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INTRODUCTION |
Thioredoxin (TRX)1 is a
small 12-kDa ubiquitous protein containing two redox-active
half-cystine residues in an active center with conserved amino acid
sequence Cys-X-X-Cys (where X
indicates various amino acids) that functions as a protein-disulfide
reductase. The two cysteine residues in the active site provide the
sulfhydryl groups involved in the thioredoxin-dependent
reducing activity. Under an oxidized form, the TRX-S2
protein contains a disulfide bridge within the active site that is
reduced to a TRX-(SH2) dithiol by NADPH and the
flavoprotein TRX reductase (for review, see Ref. 1). Under this reduced
form, thioredoxin becomes a very powerful reductant of disulfide
bridges in target proteins.
Thioredoxin was initially characterized from Escherichia
coli extracts as a hydrogen donor for ribonucleotide reductase
(2). Thioredoxins were later shown to act as hydrogen donors to
peroxiredoxins that reduce hydrogen peroxide (3) and to
methionine-sulfoxide reductases that reactivate proteins damaged by
stresses that generate reactive oxygen species (ROS) (4). They are
necessary for a number of other metabolic enzymes that form a disulfide
as part of their catalytic cycle (5). In addition, thioredoxins induce conformational changes of the targeted proteins by disulfide bond reduction and assist in the protein folding pathway (6). Thioredoxins directly or indirectly interact with several nuclear factors, such as
the mammalian transcription factor NF
B and Ref-1 (see Ref. 7 and
references therein). Thioredoxins play important roles in cell cycle
and division and embryogenesis (8) and inhibit spontaneous
apoptosis in tumoral cells (9). Mammalian thioredoxin has also
been shown to associate directly with nuclear receptors such as the
glucocorticoid receptor GR (10), whose regulation requires cooperation
between heat-shock proteins such as Hsp70 and Hsp90 (11).
Hsp70 and Hsp90 are molecular chaperones that are involved in many
important biological processes by appropriately folding nascent
polypeptides on ribosomes as well as by assembling multisubunit protein
complexes (12). Chaperones also play a pivotal role by renaturing
proteins after exposure to various stresses, driving protein
translocation across membranes, and disassembling protein complexes
prior to protein degradation. Molecular chaperones often function
together, and in this respect, members of the Hsp70 family are of prime
importance. They are highly conserved from bacteria to human and
exhibit a well defined structure as follows: the 44-kDa amino-terminal
part of the protein binds nucleotides, whereas the 28-kDa
carboxyl-terminal domain interacts with misfolded or partially unfolded
polypeptides (13).
The chaperone activity of Hsp70 is regulated by cofactors that catalyze
the interconversion between the ATP and ADP states (14). In bacteria,
cycling of the Hsp70 homologue DnaK between different nucleotide states
is regulated by the chaperone cofactors DnaJ and GrpE (15). DnaJ
stimulates the Hsp70 ATPase activity, and the conversion of Hsp70 into
the ADP-bound state allows it to interact with polypeptide substrates
(16). In contrast, GrpE binds to the ATPase domain of DnaK and triggers
the release of ADP, hence accelerating substrate dissociation upon ATP
re-binding (17). In eukaryotes, the yeast DnaJ homologue Hsp40/Zuo1
(18) and the mammalian Hsp40/Hdj (19) have also been shown to associate with Hsp70. However, structural homologues of GrpE are limited to
compartments of prokaryotic origin, i.e. mitochondria and
chloroplasts (20). Mammals contain other classes of Hsp70
co-chaperones, such as the p48/Hip
(Hsc70-interacting protein),
BAG-1/Hap (Hsp70-activating protein), and CHIP (carboxyl terminus of
Hsc70-interacting protein) (21-23)
cofactors. BAG-1 and Hop accelerate ADP release and stimulate the
re-binding of ATP. CHIP decreases net ATPase activity and reduces
chaperone efficiency, thus negatively regulating the Hsp-substrate binding cycle. In contrast, Hip stabilizes the ADP-bound form of Hsp70,
extending the time window during which Hsc70 interacts stably with a
polypeptide substrate. All these co-chaperones have a spatial
distribution that is critical to the coordination of Hsp70 functions,
because they ensure that substrates are both bound and released at an
appropriate place and time by regulating the ATP/ADP cycle (24).
In this report, we describe the molecular cloning and characterization
of a novel bipartite protein from Arabidopsis thaliana that
may be a new component of the Hsp70 chaperone system. This protein
exhibits a unique domain structure with a carboxyl-terminal thioredoxin
domain, the amino-terminal domain containing three tetratricopeptide
repeats similar to that of the rat and human Hip (21). We termed this
new protein AtTDX for Tetratricopeptide domain-containing thioredoxin. We present
evidence that AtTDX displays a disulfide reductase activity both
in vitro and in vivo due to its thioredoxin
domain, whereas its amino terminus interacts specifically with the
yeast Hsp70 Ssb2 protein. We show that the interaction between AtTDX
and Ssb2 is sensitive to the redox status, and we demonstrate that the
thioredoxin domain of AtTDX acts as a redox switch that turns the
complex with Ssb2 on and off. We also show that a conserved cysteine in
the ATPase domain of Ssb2 is required for disruption of the complex
with AtTDX. The work we present here provides the first example of a
redox-dependent interaction of a thioredoxin-like protein
with a member of the Hsp70 family.
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EXPERIMENTAL PROCEDURES |
Recombinant DNA and DNA Analyses--
The A. thaliana
two-leaf stage cDNA library used for the yeast complementation
assays was constructed in a pFL61 vector (25). PCR amplifications were
done using the Expand Long Template kit (Roche Molecular Biochemicals).
Directed mutagenesis was done by primer-mediated mutagenesis using
QuikChangeTM Site-directed Mutagenesis Kit (Stratagene).
All the constructs and PCR products were checked by sequencing. Details
concerning plasmids created in this study are available upon request.
The program Darwin used to create the phylogenic tree is at
cbrg.inf.ethz.ch. Swiss-PDB-Viewer and Rasmol programs used to
analyze the Ssb2 sequence are at www.expasy.ch/spdbv/mainpage.htm
and klaatu.oit.umass.edu:80/microbio/rasmol, respectively.
Protein Analyses and Antibodies--
Protein concentrations were
determined with a protein assay kit (Bio-Rad). Western blots were
developed by enhanced chemiluminescence. Anti-HA monoclonal 12CA5 and
anti-GST antibodies were purchased from Roche Molecular Biochemicals
and Amersham Biosciences, respectively. Polyclonal antibodies against
His-AtTDX were raised in rabbit (Eurogentec).
Yeast Strains and Media--
Functional complementation assays
were done in the strain EMY63 (MATa ade2-1 ade3-100
his3-11 leu2-3 lys2-801 trp1-1 ura3-1
trx1::TRP1,
trx2::LEU2) as described by
Bréhélin et al. (26). For two-hybrid
experiments, we used the strain YRG2 (MATa ura3-52 his3-200 ade2-100 lys2-801 trp1-901 leu2-3 112 gal4-542 gal80-538
LYS::UASGAL1-TATAGAL1-HIS3
URA::UASGAL4 17
mers(x3)- TATACYC1-LacZ, Stratagene).
Co-immunoprecipitation experiments were performed using the strain W303
(MATa ade2 his3 leu2 trp1 ura3) (27).
Saccharomyces cerevisiae was transformed after acetate
chloride treatment as described by Gietz et al. (28). Standard yeast media and B sulfur-less medium were prepared as described by Rouillon et al. (29). Transformants were
selected on solid YNB medium containing appropriate auxotrophic supplements.
For H2O2 sensitivity assays, transformed cells
were first grown on histidine-containing minimal medium to a density of
107 cells per ml and then serial diluted in histidine-less
minimal medium in the presence of increasing concentrations of
H2O2. Plates were then incubated at 30 °C
for 3 days.
Expression of Recombinant Proteins--
A pET16b vector
(Novagen) was used for the expression of His-tagged version of AtTDX
and its derivatives in E. coli, allowing the amino-terminal
introduction of a His6 tag. The coding region of AtTDX and
AtTDX
-(1-269) were subcloned as
NdeI-BamHI fragments into corresponding sites of
pET16b. The coding region of ySsb2 was cloned as a
BamHI-BamHI fragment into the pGEX-4T1 vector (Amersham Biosciences) for the expression of a amino-terminal GST-tagged version of the protein. BL21 (DE3) cells harboring the
appropriate plasmids were cultured at 37 °C in LB medium with ampicillin. Expression of the recombinant proteins was induced at
A600 of 0.2 by addition of
isopropyl-1-thio-
-D-galactopyranoside to 1 mM, and cells were further grown for 3 h. Cells were
then harvested, and total proteins were extracted from bacteria using a
hydraulic press (Carver, model 3968) according to Verdoucq et al. (30). Cells expressing His-AtTDX and derivatives were
resuspended in a 1× imidazole binding buffer (Novagen), and His-tagged
proteins were purified on Ni2+ His.Bind resin according to
the manufacturer's instructions. Cells expressing ySsb2 were
resuspended in a 1× phosphate-buffered saline buffer (Amersham
Biosciences), and GST-ySsb2 was purified on glutathione-Sepharose 4B
column according to the manufacturer's instructions.
His-tagged AtTDX and AtTDX
-(1-269) proteins used for determination
of thioredoxin activity were tested using the insulin-disulfide reduction assay as described previously (31). All assays were monitored
by addition of 1 mM dithiothreitol, and measurements were
performed at A650 for 45 min on a
spectrophotometer (model DU7400, Beckman Instruments).
Two-hybrid Experiments--
All the experiments were performed
in the yeast reporter strain YRG2 using the Gal4 Two-hybrid Phagemid
Vector kits (Stratagene). The vector pBDGal4 was used to clone AtTDX
and its derivatives as the baits, and pADGal4 was used to clone the
target protein Ssb2 and its derivatives as the targets. Two two-hybrid
cDNA libraries were screened: an A. thaliana cell
suspensions cDNA library cloned into a pADGal4 vector (gift of B. Lescure), and a yeast cDNA library cloned into a pGAD-GH vector
(32). Library screening and control/test experiments were as described
by Stratagene using 20 mM 3-aminotriazole. Double
transformants were grown on selective medium and tested by histidine
prototrophy and
-galactosidase activity.
All the fragments for both the bait and the target cDNAs, as well
as truncations, were obtained by PCR amplification, using restriction
site-containing primers on yeast genomic DNA extracted with standard
procedures. The pBD.AtTDX
-(112-213) and pAD.Ssb2
-(387-549) constructs were obtained by a two-step PCR. The clone pAD.AtHsc70-1 was
obtained by PCR amplification on clone G11F4T7 (NCBI stock center).
Co-immunoprecipitation Assays--
In vivo
interaction between HA-AtTDX and GST-Ssb2-tagged proteins was assayed
by co-immunoprecipitation experiments. Briefly, the full-length AtTDX
sequence was cloned into a pFL39 vector, whereas the full-length Ssb2
sequence was cloned into a pEG.KT vector. Cultures of yeast cells,
protein extractions, and immunoprecipitation experiments were performed
as described previously (29). Precipitates were separated on 10%
SDS-PAGE gels and analyzed by Western blotting.
Reconstitution of AtTDX-ySSB2 Complex Using Affinity
Chromatography--
Recombinant His-AtTDX and GST-ySSB2 were incubated
in a 20 mM imidazole-containing buffer (Buffer A) for 15 min at 30 °C as described previously (21). Using a batch method, 100 µl of Ni2+ His.Bind resin (Novagen) equilibrated with 1×
binding buffer was added, and the samples were further incubated for 30 min at 4 °C. After washing with Buffer A, bound proteins were eluted with 3 volumes of Buffer A, 150 mM imidazole. Proteins were
then separated on 10% SDS-PAGE gels and analyzed by Western blotting.
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RESULTS |
Cloning of a Novel TPR-containing Thioredoxin-like Protein
from A. thaliana--
To isolate new Arabidopsis genes
encoding thioredoxins or thioredoxin-like proteins, we screened an
A. thaliana cDNA library by functional complementation
of trx1
, trx2
double disrupted yeast cells. In
S. cerevisiae, the simultaneous disruption of the two
thioredoxin encoding genes, TRX1 and TRX2, leads
to several growth defects, including organic sulfur auxotrophy and
sensibility to oxygen peroxide (33). trx1
,
trx2
mutant cells were transformed with a library of
2-day-old seedlings cDNA that are expressed from the strong
PGK1 promoter on vector pFL61 (25), and uracil prototroph
clones able to grow without methionine were selected. From a screen of
5·106 independent transformants, 20 methionine prototroph
colonies were recovered. Further analyses demonstrated that these
clones correspond to the expression of two different A. thaliana cDNAs from the PGK1 promoter. The first
one encodes AtTRX3 protein, a thioredoxin shown previously to
complement the EMY63 mutant for sensibility to oxygen peroxide but not
for sulfate assimilation when expressed from a centromeric low copy
plasmid (34). In the present study, AtTRX3 is expressed from a strong
promoter on a high copy plasmid, and therefore, methionine auxotrophy
complementation is believed to result from high AtTRX3 synthesis. The
second cDNA encodes a novel thioredoxin-containing protein. This
full-length cDNA is 1365 nucleotides in length and contains an open
reading frame with the potential of coding a 380-residue polypeptide
(Fig. 1A). This
protein displays a bipartite structure with an amino-terminal part that
comprises three 34-amino acid tetratricopeptide domains (TPR, residues
112-213) and a carboxyl-terminal domain (residues 270-380) that is
highly similar to thioredoxin proteins. The two domains are separated
by a central region rich in charged residues. We named this novel
A. thaliana protein, AtTDX, for
Tetratricopeptide domain-containing
thioredoxin.

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Fig. 1.
The AtTDX cDNA and
gene. A, the nucleotide (upper line,
standard numbering) and the deduced amino acid (lower
line, italicized numbering) sequences of AtTDX
cDNA. Exons are in capital letters, and untranslated regions in lowercase letters. Residues
comprising the TPR domains are indicated by heavy
underlines; a charged central region is indicated by a
dashed line, and the beginning of thioredoxin domain is
indicated by boldface nucleotides and corresponding G
residue. The thioredoxin active site is boxed. The
doubly underlined sequences including the start and stop
(marked by an asterisk) codons correspond to
oligonucleotides used to amplify the genomic AtTDX sequence from
Arabidopsis. Two putative polyadenylation signals are
singly underlined. B, schematic representation of
the AtTDX gene structure shows intron positions.
C, alignment of AtTDX and its tobacco counterpart NtTDX with
AtHIP. Homologous residues are boxed. D, Darwin
tree of TPR-containing proteins from TDX (underlined), HIP,
PPt, Sti, and Sgt protein families. EMBL or Swiss-Prot accession
numbers and codes are indicated below. A. thaliana
(At) TDX, HIP (T04562), PPt (AC006931), Sti1a (T48150),
Sti1b (AC007190), PPtlike (AL161559); Nicotiana
tabacum (Nt) TDX; Homo sapiens
(Hs) HIP (U28918), Sti1 (BC002987) and PPt (ac007193);
Rattus norvegicus (Rn) HIP (X82021);
Caenorhabditis elegans (Ce) HIP (T24865);
Drosophila melanogaster (Dm) HIP (AE003429), HOP
(AF056198) and PPt (AE003684); Plasmodium berghei
(Pb) HIP (T10455); Schizosaccharomyces pombe
(Sp) DnaJ (AL391604) and PPt (AL022019); Leishmania
major (Lm) Sti1 (U73845); Pisum sativum
(Ps) PPt (AF179282); S. cerevisiae
(Sc) Sti1 (M28486) and Sgt2 (U43491); Oryza
sativa (Os) Sgt1 (AF192467); Xenopus laevis
(Xl) PPt (AF018263). The GenBankTM
accession numbers for AtTDX cDNA, AtTDX gene,
and NtTDX cDNA are AY064251, AY064252, and AY064253,
respectively.
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Occurrence of TDX in Plant Genomes--
To confirm the occurrence
of the AtTDX gene within the A. thaliana genome
as well as to determine the structure of this gene, we amplified by PCR
genomic fragments using two oligonucleotides flanking the
AtTDX cDNA coding sequence (Fig. 1A). The
chromosomal copy of AtTDX contains 9 introns that are all
located upstream from the thioredoxin domain, the 9th being found at
the beginning of the thioredoxin domain (Fig. 1B). Southern
blot experiments demonstrated that AtTDX is unique within
the A. thaliana genome (not shown), data confirmed by the
recent completion of the sequence of this plant genome. Northern assays
showed that AtTDX is ubiquitously expressed but at low
levels in A. thaliana tissues (data not shown). To get more
insight on the occurrence of TDX-encoding genes in plants,
we also sequenced a cDNA from tobacco encoding an AtTDX homologue (NtTDX, gift of N. Chaubet). The AtTDX and NtTDX
proteins share more than 65% of identical residues (Fig.
1C). These observations confirmed that AtTDX is a real plant
protein and does not arise from the fusion of two unrelated sequences
during the cDNA library construction. AtTDX is located
on chromosome 3 (BAC MEB5, accession number AB019230) but is
erroneously annotated, each domain being considered as an independent gene.
The amino-terminal part of AtTDX (from amino acids 1-256), which
contains the three TPR repeats, is also highly similar to the A. thaliana HIP co-chaperone (Fig. 1C). Moreover, the TPR repeats found within the AtTDX protein exhibit typical features of most
of the TPR-containing proteins. A phylogenic analysis of some
TPR-containing protein families from A. thaliana clearly shows that these proteins are grouped according to their cellular function and that the TDX family is closely related to the HIP family
(Fig. 1D). Interestingly, AtHIP is positioned within the TDX
group. On a separate Darwin tree, thioredoxin domains of AtTDX and
NtTDX form a new family, closely related to the one containing the
cytosolic thioredoxin h (data not shown).
The Thioredoxin Domain of AtTDX Is a Functional Protein-disulfide
Reductase--
The functional complementation of the yeast
trx1
, trx2
double mutant strongly suggested that AtTDX
exhibits thioredoxin activity in vivo. Indeed, AtTDX
expression not only relieved the methionine auxotrophy of
trx1
, trx2
cells but also allowed them to reduce
methionine sulfoxide (Fig.
2A). To assess further the possibility that AtTDX was indeed a disulfide reductase, the AtTDX protein was expressed in E. coli as a fusion to a
polyhistidine tag, and the recombinant protein was purified by
Ni2+ affinity chromatography. Recombinant AtTDX was assayed
in vitro for thioredoxin activity by measuring its ability
to catalyze the reduction of insulin disulfide bridges (31). Moreover,
to determine whether thioredoxin activity could be restricted to the
AtTDX carboxyl-terminal domain, we purified a truncated form of AtTDX
containing only the last 110 amino acids (AtTDX
-(1-269)). As shown
in Fig. 2B, a rapid induction of insulin reduction is observed in the presence of 5 µM recombinant AtTDX. The
in vitro insulin reduction activity displayed by AtTDX is
equivalent to the activity displayed by AtTRX3, a thioredoxin
previously shown to exhibit high TRX activity (35). Moreover, the
assays revealed that the truncated derivative AtTDX
-(1-269) was
also able to reduce insulin. Therefore, this experiment demonstrated
that AtTDX possesses disulfide reductase activity in vitro
due to the thioredoxin domain found at its carboxyl-terminal part.

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Fig. 2.
AtTDX possesses disulfide reductase
activity. A, AtTDX is able to complement the
trx1 ,trx2 mutant. Cells expressing AtTDX
and AtTRX3 from the pGK1 promoter were grown to a density of
107 cells per ml and plated on galactose agar-containing
medium at A600 = 0.2 in the presence or
absence of methionine (+Met, Met) or in the
presence of methionine sulfoxide (+MetSO) as sole source of
sulfur. Plates were incubated 6 days at 30 °C. B, both
AtTDX and AtTDX -(1-269) are capable of in vitro
disulfide reduction of bovine insulin. The thioredoxin activity was
measured as turbidity at 650 nm due to insulin precipitation by the
addition of the proteins (5 µM each) at 25 °C.
Thioredoxin AtTRX3 was used in this assay as a positive control,
whereas bovine serum albumin and dithiothreitol alone are negative
controls.
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AtTDX Interacts with the Yeast Ssb2 Hsp70 Chaperone--
To gain
more insights on the function of the AtTDX protein, we next tried to
understand what could be the role of its amino-terminal TPR-containing
domain. Most TPR-containing proteins have been shown to be associated
with large protein complexes through their TPR motifs (36, 37). We thus
tested whether proteins capable of interacting with the amino-terminal
domain of AtTDX could be identified in a two-hybrid screen. Indeed,
heterologous functional assays in S. cerevisiae have already
been successfully used to characterize the functional specificity of
thioredoxins from plants (34). Moreover, the success in the functional
complementation of the trx1
, trx2
mutant
cells by AtTDX cDNA (leading to its own characterization, this
work) suggested that AtTDX interacts with yeast proteins. We therefore
reasoned that a similar two-hybrid approach using a yeast cDNA
library could be applied for deciphering the function of AtTDX, to
determine whether yeast proteins capable of specifically interacting
with AtTDX could be identified. A first experiment was first performed
using as the bait the first 269 amino acids of AtTDX
(AtTDX
-(270-380)) fused to the Gal4 DNA-binding domain, against a
yeast cDNA library as a heterologous two-hybrid screening assay.
From a screen of 1 × 106 independent transformants,
14 histidine, aminotriazole-resistant prototroph colonies were
selected. Plasmid DNAs were extracted from the 14 selected colonies and
used to retransform the reporter strain. Only one positive clone
displaying a strong positive His+ phenotype was recovered, and the
corresponding plasmid DNA was subsequently analyzed. The sequencing of
this plasmid revealed an open reading frame of 573 amino acids fused
in-frame with the Gal4 activation domain. Data base searching showed
that the cloned DNA fragment encodes the yeast Ssb2, a member of the
heat-shock protein 70 family protein (38). Because the isolated DNA
lacked the last 40 amino acids of Ssb2, we amplified the full-length SSB2 nucleotide sequence by PCR with yeast genomic DNA, and
we tested the interaction of the entire Ssb2 with both AtTDX and AtTDX
-(270-380). As shown in Fig. 3,
strong interactions were visualized between the full-length Ssb2 and
either AtTDX or AtTDX
-(270-380).

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Fig. 3.
AtTDX interacts with the yeast Hsp70 Ssb2 in
the yeast two-hybrid system. Left panel represents
minimal medium containing histidine (+His) such that
interaction is not required for growth (control plate), and right
panel is His-free but contains 20 mM
3-aminotriazole (3AT), such that interaction is
required for YRG2 cell growth (test plate). Four independent
transformed cells were tested.
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The interaction between AtTDX
-(270-380) and Ssb2 was next assayed
by co-immunoprecipitation experiments. Wild-type yeast cells (W303-1A)
were co-transformed with plasmids expressing a hemagglutinin (HA)
epitope-tagged AtTDX and a glutathione S-transferase (GST)-tagged Ssb2 (Fig. 4A).
Proteins of the resulting transformants were extracted and
immunoprecipitated with either anti-GST (Fig. 4B) or anti-HA
(Fig. 4C) antibodies. Immunoprecipitated proteins were next
analyzed by immunoblotting with either anti-GST or anti-HA antibodies.
In both cases, co-immunoprecipitation assays revealed a specific and
strong interaction between AtTDX and Ssb2. Identical results were
obtained when the truncated AtTDX
-(270-380) protein was used (data
not shown).

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Fig. 4.
AtTDX interacts with Ssb2 in vivo
by co-immunoprecipitation and in vitro by
affinity chromatography. A, immunoblot of
AtTDX -(270-380) and Ssb2 translated proteins in W303 cells.
HA-AtTDX -(270-380) and GST-Ssb2 were expressed from the
GAL1 promoter, alone or together, in W303 cells, and
30 µg of each extract was tested for antibody recognition.
B, anti-GST immunoprecipitates were probed either with
anti-GST or anti-HA antibodies. C, anti-HA
immunoprecipitates were probed either with anti-GST or anti-HA
antibodies. + and denote the presence or absence of protein.
D, immunoblot analysis of eluate from immobilized His-AtTDX
using specific antibodies against AtTDX and GST tag, after incubation
of recombinant His-AtTDX and GST-Ssb2 proteins. A control
lacking His-AtTDX is shown. FT, flow through. E,
elution.
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We next tested the direct physical interaction between AtTDX and Ssb2,
using an in vitro affinity procedure. For this purpose, both
His-tagged AtTDX and GST-tagged Ssb2 proteins were produced in E. coli BL21 cells and purified, and formation of a protein complex
between the two recombinant proteins was assayed by monitoring their
retention on a Ni2+ column. Following incubation of the two
proteins, Ssb2 was specifically retained on the column (Fig.
4D), confirming the results of the two-hybrid screen.
AtTDX Interacts with the ATPase Domain of Ssb2 via Its TPR
Domain--
To determine which region of AtTDX mediates its binding
to Ssb2, we constructed a series of deleted derivatives in the pBDGal4 vector, and the resulting constructions were tested for interaction with Ssb2. As reported above, the thioredoxin domain of AtTDX is not
required for its interaction with Ssb2. Further dissection of the
remaining protein indicated that the TPR domain located between
residues 112 and 213 (Fig. 5A)
is necessary for the interaction between AtTDX and Ssb2.

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Fig. 5.
AtTDX-Ssb2 interaction involves the TPR
domain of AtTDX and the ATPase domain of Ssb2 in the yeast two-hybrid
system. A, schematic representation of AtTDX and its
derivative proteins fused to the Gal4 DNA-binding domain. B,
schematic representation of Ssb2 and its derivative proteins fused to
the Gal4 DNA-activating domain. Characteristic domains of AtTDX and
Ssb2 are indicated above the concerned regions. Interactions
were determined by -galactosidase lift assay as follows: +++, ++,
and indicate strong positive, medium, and no interaction,
respectively.
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Likewise, to characterize which domains of Ssb2 were necessary for its
interaction with AtTDX, we next generated deleted derivatives of
SSB2 that were expressed as fusions to the Gal4 activation domain. Two-hybrid assays revealed that the deletion of either the
central peptide binding domain (Ssb2
-(387-613)) or the 10-kDa carboxyl-terminal domain (Ssb2
-(550-613)) of Ssb2 did not affect the Ssb2-AtTDX interaction (Fig. 5B). In contrast, a
derivative of Ssb2 lacking the ATPase domain (Ssb2
-(1-386)) was no
longer capable of interacting with AtTDX.
The Interaction between AtTDX and Ssb2 Is Sensitive to Oxidative
Stress--
Eukaryotic cells continuously produce ROS from multiple
sources. Both thioredoxin and Hsp protein families are among the
cellular enzymes that protect cells against damage induced by ROS.
Because AtTDX and Ssb2 each belong to one of these families, we
wondered whether the AtTDX-Ssb2 interaction that we observed might be
regulated in response to an oxidative stress. To test such an
hypothesis, we first decided to analyze the effect of oxygen peroxide
(H2O2) that is known to induce an oxidative
stress in yeast cells (39). Second, we decided to perform again a
two-hybrid assay to test the effect of H2O2,
because the yeast two-hybrid system involving Gal4 has been shown to be
adapted to the study of protein interactions under oxidative stress
(40).
Serial dilutions of cells co-expressing Ssb2 fused to the Gal4
activation domain and AtTDX fused to the Gal4 DNA-binding domain were
plated on minimal medium containing increasing amounts of H2O2, in the presence and absence of histidine.
In the presence of histidine, the addition of
H2O2 up to 1.5 mM on
histidine-containing plates impaired cell growth only very weakly (Fig.
6A, upper panels). In the
absence of histidine, cells were able to grow in the presence of
H2O2 at concentrations as low as 0.5 mM, confirming that Ssb2 and AtTDX interact each other.
However, in a striking contrast, the addition of higher concentrations
of H2O2 severely prevented cell growth on
plates lacking histidine, no growth being observed as soon as 1 mM H2O2 was added to the medium
(Fig. 6A, lower panels). A similar result was obtained when
the thiol-oxidizing agent diethyl maleate was used (data not shown).
These results therefore suggested that in the presence of oxidizing
compounds, the Ssb2-AtTDX interaction is abolished.

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Fig. 6.
Interaction between AtTDX and Ssb2 is
sensitive to oxidative stress in a two-hybrid experiment.
A, increasing concentrations of H2O2
cause the disruption of AtTDX-Ssb2 interaction; B, the
interaction between Ssb2 and AtTDX -(270-380) remains insensitive to
H2O2. Four independent transformed cells at
serial dilutions were tested. For each type of interaction, the
upper panels contain histidine (+His) and the
lower panels contain 20 mM 3-aminotriazole
(+3AT).
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We next tried to decipher whether the thioredoxin domain of AtTDX might
be involved in the above reported regulation of Ssb2-AtTDX interaction.
We therefore used the AtTDX derivative lacking the thioredoxin domain
(AtTDX
-(270-380)) in place of the full-length AtTDX in the
two-hybrid assays, and we tested the transformed cells for their
sensitivity to H2O2. Results of the experiments showed that, in this case, the addition of H2O2
only weakly impaired the growth of the cells, in both the presence and
absence of histidine (Fig. 6B). Thus, the thioredoxin domain
of AtTDX appears to be involved in the H2O2
regulation of Ssb2-AtTDX interaction.
The Thioredoxin Active Site of AtTDX Is Involved in the Response to
Oxidative Stress--
To gain further insights on the role of the
thioredoxin domain of AtTDX in the
H2O2-dependent regulation of
Ssb2-AtTDX interaction, we constructed two new mutant derivatives of
AtTDX. In the first, we replaced the first cysteine of the thioredoxin
domain active site (Cys-304) by a serine residue
(AtTDXSer304). For all the thioredoxins tested so far, the
replacement of the first cysteine of the active site was demonstrated
to inhibit the first step of the reduction process of the target by
preventing the thioredoxin domain to bind to the target protein (41). A second mutant was generated by replacing the second cysteine (Cys-307) of the thioredoxin domain active site by a serine residue
(AtTDXSer307). In this case, the first step of the
reduction of the target by thioredoxin occurs while the second step is
abolished, therefore leading to a covalent link between thioredoxin and
its target protein, with thioredoxin being unable to be released from
the intermediate complex (42). Both AtTDX mutant alleles were
cloned in-frame downstream from the Gal4 DNA-binding domain and tested by two-hybrid assays for their interaction with Ssb2 under
H2O2 regulation. The results shown in Fig.
7A clearly demonstrated that,
unlike what was observed with the wild-type AtTDX, the addition of 1 mM H2O2 had no effect on the cells
expressing either AtTDXSer302 or AtTDXSer307
fused to the Gal4 DNA-binding domain and grown in the absence of
histidine. Thus, in two-hybrid assays, each of the two mutations mimics
the behavior of the large deletion
-(270-380) that removed the
entire thioredoxin domain from AtTDX. Taken together, these results
demonstrated that the thioredoxin active site of AtTDX is directly
responsible for the H2O2 sensitivity of the
Ssb2-AtTDX interaction and suggest that, in response to oxidative
stress, the complex formed between AtTDX and Ssb2 is disrupted via the reducing power of the thioredoxin domain of AtTDX.

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Fig. 7.
The thioredoxin domain of AtTDX and the
conserved Cys-20 in the ATPase domain of Ssb2 are determinants for
sensitivity of AtTDX/Ssb2 interaction to oxidative stress in the
two-hybrid system. A, interaction between AtTDX and
Ssb2 is restored under oxidative stress by the replacement of both
Cys-304 to Ser-304 and Cys-307 to Ser-307 in AtTDX. B,
interaction between AtTDX and Ssb2 is also restored under oxidative
stress by the replacement of the conserved Cys-20 to Ser-20 in the
ATPase domain of Ssb2. All the interactions were tested through four
serial dilutions of YRG2 transformed cells on minimal medium containing
1 mM H2O2 or not.
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The Conserved Cys-20 in Ssb2 ATPase Domain Is Involved in the
Release of AtTDX/Ssb2 Interactants--
The disulfide
reductase activity of the thioredoxin domain of AtTDX and the
involvement of the thioredoxin active site in the
H2O2-mediated regulation of AtTDX-Ssb2
interaction prompted us to investigate whether a cysteine residue
within the Ssb2 protein would be targeted by the thioredoxin active
site under oxidative stress. The Ssb2 protein contains only two
cysteines, one located in the ATPase domain at position 20 (Cys-20) and
the second in the peptide-binding domain at position 455. We noticed
that only Cys-20 of the ATPase domain is highly conserved among Ssb2
homologues from eukaryotes. To test whether the
H2O2 regulation of the Ssb2-AtTDX interaction
might rely on this residue, we constructed a mutated version of Ssb2 in
which Cys-20 was replaced by a serine residue (Ssb2Ser20).
This new Ssb2 allele was cloned in-frame downstream from the activation
domain of Gal4, and the resulting construction was used in two-hybrid
experiments performed as described above. We first checked that the
mutation did not affect the interaction between Ssb2Ser20
and AtTDX in the absence of H2O2 (data not
shown). Next we observed that the Ssb2Ser20-AtTDX
interaction was resistant to the presence of
H2O2 (Fig. 7B), in contrast to the
interaction between the wild-type Ssb2 and AtTDX. These results
therefore indicate that the oxidation of Cys-20 of Ssb2 is necessary to
release the Ssb2-AtTDX interaction under oxidative stress.
 |
DISCUSSION |
During the last 10 years, an increasing number of genes encoding
members of the thioredoxin family have been reported in all prokaryotic
and eukaryotic organisms. In plants, the complexity of the thioredoxin
reductase/thioredoxin systems was first shown by the sequencing of
A. thaliana expressed sequence tags that demonstrated the
presence of at least five thioredoxin h-encoding genes in this
organism (35). Today, more than 18 thioredoxin and thioredoxin-like
sequences have been found in A. thaliana (43). Here, we
report the cloning and the characterization of AtTDX, a novel and
striking member of the thioredoxin family from A. thaliana.
AtTDX is the first member of the thioredoxin family described to date
that possesses an extra domain with tetratricopeptide repeats. This
domain interacts strongly with the ATPase domain of Ssb2, a member of
the Hsp70 family. Moreover, the thioredoxin active site of AtTDX and a
conserved cysteine residue within the ATPase domain of Ssb2 were shown
to mediate the release of the AtTDX-Ssb2 interaction under oxidative
stress conditions.
AtTDX, a New Thioredoxin-like TPR-containing Protein--
By using
yeast mutant cells that do not express endogenous thioredoxin, we have
isolated by functional complementation AtTDX, a novel thioredoxin
containing protein from A. thaliana. The AtTDX protein
displays an interesting bipartite structure encompassing both a
thioredoxin domain and a TPR repeat containing domain is highly similar
to the co-chaperone Hip protein. Both in vitro and in
vivo assays demonstrated that AtTDX has disulfide reductase activity and therefore that AtTDX could be assigned to the thioredoxin superfamily. In both prokaryotic and eukaryotic cells, several proteins
containing a thioredoxin catalytic domain fused to an unrelated extra
domain have been described (43). However, the function of the
associated domain has been established in two instances only, the human
cytosolic PICOT, a 37-kDa protein kinase C-interacting protein with
amino-terminal thioredoxin domain (44), and the plant APS reductases,
three 50-kDa exhibiting a carboxyl-terminal thioredoxin domain (45).
The functional characterization of such proteins was of particular
interest because it demonstrated coordinated functions between each domain.
Among the members of the thioredoxin family, AtTDX is the first
described as comprising TPR repeats associated to the thioredoxin domain. TPRs are 34-amino acid motifs that were originally identified in yeast (46, 47) and then found in a large number of both prokaryotic
and eukaryotic proteins (36). TPR repeats mediate protein-protein
interactions. TPR-containing proteins play diverse roles in many
cellular processes like cell cycle regulation, transcriptional repression, heat shock response, protein kinase inhibition, and peroxisomal protein transport (36). It is noteworthy that the TPR
domain of AtTDX is more particularly related to TPR proteins known to
interact with members of the heat-shock protein family. In particular,
we found that the TPR repeats of AtTDX are highly related to those of
Hip and Sti1p (the yeast Hop counterpart), with Hip being a
co-chaperone that specifically binds to Hsp70 and Hop providing a
physical link between Hsp70 and Hsp90 (48). In addition, AtTDX displays
a significant degree of similarity with TPR domains of protein
phosphatase 5, cyclophilin CyP40, and FKBP52, all factors known to
interact with Hsp90 chaperones (11).
AtTDX Is Capable of Interacting with a Yeast Hsp70
Chaperone--
The homology of AtTDX with HIP suggested that AtTDX
could interact with heat-shock proteins through its TPR repeats. By
using a heterologous two-hybrid strategy, we indeed isolated one yeast protein capable of interacting with AtTDX, the Ssb2 protein, a member
of the yeast Hsp70 family (38).
Interaction domain mapping experiments favor the hypothesis that the
AtTDX-Ssb2 interaction is specific and does not result from the
targeting of AtTDX by Ssb2 in response to overproduction and/or
misfolding. Indeed, we show here that the ATPase domain of Ssb2
mediates its interaction with AtTDX, whereas it is well established
that misfolded proteins are selectively recognized by the
peptide-binding domain of Hsp70 chaperones (13). Furthermore, we show
that a single cysteine replacement within the thioredoxin active site
of AtTDX strongly enhances the AtTDX-Ssb2 interaction, whereas it was
shown that such a mutation has a very limited impact on thioredoxin
structure (49, 50). Finally, we searched for Hsp70 genes in
A. thaliana data bases and found that the
AtHsc70-1 gene (51) encodes the closest A. thaliana homologue of Ssb2. The two proteins Ssb2 and AtHsc70-1
share 53% identical residues (68% similarity). We cloned the
AtHsc70-1 gene into the pADGal4 vector and succeeded in
obtaining a two-hybrid interaction with AtTDX, even weaker than that
involving Ssb2 (not shown). These results, considered as a whole, are
strong evidence that AtTDX is a new Hsp70 interactant.
Surprisingly, we did not isolate any clone encoding Hsp70 proteins from
the Ssa family in our two-hybrid screens. Ssa and Ssb share 65%
identity (more than 72% of similarity) in their ATPase domain, which
is precisely the region of Ssb2 that interacts with AtTDX. In our
screens, only positive clones strongly interacting with AtTDX were
selected, and one can imagine that proteins interacting with AtTDX with
a low specificity were not retained. We also did not isolate
clones encoding members of the Hsp90 proteins, because of the high
degree of similarity between the TPR repeats of AtTDX and the Hop/Sti1
proteins. The absence of such proteins among the positives clones
suggests that AtTDX does not act as a physical link between Hsp70 and
Hsp90 proteins, as Hop does (48).
The fact that AtTDX interacts with Ssb2 and with an A. thaliana Ssb homologue raises the question as to how the AtTDX
function correlates with Hsp70 chaperone activity. Ssb proteins are
ribosome-associated chaperones that interact with nascent chain and
likely play an important role in early protein folding events (38, 52).
Several partner proteins that regulate Hsp70 function as a molecular
chaperone have already been identified, including positive (Hsp40 and
HIP) and negative (CHIP and Bag-1) regulators (18, 21-23). HIP and Bag-1, like AtTDX, interact with the Hsp70 ATPase domain (21, 54) and
functionally compete in regulating the in vivo chaperone activity of Hsp70 (55), within the cycle between the ADP- and ATP-bound
states. Interestingly, AtTDX and AtHIP are highly homologous in their
amino-terminal interacting TPR region. Because both are expressed in
A. thaliana, one could imagine that they sequentially interact with some common Hsp70 at different times and/or in different circumstances during the plant life. The next step in understanding AtTDX function will be to investigate in which of the ATP/ADP states
Ssb2 interacts with AtTDX and whether AtTDX binds to Ssb2 by displacing
Hsp70 cofactors or by synergy with other co-chaperones. This could
bring new elements to determine whether AtTDX as a new Hsp70
co-chaperone.
A Redox Switch Dissociates the AtTDX-Hsp70 Complex--
One major
result from our two-hybrid experiments indicated that the AtTDX-Ssb2
interaction is specifically released upon oxidative stress. Both the
removal of the thioredoxin domain from AtTDX and the replacement of one
cysteine from the thioredoxin active site by a serine residue renders
the AtTDX-Ssb2 interaction insensitive to oxidative stress. This is the
demonstration that the thioredoxin domain of AtTDX is directly involved
in the oxidative process that dissociates AtTDX from Ssb2. Furthermore,
we showed that Cys-20 located in the ATPase domain of Ssb2 is not
necessary for AtTDX-Ssb2 complex formation but is mandatory for its
dissociation. Taken altogether, our results demonstrate that a redox
switch governs the association/dissociation of AtTDX-Ssb2 complex.
Oxidizing conditions like exposure to H2O2 are
known to cause disulfide bonds to form and chaperone function to be
turned on. Proteins from distinct Hsp families were also shown to be affected in their activity and/or conformation by the redox status. The
murine small Hsp25 that carries a single Cys residue equilibrates between reduced protein and protein dimer, depending on the
oxidoreduction conditions (56). The E. coli Hsp33 has
been described recently (57) as a chaperone with an on-off mode of
activity that uses reactive disulfide bonds as molecular switches.
Exposure to hydrogen peroxide causes zinc to be released from Hsp33
conserved cysteines and disulfide bond formation and the chaperone
function to be turned on. Another example is given by Tsai et
al. (58) that showed that the human protein-disulfide isomerase is
capable of redox-driven chaperone activity. All these examples are
consistent with our results that show an in vivo redox
regulation of a member of the Hsp70 family by a thioredoxin.
Recently, Hsp70 chaperoning activity was shown to be increased by
environments that mimics oxidative stress (59). Because association
with peptides under oxidative conditions is not reversible by reducing
agents, it has been proposed that other chaperone-associated factors
are required for substrate release. AtTDX may be one of these
chaperone-associated factors, acting as an oxidative stress detector
through its disulfide reductase activity. In the case of AtTDX/Ssb2
dissociation, involvement of AtTDX disulfide reductase activity and
oxidation of Ssb2 cysteine 20 are two mandatory events demonstrated by
the present work. At present, the exact function of Ssb2 Cys-20 under
normal conditions and oxidative stress remains to be demonstrated. A
survey of all Hsp70 of bacteria, fungi, animals, and plants shows the
presence of a conserved cysteine in the position homologue to that of
Ssb2 Cys-20. This suggests that the mechanism of a
redox-dependent release is conserved. Moreover, analysis of
the Ssb2 sequence with Swiss-PDB-Viewer and Rasmol programs proposed
that Cys-20 is located in a small convex cavity near the protein
surface and is thus easily accessible for disulfide bond
formation/oxidation. These findings suggest that Ssb2 Cys-20 may be
engaged in a disulfide bond with a third partner, which could be
another Hsp70 molecule in a self-association (53) or another protein
with conserved cysteine. How thioredoxin activity can affect, directly
or indirectly, the redox state of Ssb2 Cys-20 under oxidative stress
remains unsolved. Several hypotheses exist that require further
experiments, including fine analysis of determinants and other partners
involved in the interaction.