The thioredoxin-thioredoxin reductase system can function in vivo as an alternative system to reduce oxidized glutathione in Saccharomyces cerevisiae

Article abstract of DOI:10.1074/jbc.M109.062844

Cellular mechanisms that maintain redox homeostasis are crucial, providing buffering against oxidative stress. Glutathione, the most abundant low molecular weight thiol, is considered the major cellular redox buffer in most cells. To better understand how cells maintain glutathione redox homeostasis, cells of Saccharomyces cerevisiae were treated with extracellular oxidized glutathione (GSSG), and the effect on intracellular reduced glutathione (GSH) and GSSG were monitored over time. Intriguingly cells lacking GLR1 encoding the GSSG reductase in S. cerevisiae accumulated increased levels of GSH via a mechanism independent of the GSH biosynthetic pathway. Furthermore, residual NADPH-dependent GSSG reductase activity was found in lysate derived from glr1 cell. The cytosolic thioredoxin-thioredoxin reductase system and not the glutaredoxins (Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in glr1 cells reduced GSSG accumulation, increased GSH levels, and reduced cellular glutathione E _h′. Conversely, deletion of TRX1 or TRX2 in the glr1 strain led to increased accumulation of GSSG, reduced GSH levels, and increased cellular E_h′. Furthermore, it was found that purified thioredoxins can reduce GSSG to GSH in the presence of thioredoxin reductase and NADPH in a reconstituted in vitro system. Collectively, these data indicate that the thioredoxin-thioredoxin reductase system can function as an alternative system to reduce GSSG in S. cerevisiae in vivo.

Full text of DOI:10.1074/jbc.M109.062844

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 9, pp. 6118–6126, February 26, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
The Thioredoxin-Thioredoxin Reductase System Can
Function in Vivo as an Alternative System to Reduce Oxidized
□
S
Glutathione in Saccharomyces cerevisiae*
Received for publication, September 6, 2009, and in revised form, November 8, 2009 Published, JBC Papers in Press, December 1, 2009, DOI 10.1074/jbc.M109.062844
Shi-Xiong Tan‡§1, Darren Greetham¶, Sebastian Raeth§, Chris M. Grant¶, Ian W. Dawes‡§2, and Gabriel G. Perrone‡§
From the ‡Ramaciotti Centre for Gene Function Analysis and the §School of Biotechnology and Biomolecular Sciences, University of
New South Wales, Sydney, New South Wales 2052, Australia and the ¶Faculty of Life Sciences, University of Manchester,
Manchester M13 9PT, United Kingdom
Cellular mechanisms that maintain redox homeostasis are
crucial, providing buffering against oxidative stress. Glutathi-
one, the most abundant low molecular weight thiol, is consid-
ered the major cellular redox buffer in most cells. To better
understand how cells maintain glutathione redox homeostasis,
cells of Saccharomyces cerevisiae were treated with extracellular
oxidized glutathione (GSSG), and the effect on intracellular
reduced glutathione (GSH) and GSSG were monitored over
time. Intriguingly cells lacking GLR1 encoding the GSSG reduc-
tase in S. cerevisiae accumulated increased levels of GSH via a
mechanism independent of the GSH biosynthetic pathway. Fur-
thermore, residual NADPH-dependent GSSG reductase activity
was found in lysate derived from glr1 cell. The cytosolic thiore-
doxin-thioredoxin reductase system and not the glutaredoxins
(Grx1p, Grx2p, Grx6p, and Grx7p) contributes to the reduction
of GSSG. Overexpression of the thioredoxins TRX1 or TRX2 in
glr1 cells reduced GSSG accumulation, increased GSH levels,
and reduced cellular glutathione Ehꢀ. Conversely, deletion of
TRX1 or TRX2 in the glr1 strain led to increased accumulation of
GSSG, reduced GSH levels, and increased cellular Ehꢀ. Further-
more, it was found that purified thioredoxins can reduce GSSG
to GSH in the presence of thioredoxin reductase and NADPH in
a reconstituted in vitro system. Collectively, these data indicate
that the thioredoxin-thioredoxin reductase system can function
as an alternative system to reduce GSSG in S. cerevisiae in vivo.
importance of redox homeostasis, it is vital to understand the
mechanisms involved in maintaining normal redox homeosta-
sis. The high abundance of glutathione (1–10 mM) in cells and
its low redox potential (Ϫ240 mV) make the glutathione system
a major intracellular redox buffer in most cells (1, 4, 5). Previous
studies have indicated that cells lacking Cu,Zn-superoxide dis-
mutase, genes involved in glutathione homeostasis or those
involved in the pentose phosphate pathway can alter intracel-
lular the glutathione redox balance (6–9), demonstrating the
complexity of redox systems. Therefore, understanding the fac-
tors that affect cellular glutathione homeostasis contributes
importantly to our overall understanding of how cells maintain
intracellular redox homeostasis.
In Saccharomyces cerevisiae, reduced glutathione (GSH)3
biosynthesis occurs in the cytosol via two ATP-dependent
steps. The first is catalyzed by ␥-glutamylcysteine synthetase
encoded by GSH1 (10), which catalyzes condensation of gluta-
mate and cysteine. Glutathione synthetase encoded by GSH2
(11) catalyzes the addition of glycine to ␥-glutamylcysteine to
form GSH (12). GSH is an important antioxidant, because yeast
strains altered in their GSH redox state are sensitive to oxidant-
induced stress (13–16).
Several cellular processes use the reducing power of GSH
including glutathione peroxidases, which mediates the reduc-
tion of hydrogen peroxide to water, generating stoichiometric
quantities of oxidized glutathione (GSSG) in the process. GSSG
can be recycled to GSH by glutathione reductase using reducing
equivalents supplied by NADPH (2). GLR1 encodes the only
known GSSG reductase in S. cerevisiae. Mutants deleted for
GLR1 are viable, overaccumulate GSSG, and are hypersensitive
to oxidants (17, 18). Besides glutathione, cells have other redox-
active molecules such as the thioredoxins and glutaredoxins
that participate in oxidative stress defense.
Aerobic organisms maintain a reducing intracellular envi-
ronment to facilitate appropriate functioning of numerous pro-
cesses, including the proper folding of proteins and mainte-
nance of their activity. Cellular mechanisms that maintain
redox homeostasis are crucial, because they provide a buffer
against conditions that may perturb the redox environment of
cells and/or induce oxidative stress (1, 2). Changes to redox
state such as exposure to reactive oxygen species (ROS) can lead
to detrimental effects including oxidation of sulfhydryl groups
in proteins that can result in loss of activity (3). Given the
Thioredoxins are low molecular weight thiol-disulfide oxi-
doreductases with a conserved CGPC active site and are
involved in the reduction of enzymes that are oxidized to form
a disulfide (19). These enzymes include 3Ј-phosphoadenosine
5Ј-phosphosulfate (PAPS) reductase required for sulfur assim-
ilation and ribonucleotide reductase (RNR) required for
maintaining dNTPs for DNA synthesis (3). S. cerevisiae con-
tains two cytoplasmic thioredoxins encoded by TRX1 and
* This work was supported by grants from the Australian Research Council
and the Cancer Institute New South Wales.
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1.
1 Recipient of an Endeavor International Postgraduate Research Scholarship
from the Australian Government.
2 To whom correspondence should be addressed. Tel.: 61-2-9385-2089; Fax: 3 The abbreviations used are: GSH, reduced glutathione; GSSG, oxidized glu-
61-2-9385-1050; E-mail: i.dawes@unsw.edu.au.
tathione; HA, hemagglutinin.
6118 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 9•FEBRUARY 26, 2010

Alternative System to Reduce GSSG
TABLE 1
S. cerevisiae strains used in this study
Strain
Genotype
Source
W303
MATa ura3–52 leu2–3,112 trp1–1 ade2–1 his3–11
Chris M. Grant
Grant et al. (14)
Grant et al. (17)
This study
JL3
gsh1::LEU2 in W303
CY7
glr1::TRP1 in W303
ST201
gsh1::LEU2 glr1::TRP1 in W303
ST203
glr1::TRP1 GSH1-HA::kanMX in W303
grx6:: natMT1 grx7:: hphNT1 glr1:: TRP1 in W303
grx1::LEU2 grx2::HIS3 glr1::TRP1 in W303
grx1::LEU2 grx2::HIS3 grx6:: natMT1 grx7:: hphNT1 glr1:: TRP1 in W303
trx1::hphNT1 glr1::TRP1 in W303
This study
ST204
This study
ST208
This study
ST209
This study
ST301
This study
ST302
trx2::hphNT1 glr1::TRP1 in W303
This study
BY4743
BY4743 gsh1)
BY4743 (glr1)
BY4743 (hgt1)
MATa/MAT␣ his3⌬1/ his3⌬1 leu2⌬0/leu2⌬0 met15⌬0/MET15 LYS2/lys⌬0 ura3⌬0/ura3⌬0
EUROSCARF
EUROSCARF
EUROSCARF
EUROSCARF
gsh1::kanMX in BY4743
glr1::kanMX in BY4743
hgt1::kanMX in BY4743
TRX2. Mutants deleted for both TRX1 and TRX2 are defective (34, 35). However, cells deleted for TRR1 over-accumulate both
in sulfate assimilation, consistent with the role of the two thio-
redoxins as hydrogen donors for PAPS reductase (20). Trx2p is
involved in oxidative stress defense because the trx2 mutant is
sensitive to H2O2 (21, 22). Thioredoxin reductase encoded by
TRR1 reduces oxidized thioredoxins directly. Mutants deleted
for TRR1 are sensitive to hydrogen peroxide and are auxotro-
phic for methionine (23, 24).
GSH and GSSG (18). Furthermore, the trr1 glr1, and the trx1
trx2 glr1 mutants are inviable (18, 36), indicating a potential
link between these two systems.
To further understand how cells maintain intracellular glu-
tathione redox balance, we investigated the mechanisms of how
cells respond to increased levels of GSSG in vivo. Surprisingly,
we found that cells deleted for GLR1, encoding the only known
glutathione reductase in S. cerevisiae, are able to convert GSSG
to GSH. This led us to investigate possible alternative mecha-
nisms that can reduce GSSG in cells.
Glutaredoxins are small proteins that act as thiol oxi-
doreductases involved in the reduction of protein disulfides or
glutathione-protein mixed disulfides (25). In S. cerevisiae, the
dithiol glutaredoxins are encoded by GRX1 and GRX2 and con-
tain CPYC at their active site (26). Cells lacking GRX1 are sen-
sitive to superoxide generating agents and hydroperoxides,
whereas cells lacking GRX2 are sensitive to hydroperoxides but
not superoxides, indicating that the functions of GRX1 and
GRX2 are only partially overlapping (26), and the two genes are
differentially regulated (27).
EXPERIMENTAL PROCEDURES
Yeast Strains and Plasmids—The S. cerevisiae strains used
(Table 1) were derived from BY4743 (Open Biosystems) or
from W303 as described previously (14). Single gene deletion
strains in the W303 background were generated using PCR to
amplify the disrupted allele and flanking region (ϳ200–400 bp)
from the strains available from the S. cerevisiae Genome Dele-
tion Project (37) or using the appropriate plasmids from the
PCR tool kit (38). The amplified DNA product was transformed
into W303 with selection on YEPD medium containing 220
␮g/ml geneticin (for KanMX selection, Invitrogen), 100 ␮g/ml
nourseothricin (for natMT1 selection, Sigma) or 300 ␮g/ml
hygromycin (for hphNT1 selection, Invitrogen). Transforma-
tion was performed using the lithium acetate-based method
(39). Each deletant was confirmed by PCR analysis on genomic
DNA isolated from the respective strains. Chromosomal HA-
tagging of GSH1 was performed using the PCR-based targeted
gene insertion method described (38). The resulting PCR frag-
ment was transformed into W303 to generate strains contain-
ing an in-frame GSH1 allele with a C-terminal chromosomal
HA tag. Multiple mutants were generated by mating and spor-
ulation using standard yeast genetic methods.
S. cerevisiae contains five monothiol glutaredoxins. Grx3p,
Grx4p, and Grx5p contain the CGFS motif in their active site.
Grx3p and Grx4p regulate the nuclear localization of the Aft1p
transcription factor involved in iron homeostasis (28, 29),
whereas Grx5p participates at the late stages of the biosynthesis
of Fe/S clusters in the mitochondrion (30, 31). GRX6 and GRX7
encode two monothiol glutaredoxins that contain active site
sequences CSYS and CPYS, respectively. Both Grx6p and
Grx7p are localized in the Golgi, but are not involved in oxida-
tive protein folding (32). Cells lacking GRX6 and/or GRX7 were
shown to be sensitive to oxidants hydrogen peroxide and di-
amide in one study (32), but showed no obvious phenotype,
except for mild resistance to the glycosylation inhibitor tunica-
mycin in another (33). The biological role of Grx6p and Grx7p
remains unknown.
Although the thioredoxins and glutaredoxins are similar in
structure and have overlapping functions (22), they are regu-
lated in a different manner. The oxidized disulfide form of thi-
oredoxin is reduced directly by thioredoxin reductase using
NADPH as the electron donor, whereas glutaredoxin is reduced
by glutathione (GSH), and the oxidized glutathione (GSSG) is
in turn reduced by glutathione reductase using electrons
donated by NADPH (25). Glr1p is highly specific for the reduc-
tion of GSSG. Alternatively, while thioredoxins have been
found to reduce a broad spectrum of substrates, GSSG was
The N-terminally HA-tagged galactose-inducible TRR1,
TRX1, and TRX2 constructs were generated using the Gateway
system (Invitrogen) according to the manufacturer’s instruc-
tions. Plasmids carrying the respective genes from the S. cerevi-
siae ORF collection (40) were shuffled into pDonr-221 using
the BP reaction. The entry clones obtained were then used to
introduce the respective genes into pAG416GAL1-ccdB (41)
using the LR reaction. The resulting clones were sequenced for
generally not considered to be a good substrate for thioredoxins verification.
FEBRUARY 26, 2010•VOLUME 285•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6119

Alternative System to Reduce GSSG
Growth Conditions—Yeast strains were grown in YEPD ϫ100 magnification using a graticule calibrated against a slide
medium containing 1% (w/v) yeast extract, 2% (w/v) peptone, micrometer grating. For each strain, at least 100 cells were mea-
2% (w/v) glucose, or synthetic defined medium (SD) containing sured and the average cell volume determined.
0.17% yeast nitrogen base without amino acids and ammonium
Analysis of Glutathione Reductase Activity in Cell Lysates—
sulfate, 0.5% ammonium sulfate, and supplemented with min- Cells were harvested according to the procedure used for West-
imal auxotrophic requirements. For induction of genes under ern blot analysis. Cell lysates were clarified by ultracentrifuga-
the control of the GAL1 promoter, cells were grown for 2 days tion (100,000 ϫ g, 4 °C, 20 min), and proteins present in each
in SD medium and re-inoculated to synthetic defined medium soluble fraction were concentrated using an Amicon Ultra cen-
containing 2% (w/v) galactose (SD-GAL) instead of glucose. trifugal filters (3 kDa cutoff; Millipore) at 4 °C. Cell lysates were
Auxotrophic supplements were added as follow: 76 mg/liter of mixed with an equal volume of ice-cold phosphate-buffered
L-tryptophan, L-isoleucine, and L-valine, 260 mg/liter L-leucine, saline and re-concentrated using the above columns. This proc-
380 mg/liter L-histidine, 26 mg/liter uracil, and 18 mg/liter ade- ess was repeated twice to reduce the levels of small molecular
nine. Appropriate supplements were omitted for the purpose of weight compounds including GSH and GSSG. Lysates prepared
selecting plasmids.
in this manner were assayed immediately. Glutathione reduc-
Western Blot Analysis—Cells were grown as stated, and ϳ40 tase activity was determined by monitoring oxidation of
ml of cells were harvested and lysed in lysis buffer (0.1 M Tris- NADPH (340 nm). Complete (total) reaction mixtures con-
HCl, pH 8, 20% (v/v) glycerol, 2 mM phenylmethylsulfonyl fluo- tained 20 ␮l of cell lysate in phosphate-buffered saline and
ride, 1% SDS). Protein content of cell lysate was determined and NADPH (600 ␮M). Reactions were initiated by addition of 1 mM
adjusted to 2 ␮g/␮l in sample-loading buffer (5% (v/v) ␤-mer- GSSG, and reduction of GSSG was estimated by monitoring
captoethanol, 40% (v/v) glycerol, Tris-HCl, pH 6.8). Samples GSSG-dependent NADPH oxidation, which was reflected by a
were boiled for 10 min, and 30 ␮g of each sample were loaded decrease in absorbance at 340 nm. The background level of
onto a 4–12% NuPAGE gel (Invitrogen). Anti-HA was diluted NADPH oxidation was also determined by monitoring
to 1:1000, and anti-Pgk1p was diluted to 1:5000.
NADPH oxidation by the corresponding cell lysate in the
␤-Galactosidase Assay—Cells were grown to exponential absence of added GSSG. GSSG-dependent oxidation of
phase (A600 ϳ 0.5), harvested, and analyzed for ␤-galactosidase NADPH was calculated by subtracting the values obtained for
activity as described (42). ␤-Galactosidase activity was GSSG-independent NADPH oxidation from the value obtained
expressedasunitsofONPG(O-nitrophenyl-␤-D-galactopyrano- for the “complete” reaction mixture. Activity was normalized to
side) hydrolyzed (nmol) per min over total protein (mg), and the protein concentration present in each sample and
the extent of induction was the ratio of the ␤-galactosidase expressed as nmol of NADPH oxidized per minute per mg of
activity of the treated sample to that of the untreated sample. protein (nmol minϪ1 mgϪ1).
For each strain, three independent transformants were assayed.
Reduction of GSSG by Purified Trx1p or Trx2p and Trr1p in
Determination of Intracellular Glutathione Levels—Reduced Vitro—Plasmids expressing six-histidine residue-tagged ver-
(GSH) and oxidized glutathione (GSSG) were estimated as sions of Trx1p (pBAD-YTRX1) and Trr1p (pBAD-YTRR1)
described previously (14, 43). Cells were grown in SD medium were a kind gift from Barry Rosen (45). Trx2p was amplified by
(40 ml) to an A600 of 1 (ϳ2 ϫ 107 cells/ml) and harvested by PCR and cloned into the pBAD expression vector (Invitrogen).
centrifugation (10,000 ϫ g, 20 s), washed in 3 ml of ice-cold PBS Histidine-tagged proteins were purified by Ni2ϩ-nitrilotriace-
buffer, resuspended in 350 ␮l of ice-cold 8 mM HCl containing tic acid chromatography and protein purity checked on SDS-
3% (w/v) 5-sulfosalicilic acid and disrupted using a mini-bead PAGE gels. Glutathione reductase (Glr1p) derived from S. cer-
beater (1-min high speed, 4 °C). The lysate was clarified by evisiae was purchased from Sigma. Reduction of GSSG by
centrifugation (5 min, 10,000 ϫ g, 4 °C), and the supernatant Glr1p or Trr1p was measured in vitro with purified proteins.
used to determine total glutathione. GSSG was determined Reaction mixes contained NADPH (600 ␮M) and either Glr1p
by reacting GSH in samples with 2-vinylpyridine prior to the (6.67 nM), or Trr1p (0.25 ␮M) and Trx1p (1.5 ␮M), or, Trr1p
assay. Levels of GSH and GSSG were calculated as described (0.25 ␮M) and Trx2p (1.5 ␮M). Reactions were started by addi-
in Ref. 43.
tion of 1 mM GSSG and reduction of GSSG to GSH estimated by
Estimation of Intracellular Redox Environment—Intracellu- following the decrease in oxidation of NADPH (340 nm).
lar values of the GSSG/2GSH half-cell redox potential (gluta-
RESULTS
thione EhЈ) were determined as an indicator of the cellular
redox environment (1). These were calculated from intracellu-
Treatment of Cells with Extracellular GSSG Leads to Accu-
lar concentrations of GSH and GSSG using the Nernst equa- mulation of Intracellular GSH—To investigate if perturbation
tion: EhЈ ϭ E0 Ϫ 2.303(RT/nF)log10[(GSH)2/(GSSG)]. Glutathi- in cellular glutathione redox environment can lead to a change
one EhЈ values are expressed in mV; E0 is the standard potential in cellular EhЈ, intracellular GSSG concentration was increased
for reduced glutathione (Ϫ0.24 mV) at pH 7, R is the gas con- by adding GSSG to the medium, and the changes in intracellu-
stant (8.31 J molϪ1 KϪ1), T is the absolute temperature (303 K), lar glutathione concentration and cellular EhЈ of the glutathione
n is the number of electrons transferred (2), and F is the Faraday redox couple monitored over time.
constant (96,406 J VϪ1 molϪ1). To estimate intracellular gluta-
In wild-type and glr1 cells, addition of 100 ␮M GSSG extra-
thione concentration, cell volumes were estimated from cell cellularly led to an increase in intracellular glutathione concen-
major (a) and minor (b) axes using the formula (44): V ϭ tration from 1 to 3 mM in 20 min and reaching a maximum of 6
␲a2b/6. Cell dimensions were determined microscopically at mM after 2 h of treatment (Fig. 1A). Increased intracellular glu-
6120 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 9•FEBRUARY 26, 2010

Alternative System to Reduce GSSG
Treatment of Cells with Extracel-
lular GSSG Leads to Accumulation
of GSH in a Gsh1p- and Glr1p-inde-
pendent Manner—The first com-
mitted step for de novo synthesis of
GSH is catalyzed by ␥-glutamylcys-
teine synthetase encoded by GSH1
(10). To determine whether ele-
vated GSH accumulation in the glr1
cells treated with GSSG was associ-
ated with the expression of GSH1,
GSH1 expression was monitored in
the glr1 mutant over time using a
GSH1::lacZ construct. The protein
level of Gsh1p was also monitored
using Western blot analysis of a
glr1 strain harboring a chromo-
somally HA-tagged GSH1 under
the control of its native promoter
(Fig. 2, A and B).
The transcript level of GSH1 and
the level of Gsh1-HAp did not
increase in glr1 cells after exposure
FIGURE1. EffectofextracellularGSSGonintracellularglutathioneredoxhomeostasis. Thewild-type, hgt1
to GSSG for 60 min (Fig. 2, A and B),
despite a significant increase in
and glr1 cells were grown to exponential phase (A600, 0.5) in SD medium exposed to 100 ␮M GSSG, and
intracellular glutathione was determined over the indicated time course. A, total intracellular glutathione;
B, GSH; and C, GSSG of the indicated strains were determined. D, cellular glutathione EhЈ was determined over
the time course. Error bars indicate the S.E. of four samples from two independent experiments.
intracellular concentration of GSSG
and GSH (Fig. 1). From these data, it
is unlikely that transcriptional regu-
tathione accumulation was dependent on the high affinity glu-
tathione transporter Hgt1p/Opt1p (encoded by YJL212c),
because cells lacking HGT1 exhibited a minimal increase in
intracellular glutathione upon GSSG treatment (Fig. 1A). Upon
GSSG treatment, an accumulation of total glutathione in wild-
type and glr1 cells was therefore largely due to uptake of GSSG
by Hgt1p. Previous studies found that Hgt1p is the sole high
affinity GSH transporter in S. cerevisiae (46). Here it was dem-
onstrated that Hgt1p could also transport GSSG into cells,
because a minor increase in glutathione level was observed in
the hgt1 mutant treated with GSSG. This indicates the presence
of another less efficient route for GSSG uptake in S. cerevisiae.
Upon GSSG treatment, the glutathione accumulated by
wild-type cells was predominantly in the reduced form GSH
(Fig. 1B). This increase in GSH was likely to be due to the uptake
of GSSG, followed by conversion of GSSG to GSH by GSSG
reductase Glr1p, because glr1 cells accumulated predominantly
GSSG upon GSSG treatment (Fig. 1C). Interestingly, the level of
GSH in the glr1 mutant increased by up to 2-fold after exposure
to GSSG for 1 h (Fig. 1B). Cells deleted for GLR1 have a more
oxidizing intracellular environment as reflected from the EhЈ
determination (Fig. 1D). Although exposure of wild-type and
glr1 cells to GSSG led to a change in intracellular GSH and
GSSG concentration (Fig. 1, B and C), cellular EhЈ remained
relatively constant over the time of treatment. These data indi-
cate that in the presence of excess GSSG, cells increase the level
of GSH possibly to maintain cellular EhЈ. Increased GSH accu-
mulation in the glr1 cells treated with GSSG could have been
due to elevated GSH synthesis and/or reduction of GSSG by a
Glr1p-independent mechanism.
lation of GSH1 is involved but it is not possible to exclude that
elevated GSH accumulation in glr1 cells treated with GSSG may
have been due to post-translational regulation of Gsh1p activ-
ity. To explore this issue and to determine whether increased
GSH in glr1 cells treated with GSSG was dependent on GSH
synthesis per se, GSH1 was deleted in the glr1 mutant.
The gsh1 glr1 mutant is auxotrophic for glutathione but was
found to be capable of utilizing GSSG as a sole source of gluta-
thione (Fig. 2D). In exponential phase, gsh1 glr1 cells grown in
medium supplemented with 25 ␮M GSH accumulated intracel-
lular GSSG to a similar level as that of glr1 cells (Fig. 2C). The
gsh1 glr1 mutant treated with GSSG accumulated total gluta-
thione (Fig. 2E), and GSSG increased by more than 4-fold rela-
tive to untreated cells at time 0 (Fig. 2G). Surprisingly, upon
GSSG treatment the gsh1 glr1 mutant also displayed an accu-
mulation of intracellular GSH (Fig. 2F). To exclude any possi-
bility of GSH contamination being present in the GSSG stock
solution used for this experiment, a separate GSSG stock solu-
tion was treated with 2-vinylpyridine to derivatize any GSH
that may have been present at trace levels. Comparable data
were obtained for GSSG pretreated with 2-vinylpyridine (Fig. 2,
E and F). The lower level of GSH and total glutathione, but
similar level of GSSG in the gsh1 glr1 mutant may indicate that
cells degrade excess GSSG, and/or in the gsh1 glr1 mutant
uptake of GSH cannot keep pace with oxidation of GSH to
GSSG followed by degradation of GSSG. These data indicate
that the increase in GSH level observed in the glr1 mutant
treated with GSSG was due to the presence of a Glr1p-indepen-
dent system capable of reducing GSSG to GSH.
FEBRUARY 26, 2010•VOLUME 285•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6121

Alternative System to Reduce GSSG
Presence of Residual GSSG Reduc-
tase Activity in the glr1 Mutant—To
further examine the Glr1p-inde-
pendent GSSG reductase activity in
vitro, whole cell lysates of the wild-
type and glr1 strains were assayed
for their ability to reduce GSSG
using an NADPH oxidation assay.
The cell lysate derived from glr1
cells displayed some residual GSSG-
dependent oxidation of NADPH
(ϳ2% of wild-type cells; Fig. 2H),
indicating the presence of residual
GSSG reductase activity. This re-
sidual Glr1p-independent GSSG
reductase activity was NADPH-de-
pendent because
a
GSSG-de-
pendent oxidation of NADH was
not observed (data not shown). Cell
lysates derived from glr1 cells that
were subjected to heating (100 °C,
10 min) did not exhibit residual
GSSG reduction activity (Fig. 2H),
indicating that the residual Glr1p-
independent GSSG reductase activ-
ity was heat labile and may be due to
the presence of a protein in cells,
other than Glr1p, with the capacity
to reduce GSSG to GSH.
The in vivo data and the total cell
lysate analysis (Fig. 2) indicate that
S. cerevisiae has an alternative sys-
tem to Glr1p that reduces GSSG
in a NADPH-dependent manner.
Although the residual GSSG reduc-
tase activity was relatively low,
based on the in vitro assay, it still
provided sufficient activity to
reduce GSSG in vivo, as observed in
the gsh1 glr1 cells (Fig. 2). We sur-
mised that there are two Glr1p-in-
dependent systems that could possi-
bly reduce GSSG to GSH in
S. cerevisiae, both of which are de-
pendent on NADPH. These are the
glutaredoxin and the thioredoxin
systems.
Grx1p, Grx2p, Grx6p, and Grx7p
Do Not Contribute to Reduction of
GSSG in Cells Deleted of GLR1—Re-
cently, it was demonstrated that
mammalian glutaredoxin 2 (Grx2)
FIGURE 2. Extracellular GSSG leads to an increase in intracellular GSH in a GSH1- and GLR1-independent
manner. A, exponentially growing glr1 cells transformed with the GSH1::lacZ construct were treated with 100
␮M GSSG and assayed for ␤-galactosidase activity. B, chromosomal copy of GSH1 in the glr1 mutant was tagged
withHA. Exponentiallygrowingcellsweretreatedwith100 ␮M GSSGovertheindicatedtime, andthecelllysate
analyzedforGsh1-HApusinganti-HAantibodies. Pgk1pwasdetectedusinganti-Pgk1pantibodiesasaloading
control. C, indicated mutants were grown to A600 0.5 in SD medium supplemented with 25 ␮M GSH. Cells were
harvested and analyzed for intracellular GSH and GSSG levels. D, indicated strains were grown for 2 days and can use thioredoxin reductase to
A600 adjusted to 1.0 before 5 ␮l of the diluted cultures were spotted onto SD plates containing no glutathione,
reduce several substrates, including
GSSG. This indicates that apart
from the conventional glutathione
reductase pathway, the glutare-
doxin-thioredoxin reductase path-
25 ␮M GSH, or 25 ␮M GSSG. E–G, cells of gsh1 glr1 were treated with 100 ␮M GSSG (diamond), 100 ␮M GSSG
treated with 2-vinyl-pyridine (square), or untreated (triangle). E, intracellular total glutathione; F, GSH; and G,
GSSG were determined at the indicated time. Error bars indicate the S.E. of four samples of two independent
experiments. H, GSSG reductase activity in glr1 cell lysate. Wild-type and glr1 cells were grown to exponential
phase, and the cell lysate of each strain was assayed for GSSG reductase activity.
6122 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 9•FEBRUARY 26, 2010

Alternative System to Reduce GSSG
way may be able to act as an alternate system to regenerate GSH
from GSSG. Grx2 has a CSYC motif at its active site, and muta-
tion of the second cysteine residue to serine (CSYC to CSYS)
does not abolish Grx2 activity (47). In S. cerevisiae, GRX6
encodes a monothiol glutaredoxin containing the CSYS motif.
Furthermore it was shown that glutaredoxins Grx1p, Grx2p,
Grx6p, and Grx7p displayed activity toward the artificial disul-
fide substrate hydroxyethyl disulfide (HEDS) (26, 32), indicat-
ing that these proteins may be able to reduce GSSG. Therefore,
to investigate the possible involvement of these glutaredoxins
in reducing GSSG, their respective genes were deleted in the
glr1 strain background. Monothiol glutaredoxins Grx3p,
Grx4p, and Grx5p were not included in these analyses because
they do not contain the CSYS motif and do not display activity
toward HEDS (48, 49).
The grx6 grx7 glr1, grx1 grx2 glr1, and grx1 grx2 grx6 grx7 glr1
cells did not show any obvious growth defect in SD medium
(supplemental Fig. S1), and cell lysates of these strains still
retained GSSG reductase activity at a level comparable to that
of the glr1 strain (supplemental Fig. S1) indicating that the
Grx1p, Grx2p, Grx6p, and Grx7p do not contribute signifi-
cantly to the residual reductase activity observed in glr1 cells.
The Thioredoxin-Thioredoxin Reductase System Can Reduce
GSSG to GSH—Cells deleted for TRR1 or TRX1 and TRX2 over-
accumulate oxidized as well as reduced glutathione (18, 36).
Furthermore, the trr1 glr1 and the trx1 trx2 glr1 mutants are
inviable (18, 50), indicating that there is a close relationship
between the thioredoxin and the glutathione systems. It was
recently demonstrated that Trx1p of S. cerevisiae is able to
reduce GSSG in vitro (51), indicating a potential role of the
thioredoxin in reducing GSSG to GSH in vivo. To investigate
the possible role of the thioredoxin-thioredoxin reductases in
GSSG reduction in vivo, the TRX1, TRX2, or TRR1 genes were
separately placed under the control of the GAL1 promoter,
overexpressed in the glr1 mutant, and cell lysates were analyzed
for GSSG reductase activity, intracellular GSH/GSSG levels,
and the cellular glutathione EhЈ were estimated.
Overexpression of TRR1, TRX1, and TRX2 was each verified
using Western blot analysis (Fig. 3A). Overexpression of TRR1,
TRX1, or TRX2 led to a significant (p value Ͻ 0.05) reduction in
cellular GSSG level, and there was a significant increase (p
value Ͻ 0.05) in GSH levels when TRX1 and TRX2 were over-
expressed (Fig. 3B). The glr1 cells overexpressing TRX1 or
TRX2 displayed a more reducing environment as indicated by
the glutathione EhЈ (Fig. 3B, bottom panel).
To further examine the relative contribution of Trx1p and
Trx2p in reduction of GSSG in vivo, the glr1, trx1 glr1, and trx2
glr1 mutants were treated with extracellular GSSG and exam-
ined for intracellular glutathione concentration. The trx1 glr1
pGAL1-TRR1–3XHA, pGAL1-TRX1–3XHA, or pGAL1-TRX2–3XHA. The trans-
formed cells were grown to stationary phase in SD minus uracil medium and
re-inoculated into SD-GAL minus uracil medium, and the cultures were
allowed to grow for 18 h to A600 0.5–1.0 before harvesting. A, Western blot
analysis (top) of cell lysate using anti-HA antibody. B, intracellular concentra-
tion of GSSG, GSH, and glutathione EhЈ of the indicated strains. Error bars
FIGURE 3. The thioredoxin-thioredoxin reductase system can reduce indicate S.E. from six replicates from two independent experiments. Asterisks
GSSG in vivo. A and B, overexpression of TRR1, TRX1, and TRX2 in glr1 cells (*) indicate significant differences (p value Ͻ0.05) compared with the glr1
reduces intracellular GSSG. The glr1 mutant was transformed with mutant harboring the control vector.
FEBRUARY 26, 2010•VOLUME 285•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6123

Alternative System to Reduce GSSG
FIGURE 5. Trx1p and Trx2p can facilitate reduction of GSSG to a similar
level in vitro. Purified proteins were reconstituted with the indicated com-
pounds, and the rate of NADPH oxidation to NADPϩ was monitored at 340
nm. Error bars indicate the S.D. of three replicates.
and the trx2 glr1 mutant had a small but significantly higher
level of intracellular GSSG relative to the glr1 mutant in
untreated conditions (Time 0, Fig. 4A). Upon GSSG treatment,
the trx1 glr1 and the trx2 glr1 mutant accumulated slightly
more GSSG at 60 min (Fig. 4A) and displayed less GSH accu-
mulation compared with the glr1 mutant at 20 and 60 min (Fig.
4B). Furthermore, glr1 cells deleted for TRX1 or TRX2 dis-
played a more oxidizing environment as determined using the
glutathione EhЈ compared with the glr1 single mutant (Fig. 4C).
To investigate the relative efficacy of Trx1p and Trx2p in
GSSG reduction, in vitro reduction of GSSG was examined in
the presence of purified Trr1p, NADPH, Trx1p, or Trx2p. To
compare the efficacy of GSSG reduction by the thioredoxin-
thioredoxin reductase system with Glr1p, purified Glr1p was
also examined.
The data from Fig. 5 indicate that Trr1p cannot reduce GSSG
without the presence of Trx1p or Trx2p. The Trx1p-Trr1p and
Trx2p-Trr1p combinations reduced GSSG with similar efficacy
(ϳ18–19 nmol/min/mg), although at a much lower rate com-
pared with Glr1p (52 nmol/min/mg). The respective Vmax
(␮mol/min/mg) and Km (mM) values for GSSG reduction were
as follows: Trx1p (Vmax ϭ 101.4; Km 2.6), Trx2p (Vmax ϭ 101;
K
m ϭ 2.5), and Glr1p (Vmax ϭ 118; Km 0.08). These data indi-
cate that the thioredoxin-thioredoxin system is able to reduce
GSSG, although Glr1p is much more efficient in doing so.
The above data support the role of thioredoxin in reducing
GSSG in vivo. A single thioredoxin is able to support the con-
version of GSSG to GSH in vivo, because both the trx1 glr1 and
the trx2 glr1 mutants were still able to accumulate GSH in the
presence of excess GSSG, and the in vitro data indicate that
Trx1p and Trx2p reduces GSSG at a similar efficacy.
indicated time. A, GSSG and B, GSH of the indicated strains were determined.
FIGURE 4. The trx1 glr1 and trx2 glr1 strains have a decreased capacity to C, cellular glutathione EhЈ was determined over the indicated time. Error bars
convert GSSG to GSH compared with the glr1 strain in vivo. The indicated indicate the S.E. of six biological replicates from two independent experi-
strains were grown to exponential phase (A600, 0.5), in SD medium treated ments. Asterisks (*) indicate significant differences (p value Ͻ 0.05) compared
with 100 ␮M GSSG, and intracellular glutathione was determined over the with the glr1 mutant at the indicated time.
6124 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 9•FEBRUARY 26, 2010

Alternative System to Reduce GSSG
comparable to that of D. melanogaster (10 mMϪ1 minϪ1) (51–
53). These rate constants are at least ϳ100–400 lower when
comparing the reduction of GSSG to the reduction of insulin as
a substrate by the thioredoxin-thioredoxin reductase system
(19). However, given that GSSG may accumulate in the milli-
molar range, it was proposed that this rate is physiologically
significant, and the reaction may occur in vivo (53). Addition-
ally, the activity for GSSG reduction by the thioredoxins versus
glutathione reductase measured in vitro using recombinant
forms of the enzymes, or native forms isolated from cells may
also differ to that of the native forms of the respective enzymes
in intact cells. Differences in the availability of NADPH, GSSG,
and/or other factors, and the presence of competing side reac-
tions may also influence the relative contribution of the gluta-
thione reductase and thioredoxin systems for reduction of
GSSG in vivo. The contribution of the Trr1p/Trx1,2p system is
likely to become more important when cells experience certain
stress conditions (e.g. oxidative) where the burden on the glu-
tathione redox system is known to increase and GSSG overac-
cumulation may ensue (7, 8, 14, 15). The observations that: 1)
the gsh1 glr1 mutant can utilize GSSG as the sole source of
glutathione; 2) the gsh1 glr1 strain accumulates GSH when
challenged with exogenous GSSG and that overexpression of
thioredoxins or thioredoxin reductase can reduce GSSG accu-
mulation in glr1 cells; and 3) deletion of a single thioredoxin
reduces the accumulation of GSH in the presence of excess
GSSG all provide further support that the thioredoxin-thiore-
doxin reductase system can function to reduce GSSG to GSH in
vivo. In a recent study of Arabidopsis, it was demonstrated that
mutants lacking an isoform of glutathione reductase still
exhibit dynamic reduction of GSSG in vivo under oxidative
stress conditions (54). It was also demonstrated that the Arabi-
dopsis thioredoxins were capable of reducing GSSG in vitro.
These findings led to the proposal that the thioredoxin-thiore-
doxin reductase system may function as a backup for cytosolic
glutathione reductase in Arabidopsis. Therefore, it is likely that
the thioredoxins-thioredoxin reductase system and the gluta-
thione reductase system function to reduce GSSG not only in
plants and yeast, but also in a similar manner in other species.
DISCUSSION
Glutathione is the major low molecular weight redox buffer
in most cells (1). Understanding the mechanism that controls
the production of GSH and the recycling of GSSG in cells will
provide further insight into how cellular redox homeostasis is
maintained. Here, it was demonstrated that cells have robust
mechanisms to maintain cellular glutathione redox homeosta-
sis. Excess oxidized glutathione is readily converted to GSH,
mainly through the activity of the glutathione reductase Glr1p.
The in vivo data presented here also strongly indicate that
excess GSSG leads to an increase in cellular GSH level, and this
increase is independent of GSH1 and can occur in the absence
of glutathione reductase encoded by GLR1. The presence of an
alternative system to reduce GSSG in S. cerevisiae was demon-
strated, and this system is likely to be the cytosolic thioredoxin-
thioredoxin reductase system.
Several pieces of evidence support the role of GSSG reduc-
tion to GSH by the thioredoxin-thioredoxin reductase system.
First, it was demonstrated that overexpression of either of the
cytosolic thioredoxins Trx1p or Trx2p, or the thioredoxin
reductase Trr1p led to a decrease in intracellular GSSG level
in the glr1 mutant in vivo. Second, deletion of TRX1 or TRX2 in
the glr1 mutant treated with GSSG reduced the accumulation
of GSH compared with the glr1 mutant alone. Furthermore, it
was previously shown that in S. cerevisiae, the trx1 trx2 double
mutant and the trr1 mutant overaccumulate both oxidized and
reduced glutathione. In addition, the trr1 glr1 double mutant
and the trx1 trx2 glr1 triple mutants are inviable (18), indicating
that the thioredoxin-thioredoxin reductase system overlaps
with the glutathione reductase system. These data indicate that
the cytosolic thioredoxin-thioredoxin reductase system can
prevent the overaccumulation of GSSG in cells lacking the glu-
tathione reductase Glr1p. The synthetic lethality associated
with simultaneous disruption of the thioredoxin-thioredoxin
reductase and glutathione reductase systems makes it difficult
to determine the extent to which, or indeed whether or not,
other systems contribute in reduction of GSSG in vivo. In
addition the role that additional factors including GSH syn-
thesis, GSH/GSSG degradation, transport of GSH/GSSG
between cellular compartments, and/or formation/turnover
of glutathione-protein mixed disulfides play in maintenance
of overall cellular and organellar redox homeostasis requires
further investigation.
Acknowledgments—We thank Anita Ayer for providing the grx6 and
grx7 mutants and Geoff Kornfeld for helpful suggestions.
Although thioredoxins have been shown to reduce a broad
spectrum of substrates, GSSG is generally not considered to be
a good substrate for thioredoxins (19, 35). It has been demon-
strated that the thioredoxin-thioredoxin reductase system in
the malaria parasite Plasmodium falciparum and fruit fly Dro-
sophila melanogaster can convert GSSG to GSH in vitro (52,
53). Recently the second order rate constant of GSSG reduction
by thioredoxin 1 and thioredoxin reductases in S. cerevisiae was
determined by in vitro reconstitution of the purified proteins
(51). It was shown that purified yeast thioredoxin 1 and thiore-
doxin reductase can reduce GSSG with a second order rate
REFERENCES
1. Schafer, F. Q., and Buettner, G. R. (2001) Free Radic Biol. Med. 30,
1191–1212
2. Perrone, G. G., Tan, S. X., and Dawes, I. W. (2008) Biochim. Biophys. Acta
1783, 1354–1368
3. Rietsch, A., and Beckwith, J. (1998) Annu. Rev. Genet. 32, 163–184
4. Meister, A., and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 711–760
5. Hwang, C., Sinskey, A. J., and Lodish, H. F. (1992) Science 257, 1496–1502
6. Drakulic, T., Temple, M. D., Guido, R., Jarolim, S., Breitenbach, M., At-
tfield, P. V., and Dawes, I. W. (2005) FEMS Yeast Res. 5, 1215–1228
7. Ng, C. H., Tan, S. X., Perrone, G. G., Thorpe, G. W., Higgins, V. J., and
Dawes, I. W. (2008) Free Radic Biol. Med. 44, 1131–1145
8. Tan, S. X., Teo, M., Lam, Y. T., Dawes, I. W., and Perrone, G. G. (2009)
Mol. Biol. Cell 20, 1493–1508
constant of 6 mM minϪ1. This rate constant of the thiore-
Ϫ1
doxin-thioredoxin reductase system in S. cerevisiae is similar to
that established for the analogous system in Escherichia coli and
9. Perrone, G. G., Grant, C. M., and Dawes, I. W. (2005) Mol. Biol. Cell 16,
218–230
FEBRUARY 26, 2010•VOLUME 285•NUMBER 9
JOURNAL OF BIOLOGICAL CHEMISTRY 6125

Alternative System to Reduce GSSG
10. Ohtake, Y., and Yabuuchi, S. (1991) Yeast 7, 953–961
11. Inoue, Y., Sugiyama, K., Izawa, S., and Kimura, A. (1998) Biochim. Biophys.
Acta 1395, 315–320
Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M.,
Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F.,
Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub,
M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier,
M., M’Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Re-
vuelta, J. L., Riles, L., Roberts, C. J., Ross-MacDonald, P., Scherens, B.,
Snyder, M., Sookhai-Mahadeo, S., Storms, R. K., Ve´ronneau, S., Voet, M.,
Volckaert, G., Ward, T. R., Wysocki, R., Yen, G. S., Yu, K., Zimmermann,
K., Philippsen, P., Johnston, M., and Davis, R. W. (1999) Science 285,
901–906
12. Grant, C. M., MacIver, F. H., and Dawes, I. W. (1997) Mol. Biol. Cell 8,
1699–1707
13. Izawa, S., Inoue, Y., and Kimura, A. (1995) FEBS Lett. 368, 73–76
14. Grant, C. M., MacIver, F. H., and Dawes, I. W. (1996) Curr. Genet. 29,
511–515
15. Grant, C. M., Perrone, G., and Dawes, I. W. (1998) Biochem. Biophys. Res.
Commun. 253, 893–898
16. Stephen, D. W., and Jamieson, D. J. (1996) FEMS Microbiol. Lett. 141, 38. Janke, C., Magiera, M. M., Rathfelder, N., Taxis, C., Reber, S., Maekawa,
207–212
H., Moreno-Borchart, A., Doenges, G., Schwob, E., Schiebel, E., and Knop,
M. (2004) Yeast 21, 947–962
17. Grant, C. M., Collinson, L. P., Roe, J. H., and Dawes, I. W. (1996) Mol.
Microbiol. 21, 171–179
39. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153,
163–168
18. Muller, E. G. (1996) Mol. Biol. Cell 7, 1805–1813
19. Holmgren, A. (1985) Annu. Rev. Biochem. 54, 237–271
20. Muller, E. G. (1991) J. Biol. Chem. 266, 9194–9202
21. Kuge, S., and Jones, N. (1994) EMBO J. 13, 655–664
22. Draculic, T., Dawes, I. W., and Grant, C. M. (2000) Mol. Microbiol. 36,
1167–1174
40. Gelperin, D. M., White, M. A., Wilkinson, M. L., Kon, Y., Kung, L. A.,
Wise, K. J., Lopez-Hoyo, N., Jiang, L., Piccirillo, S., Yu, H., Gerstein, M.,
Dumont, M. E., Phizicky, E. M., Snyder, M., and Grayhack, E. J. (2005)
Genes Dev. 19, 2816–2826
41. Alberti, S., Gitler, A. D., and Lindquist, S. (2007) Yeast 24, 913–919
23. Machado, A. K., Morgan, B. A., and Merrill, G. F. (1997) J. Biol. Chem. 272, 42. Rose, M., and Botstein, D. (1983) Methods Enzymol. 101, 167–180
17045–17054
43. Vandeputte, C., Guizon, I., Genestie-Denis, I., Vannier, B., and Lorenzon,
G. (1994) Cell Biol. Toxicol. 10, 415–421
24. Pearson, G. D., and Merrill, G. F. (1998) J. Biol. Chem. 273, 5431–5434
25. Holmgren, A. (1989) J. Biol. Chem. 264, 13963–13966
44. Wheals, A. E. (1982) Mol. Cell Biol. 2, 361–368
26. Luikenhuis, S., Perrone, G., Dawes, I. W., and Grant, C. M. (1998) Mol. 45. Mukhopadhyay, R., Shi, J., and Rosen, B. P. (2000) J. Biol. Chem. 275,
Biol. Cell 9, 1081–1091
21149–21157
27. Grant, C. M., Luikenhuis, S., Beckhouse, A., Soderbergh, M., and Dawes,
I. W. (2000) Biochim. Biophys. Acta 1490, 33–42
46. Bourbouloux, A., Shahi, P., Chakladar, A., Delrot, S., and Bachhawat, A. K.
(2000) J. Biol. Chem. 275, 13259–13265
28. Ojeda, L., Keller, G., Muhlenhoff, U., Rutherford, J. C., Lill, R., and Winge, 47. Johansson, C., Lillig, C. H., and Holmgren, A. (2004) J. Biol. Chem. 279,
D. R. (2006) J. Biol. Chem. 281, 17661–17669
29. Pujol-Carrion, N., Belli, G., Herrero, E., Nogues, A., and de la Torre-Ruiz, 48. Tamarit, J., Belli, G., Cabiscol, E., Herrero, E., and Ros, J. (2003) J. Biol.
M. A. (2006) J. Cell Sci. 119, 4554–4564 Chem. 278, 25745–25751
30. Rodríguez-Manzaneque, M. T., Tamarit, J., Bellí, G., Ros, J., and Herrero, 49. Herrero, E., and de la Torre-Ruiz, M. A. (2007) Cell Mol. Life Sci. 64,
E. (2002) Mol. Biol. Cell 13, 1109–1121 1518–1530
7537–7543
31. Mu¨hlenhoff, U., Gerber, J., Richhardt, N., and Lill, R. (2003) EMBO J. 22, 50. Trotter, E. W., and Grant, C. M. (2003) EMBO Rep. 4, 184–188
4815–4825
51. Bao, R., Zhang, Y., Lou, X., Zhou, C. Z., and Chen, Y. (2009) Biochim.
Biophys. Acta 1794, 1218–1223
32. Mesecke, N., Spang, A., Deponte, M., and Herrmann, J. M. (2008) Mol.
Biol. Cell 19, 2673–2680
52. Kanzok, S. M., Fechner, A., Bauer, H., Ulschmid, J. K., Mu¨ller, H. M.,
Botella-Munoz, J., Schneuwly, S., Schirmer, R., and Becker, K. (2001) Sci-
ence 291, 643–646
33. Izquierdo, A., Casas, C., Mu¨hlenhoff, U., Lillig, C. H., and Herrero, E.
(2008) Eukaryot. Cell 7, 1415–1426
34. Holmgren, A., and Bjo¨rnstedt, M. (1995) Methods Enzymol. 252, 199–208 53. Kanzok, S. M., Schirmer, R. H., Turbachova, I., Iozef, R., and Becker, K.
35. Arne´r, E. S., Zhong, L., and Holmgren, A. (1999) Methods Enzymol. 300,
226–239
(2000) J. Biol. Chem. 275, 40180–40186
54. Marty, L., Siala, W., Schwarzla¨nder, M., Fricker, M. D., Wirtz, M., Sweet-
love, L. J., Meyer, Y., Meyer, A. J., Reichheld, J. P., and Hell, R. (2009) Proc.
Natl. Acad. Sci. U.S.A. 106, 9109–9114
36. Trotter, E. W., and Grant, C. M. (2002) Mol. Microbiol. 46, 869–878
37. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K.,
6126 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 9•FEBRUARY 26, 2010

Cell Biology:
The Thioredoxin-Thioredoxin Reductase
System Can Function in Vivo as an
Alternative System to Reduce Oxidized
Glutathione in Saccharomyces cerevisiae
Shi-Xiong Tan, Darren Greetham, Sebastian
Raeth, Chris M. Grant, Ian W. Dawes and
Gabriel G. Perrone
J. Biol. Chem. 2010, 285:6118-6126.
doi: 10.1074/jbc.M109.062844 originally published online December 1, 2009
Access the most updated version of this article at doi: 10.1074/jbc.M109.062844
Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites.
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
Supplemental material:
http://www.jbc.org/content/suppl/2009/12/01/M109.062844.DC1.html
This article cites 54 references, 30 of which can be accessed free at
http://www.jbc.org/content/285/9/6118.full.html#ref-list-1