequences form the active site of protein disulfide oxidoreductases such as thioredoxin, protein-disulfide isomerase, and DsbA(4, 5, 7) . The Cys-X-X-Cys sequences of DnaJ might be involved in oxidoreduction processes (the sequence Cys-Pro-His-Cys of DnaJ is identical to the active redox site of DsbA(7) ). In fact, DnaJ appears to contain a redox-active disulfide reducible by dithiothreitol. Reduced DnaJ was prepared by reduction of DnaJ with 10 mM dithiothreitol followed by filtration through a gel permeation column equilibrated with oxygen-free buffer, as described for thioredoxin(18) . Reduced DnaJ contains 1.9 -SH groups per mol as determined with 5,5`-dithiobis(2-nitrobenzoic acid) (Fig. 4B), while oxidized DnaJ (obtained after a 72-h incubation in air-containing buffer as described under ``Experimental Procedures'') contained less than 0.3 mol of -SH per mol (not shown). The free thiol groups in reduced and oxidized DnaJ could be visualized after alkylation of DnaJ by radiolabeled iodoacetamide in the absence or presence of dithiothreitol. Oxidized DnaJ incorporates little iodoacetamide, whereas reduced DnaJ incorporates iodoacetamide in its exposed dithiol (Fig. 4C).

Dithiothreitol Sensitivity of dnaJ Mutant

Several genes involved in protein oxidoreduction have been discovered by isolation of dithiothreitol-sensitive mutants(22) . These mutants include trxA (thioredoxin), trxB (thioredoxin reductase), dsbA, and dsbB mutants. A dnaJ mutant displays sensitivity to dithiothreitol. The colony-forming ability of the dnaJ mutant RH7742 (del(dnaJ::kn)) on minimal medium agar plates (63 glucose B(1) agar) containing 7 mM dithiothreitol at 30 °C was 2% of the parental strain MC4100 (not shown).

Implications

DnaJ displays protein-disulfide isomerase activity in vitro. Like other protein disulfide oxidoreductases (thioredoxin, protein-disulfide isomerase, DsbA), it catalyzes disulfide bond formation, isomerization, and reduction of proteins. Thus, while DnaK seems mainly involved in the control of hydrophobic interactions in folding or denatured proteins(2, 3, 23) , DnaJ, in addition to its chaperone function(3, 24) , could be implicated in controlling their dithiol/disulfide state. The tendency to form disulfides in proteins is not only dependent on the redox potential of the environment but also on the conformation of the polypeptide(6, 25) , and some folding intermediates (or some denatured polypeptides) might possess conformations that favor the formation of disulfides despite the reducing environment of the cytoplasm. A chaperone with disulfide oxidoreductase activity could destabilize these conformations and reduce their otherwise irreversible disulfides. It has been reported that thioredoxin and protein-disulfide isomerase possess chaperone-like functions in addition to their protein disulfide oxidoreductase activities (reviewed in (4) and (5) ). The involvement of DnaK/DnaJ in protein export (26) suggests that DnaJ could maintain exported proteins in a reduced state prior to membrane translocation and control the redox state of inner membrane proteins before (or during) membrane insertion. The thioredoxin system (NADPH, thioredoxin reductase, and thioredoxin) has been characterized as a hydrogen donor for ribonucleotide reductase and for enzymes reducing sulfate or methionine(5) . Recent genetic studies implicate thioredoxin reductase (but not thioredoxin) in the destruction of cytoplasmic protein disulfides and suggest that thioredoxin reductase might reduce proteins through an unidentified protein(8, 9) . It will be interesting to test whether DnaJ (as thioredoxin (5) and eukaryotic protein-disulfide isomerase(19) ) can interact with thioredoxin reductase. The viability of thioredoxin(5) , thioredoxin reductase(5) , DnaJ(27) , or glutathione (28) deficient mutants might be explained by a functional redundancy of the enzymatic systems that control protein dithiol/disulfides, similar to the redundancy of the enzymatic systems for the reduction of ribonucleotide reductase(29) . Finally, DnaJ and its eukaryotic homologs containing Cys-X-X-Cys sequences, in addition to their role in protein folding and protection against stress, could be involved as chloroplast thioredoxins in the redox controls of several cellular enzymatic activities(10) .


FOOTNOTES

*
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§
To whom correspondence should be addressed. Tel.: 33-1 44 27 50 98; Fax: 33-1 44 27 35 80.


ACKNOWLEDGEMENTS

We thank Dr. H. Berges and Dr. O. Fayet (Laboratoire de Microbiologie et Génétique Moléculaire, CNRS, Toulouse, France) for the gift of the DnaK/DnaJ hyperproducing strain.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.