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
-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
,
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 [
H]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
= 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.
[
H]ATP was obtained from Amersham Corp. and was
used at 1.5 Ci/mmol. [
C]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
-equilibrated potassium phosphate, pH 6.6, 0.3 mM EDTA, 0.13 mM bovine insulin, and 0.3 mM dithiothreitol alone (
) or in the presence of 2 µM DnaJ (
), 1 µM thioredoxin (
), or 2
µM DnaK (
). 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 (
) or in the
presence of 40 µM DnaJ (
), 40 µM
thioredoxin (
), or 40 µM DnaK (
) 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
,
50 mM Tris, pH 8.2) containing 30 µM reduced,
denatured RNase alone (
) or in the presence of 30 µM DnaJ (
) or 30 µM DnaK (
) 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 (
) or in the presence of 30 µM DnaJ (
), 5 µM DnaJ (
), 12 µM
thioredoxin (
), or 5 µM DnaK (
) 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
5 ml) of
0-0.3 M KCl in the same buffer. The fractions were
analyzed for protein content (expressed in mg/ml) (
), stimulation
of the DnaK ATPase (
), and stimulation of insulin precipitation
(
). 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) (
) or 40 µl of buffer
(
) 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 [
C]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 [
C]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
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) .