Oxidation of the Yeast Mitochondrial Thioredoxin Promotes Cell Death.
ABSTRACT Abstract Aims: Yeast, like other eukaryotes, contains a complete mitochondrial thioredoxin system comprising a thioredoxin (Trx3) and a thioredoxin reductase (Trr2). Mitochondria are a main source of reactive oxygen species (ROS) in eukaryotic organisms, and this study investigates the role of Trx3 in regulating cell death during oxidative stress conditions. Results: We have previously shown that the redox state of mitochondrial Trx3 is buffered by the glutathione redox couple such that oxidized mitochondrial Trx3 only accumulates in mutants simultaneously lacking Trr2 and a glutathione reductase (Glr1). We show here that the redox state of mitochondrial Trx3 is important for yeast growth and its oxidation in a glr1 trr2 mutant induces programmed cell death. Apoptosis is dependent on the Yca1 metacaspase, since loss of YCA1 abrogates cell death induced by oxidized Trx3. Our data also indicate a role for a mitochondrial 1-cysteine (Cys) peroxiredoxin (Prx1) in the oxidation of Trx3, since Trx3 does not become oxidized in glr1 trr2 mutants or in a wild-type strain exposed to hydrogen peroxide in the absence of PRX1. Innovation: This study provides evidence that the redox state of a mitochondrial thioredoxin regulates yeast apoptosis in response to oxidative stress conditions. Moreover, the results identify a signaling pathway, where the thioredoxin system functions in both antioxidant defense and in controlling cell death. Conclusions: Mitochondrial Prx1 functions as a redox signaling molecule that oxidizes Trx3 and promotes apoptosis. This would mean that under conditions where Prx1 cannot detoxify mitochondrial ROS, it induces cell death to remove the affected cells. Antioxid. Redox Signal. 00, 000-000.
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ABSTRACT: Previous reports described thioredoxin (Trx) as a very poor reductant for mammalian MsrB2 and MsrB3, which lack a resolving Cys residue. In contrast, we here report that Trx could reduce both MsrB2 and MsrB3 enzymes, similarly to the reduction of mammalian MsrA. We demonstrated that functional Trx is required for the reduction of these enzymes. We further identified MsrB2- or MsrB3-Trx complexes formed through intermolecular disulfide bonds involving catalytic residue of Trx. The present study provides evidence that the sulfenic acid intermediate of oxidized MsrBs lacking resolving Cys could interact with Trx and be directly reduced by this protein.Biochemical and Biophysical Research Communications 08/2008; 371(3):490-4. · 2.41 Impact Factor
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ABSTRACT: A grande gsh1 disruptant mutant of Saccharomyces cerevisiae was generated by crossing a petite disruptant to a wild-type grande strain. This strain was relatively stable, but generated petites at an elevated frequency, illustrating the ancillary role of glutathione (GSH) in the maintenance of the genetic integrity of the mitochondrial genome. The availability of the grande gsh1 deletant enabled an evaluation of the role of GSH in the cellular response to hydrogen peroxide independent of the effects of a petite mutation. The mutant strain was more sensitive to hydrogen peroxide than the wild-type strain but was still capable of producing an adaptive stress response to this compound. GSH was found to be essential for growth and sporulation of the yeast, but the intracellular level needed to support growth was at least two orders of magnitude less than that normally present in wild-type cells. This surprising result indicates that there is an essential role for GSH but only very low amounts are needed for growth. This result was also found in anaerobic conditions, thus this essential function does not involve protection from oxidative stress. Suppressors of the gsh1 deletion mutation were isolated by ethylmethanesulfonate mutagenesis. These were the result of a single recessive mutation (sgr1, suppressor for glutathione requirement) that relieved the requirement for GSH for growth on minimal medium but did not affect the sensitivity to H(2)O(2) stress. Interestingly, the gsh1 sgr1 mutant generated petites at a lower rate than the gsh1 mutant. Thus, it is suggested that the essential role of GSH is involved in the maintenance of the mitochondrial genome.FEMS Yeast Research 05/2001; 1(1):57-65. · 2.46 Impact Factor
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ABSTRACT: Initial observations that the budding yeast Saccharomyces cerevisiae can be induced to undergo a form of cell death exhibiting typical markers of apoptosis has led to the emergence of a thriving new field of research. Since this discovery, a number of conserved pro- and antiapoptotic proteins have been identified in yeast. Indeed, early experiments have successfully validated yeasts as a powerful genetic tool with which to investigate mechanisms of apoptosis. However, we still have little understanding as to why programmes of cell suicide exist in unicellular organisms and how they may be benefit such organisms. Recent research has begun to elucidate pathways that regulate yeast apoptosis in response to environmental stimuli. These reports strengthen the idea that physiologically relevant mechanisms of programmed cell death are present, and that these function as important regulators of yeast cell populations.Molecular Microbiology 01/2007; 62(6):1515-21. · 4.96 Impact Factor
ORIGINAL RESEARCH COMMUNICATION
Oxidation of the Yeast Mitochondrial Thioredoxin
Promotes Cell Death
Darren Greetham,* Paraskevi Kritsiligkou, Rachel H. Watkins, Zorana Carter,
Jill Parkin, and Chris M. Grant
Aims: Yeast, like other eukaryotes, contains a complete mitochondrial thioredoxin system comprising a thior-
edoxin (Trx3) and a thioredoxin reductase (Trr2). Mitochondria are a main source of reactive oxygen species
(ROS) in eukaryotic organisms, and this study investigates the role of Trx3 in regulating cell death during
oxidative stress conditions. Results: We have previously shown that the redox state of mitochondrial Trx3 is
buffered by the glutathione redox couple such that oxidized mitochondrial Trx3 only accumulates in mutants
simultaneously lacking Trr2 and a glutathione reductase (Glr1). We show here that the redox state of mito-
chondrial Trx3 is important for yeast growth and its oxidation in a glr1 trr2 mutant induces programmed cell
death. Apoptosis is dependent on the Yca1 metacaspase, since loss of YCA1 abrogates cell death induced by
oxidized Trx3. Our data also indicate a role for a mitochondrial 1-cysteine (Cys) peroxiredoxin (Prx1) in the
oxidation of Trx3, since Trx3 does not become oxidized in glr1 trr2 mutants or in a wild-type strain exposed to
hydrogen peroxide in the absence of PRX1. Innovation: This study provides evidence that the redox state of a
mitochondrial thioredoxin regulates yeast apoptosis in response to oxidative stress conditions. Moreover, the
results identify a signaling pathway, where the thioredoxin system functions in both antioxidant defense and in
controlling cell death. Conclusions: Mitochondrial Prx1 functions as a redox signaling molecule that oxidizes
Trx3 and promotes apoptosis. This would mean that under conditions where Prx1 cannot detoxify mitochondrial
ROS, it induces cell death to remove the affected cells. Antioxid. Redox Signal. 18, 376–385.
tems. Compartmentalization can protect against oxidative
stress by allowing cells to independently maintain their reg-
ulatory systems within individual organelles. Mitochondrial
redox regulation is particularly important in this context, es-
pecially, since during respiration, mitochondria are a primary
source of ROS and mitochondrial thiols are major ROS targets
(5). Glutathione (GSH) is a low molecular weight thiol com-
pound, that is found at high concentrations in most organ-
isms. It is synthesized in the cytosol and must be transported
into mitochondria via an active energy requiring process (14).
Once inside mitochondria, GSH provides reducing power,
but the resulting oxidized glutathione (GSSG) is unable to exit
this compartment and must be reduced by the glutathione
n oxidative stressoccurs when reactive oxygen species
(ROS) overwhelm the cellular antioxidant defense sys-
reductase (28). The yeast glutathione reductase (Glr1) colo-
calizes to the cytosol and mitochondria (29, 35). Nuclear genes
to be conserved for the glutathione reductase in mammalian
cells. Mitochondrial GSH is particularly important, since GSH
deficiency in mammalian cells results in widespread mito-
chondrial damage (26) and yeast strains lacking GSH are un-
damage to mitochondrial DNA (22). However, GSH is not
thought to play a major function as an antioxidant in cells (21),
although it is required to maintain the redox status of the mi-
tochondrial thioredoxin system (13, 40)
Thioredoxins are key oxidoreductases, which have been
(16). Yeast, like other eukaryotes, contains a complete mito-
and a thioredoxin reductase (TRR2) (30). The redox state of the
Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
*Current affiliation: Division of Food Sciences, School of Biosciences, University of Nottingham, Loughborough, United Kingdom.
ANTIOXIDANTS & REDOX SIGNALING
Volume 18, Number 4, 2013
ª Mary Ann Liebert, Inc.
cytoplasmicandmitochondrial thioredoxin systems appearsto
be independently maintained and cells can survive in the
absence of both systems (40). Mitochondrial Trx3 is maintained
in a reduced form in wild-type cells, but surprisingly, is
unaffected by the loss of Trr2 (40). In comparison, loss of the
cytoplasmic thioredoxin reductase (Trr1) causes cytoplasmic
thioredoxins (Trx1, Trx2) to accumulate in an oxidized form
(38). Unlike cytoplasmic thioredoxins, the redox state of mito-
chondrial Trx3 is buffered by the GSSG/2GSH redox couple,
such that oxidized mitochondrial Trx3 only accumulates in
mutants simultaneously lacking TRR2 and the glutathione
reductase (GLR1) (40). This is important, since it suggests that
the mitochondrial thioredoxin function can be regulated by the
GSH system. However, the requirement for the yeast mito-
chondrial Trx3 is as yet unknown, since it is dispensable for
growth under normal and oxidative stress conditions (30, 40).
This is surprising, since mammalian mitochondrial Trx2 is re-
quired for normal development of the mouse embryo and lack
of Trx2 results in embryonic lethality (27).
Yeast contains a single 1-cysteine (Cys) peroxiredoxin
(Prx1), which localizes to mitochondria (31). Prx’s are ubiq-
uitous, thiol-specific proteins that have multiple functions in
stress protection (41). They are divided into 1-Cys and 2-Cys
Prx’s, based on the number of Cys residues directly involved
in catalysis (41). Typical 2-Cys Prx’s are active as a dimer and
contain two redox active Cys residues that are directly in-
volved in enzyme activity. 1-Cys Prx’s contain a peroxidatic
Cys, but do not contain a resolving Cys residue. Since 1-Cys
Prx’s cannot form a disulfide, the reduction reaction is
thought to require a thiol-containing electron donor (36, 41).
Cys91 is the peroxidatic Cys residue in Prx1, which is highly
conserved in 1-Cys Prx’s from bacterial, plant, and mamma-
reduced form unless cells are subjected to oxidative stress.
Hydroperoxides oxidize Cys91 to the sulfenic acid form,
which can then be glutathionylated through a reaction with
GSH (13). This mixed disulfide is a substrate for reduction by
Trr2, in a reaction that proceeds through the formation of a
Prx1-Trr2 disulfide-bonded intermediate, regenerating re-
duced active Prx1. An additional role has been suggested for
mitochondrial glutaredoxin (Grx2) based on the observation
that Grx2 can reduce glutathionylated Prx1 in vitro (32). Trx3
in cells, although it can reduce oxidized Prx1 in vitro (13, 31).
We have suggested that Trx3 can directly reduce the sulfenic
acid form of the Prx1 peroxidatic Cys residue. This is similar
to the reduction by thioredoxin of the sulfenic acid interme-
methionine sulfoxide reductase (20). This raises the question
as to why the mitochondrial thioredoxin does not appear to
of Trx3 with Prx1 may only become important during par-
ticular growth or stress conditions.
An indication of a possible requirement for yeast Trx3 has
come from the finding that simultaneous loss of Trr2 and Glr1
causes slow growth under respiratory conditions; this growth
defect is mediated by oxidized Trx3, since deletion of TRX3
improves the growth of the glr1 trr2 mutant (40). In this cur-
rent study, we have characterized the slow growth caused by
oxidized Trx3 and we show that oxidized Trx3 promotes
programmed cell death (PCD) in a glr1 trr2 mutant. Further-
more, analysis of the Trx3 redox state in prx1 mutants shows
that Prx1 is required to oxidize Trx3 and regulates cell death
by oxidizing Trx3.
Oxidized Trx3 promotes apoptosis
We have previously shown that oxidized Trx3 causes slow
growth in a glr1 trr2 mutant (40). We examined whether Trx3
plays a role in yeast cell death in the glr1 trr2 mutant, which
might explain this growth defect. Cells were grown under
fermentative or respiratory conditions and the percentage of
apoptotic cells determined using annexin V staining of phos-
phatidylserine on the external plasma membrane, which is as
an early morphological marker of apoptosis (37). Loss of TRR2
or GLR1 alone, modestly increased the rate of apoptosis com-
pared with a wild-type strain under respiratory conditions
(Fig. 1A). In contrast, simultaneous loss of TRR2 and GLR1
caused a significant six-fold increase in apoptosis with ap-
proximately 30% of cells displaying annexin V staining. Cell
death appears to mainly be mediated by Trx3 in the glr1 trr2
mutant, since deletion of TRX3 significantly reduced an-
nexin V staining (Fig. 1A). Increased apoptosis is mainly a
response to respiratory growth conditions, since more
modest increases were detected under fermentative growth
conditions (Fig. 1A). Apoptosis was significantly increased
in all redox mutants examined under fermentative growth
conditions, but no Trx3-dependent increase was detected in
the glr1 trr2 mutant (Fig. 1A, compare glr1 trr2 and glr1 trr2
detected using the terminal dUTP nick end-labeling (TU-
NEL) method as another indicator of fungal apoptosis. Sig-
nificant TUNEL staining was detected in the glr1 trr2 mutant
grown under respiratory conditions, which was abrogated
by deletion of TRX3 (Fig. 1B).
Staining with propidium iodide (PI) was used as a control
to differentiate cell death in necrotic cells with disrupted cell
membranes. Necrosis was generally elevated under respira-
tory conditions compared with fermentative growth condi-
GLR1 (Fig. 1A). However, no Trx3-dependent increase in
necrosis was detected in the glr1 trr2 mutant. Since loss of
TRX3 did not affect apoptosis in a wild-type background,
these data indicate that a gain of Trx3 function, rather than a
simple loss of Trx3 activity, most likely promotes apoptosis in
the trr2 glr2 mutant.
Glutathione (GSH) was previously shown to maintain
the redox state of mitochondrial Trx3 (40). Our current
data suggest that GSH regulates Prx1 activity rather than
acting directly on Trx3. This provides a mechanism to
couple the redox state of mitochondrial GSH with a redox
sensor that regulates programmed cell death. Yeast Prx1
is highly conserved with the mammalian 1-cysteine (Cys)
peroxiredoxin (Prx6) and both are active as peroxidases,
which are reduced by GSH rather than thioredoxin (13,
18). The finding that Prx1 can regulate apoptosis through
modulating the oxidation state of thioredoxin may
therefore aid in understanding 1-Cys Prx reaction mech-
anisms in eukaryotic organisms.
YEAST MITOCHONDRIAL THIOREDOXIN ACTIVATES CELL DEATH 377
Trx3 promotes apoptosis in a Yca1-dependent manner
The yeast metacaspase Yca1 is known to mediate cell death
in response to various stimuli (19, 25). We therefore tested
whether apoptosis in the glr1 trr2 mutant requires Yca1. The
proportion ofapoptotic cells wasdetermined using annexin V
staining and deletion of YCA1 was found to reduce the high
rate of apoptosis in the glr1 trr2 mutant to wild-type levels
(Fig. 2A). Many different stimuli are known to induce yeast
apoptosis, and so we examined whether the glr1 trr2 mutant
shows any increased sensitivity to conditions that promote
apoptosis. Acetic acid is one such stimulus that is commonly
used to induce yeast apoptosis (4, 23). Wild-type and mutant
strains were grown to a stationary phase and spotted onto
respiratory growth media containing acetic acid (Fig. 2B).
Under these conditions, the glr1 trr2 mutant displayed a slow
growth, which was rescued by deletion of TRX3 or YCA1
(Fig. 2B). Taken together, these data indicate that oxidation of
Trx3 induces PCD in a Yca1-dependent manner.
Trx3-mediated apoptosis is not a general response
to oxidized mitochondria
One possible explanation to account for the high rate of
apoptosis in glr1 trr2 mutants is that it is a response to the
type glr1, trr2, trx3, glr1 trr2, and glr1 trr2 trx3 mutant strains
were grown to an exponential phase in fermentative (YEPD) or
respiratory (YEPGE) media. The percentage of apoptotic (An-
nexin V staining) and necrotic (propidium iodide staining) cells
is indicated. *p<0.05, **p<0.01, ***p<0.001 compared with the
wild-type control strain. (B) The proportion of apoptotic cells
was determined using the TUNEL assay in the same strains as
described above grown under respiratory conditions. *p<0.05
compared with the wild-type control strain. TUNEL, terminal
dUTP nick end-labeling; YEPD, yeast extract, peptone dex-
trose; YEPGE, yeast extract, peptone glycerol, ethanol.
Oxidized Trx3 promotes apoptosis. (A) The wild-
manner. (A) The wild-type yca1, glr1 trr2, glr1 trr2 trx3, and
glr1 trr2 yca1 mutant strains were grown to an exponential
phase in respiratory (YEPGE) media. The percentage of
apoptotic cells (Annexin V staining) is indicated. ***p<0.001
compared with the wild-type control strain. (B) Deletion of
YCA1 restores the growth of a glr1 trr2 mutant under re-
spiratory and acetic acid stress conditions. The indicated
strains were grown to a stationary phase and spotted onto
respiratory (YEPGE) media containing acetic acid.
Trx3 promotes apoptosis in a Yca1-dependent
378 GREETHAM ET AL.
oxidation state of mitochondria in this mutant. We therefore
tested whether loss of TRX3 in the glr1 trr2 mutant influ-
ences the cytoplasmic or mitochondrial GSH redox couple,
which is considered an indicator of the intracellular redox
environment. Wild-type and mutant strains were grown un-
der respiratory conditions and total GSH levels were found to
be comparable in wild-type, glr1, trr2, trx3, and glr1 trr2
mutants in agreement with previous observations (29, 40).
Importantly, deletion of TRX3 in the glr1 trr2 mutant did
not affect total GSH levels in cytoplasmic or mitochondrial
fractions (Fig. 3A). Oxidized GSSG levels are significantly
elevated in both mitochondrial and cytoplasmic fractions
from the glr1 mutant consistent with the important role of
glutathione reductase in recycling oxidized GSSG to reduced
GSH (29). Deletion of TRR2 in the glr1 mutant did not further
alter GSSG levels indicating that the glr1 trr2 mutant does not
have a more oxidized mitochondrial redox environment
compared with a glr1 mutant (Fig. 3A). Additionally, loss of
TRX3 did not affect GSSG levels in the glr1 trr2 mutant indi-
cating that the GSH redox state does not correlate with the
rates of apoptosis in these mutants.
Accurately measuring GSH levels in organelles, such as
mitochondria, can be problematic, since it involves disruptive
techniques, including cell breakage and fractionation. We vali-
dated our GSH assays using genetically encoded reduction–
oxidation-sensitive green fluorescent proteinprobes, whichhave
been designed to analyze redox states in the cytosol, mitochon-
drial intermembrane space (IMS), and matrix (17). These sensors
equilibrate with the local GSH pool and register thiol redox
changes via disulfide bond formation allowing comparisons of
the redox state in different intracellular compartments. The cy-
and the IMS rxYFP was predominantly oxidized in a wild-type
strain in agreement with previous observations (17). The IMS
rxYFP was unaffected in glr1 trr2 and glr1 trr2 trx3 mutants
compared with the wild-type strain in agreement with the idea
between the general mitochondrial redox state and apoptosis in
these mutants (Fig. 3B). We therefore examined whether the re-
dox state of Trx3 itself influences the rate of cell death.
Requirement for Trx3 Cys residues to promote
Mitochondrial Trx3 contains two redox active Cys residues
(Cys55, Cys58) within the Cys-Gly-Pro-Cys motif that is
conserved among thioredoxins from all species (Fig. 4A). In
addition, Trx3 contains two extra Cys residues (Cys80,
Cys92), which are not present in cytoplasmic Trx1 and Trx2
(30). The requirement for these extra Cys residues is unclear,
but mutation of Cys80 or Cys92 has been shown to reduce the
catalytic activity of purified Trx3 proteins in an insulin re-
duction assay by approximately 28% (2).
We constructed mutant versions of Trx3 to determine
which Cys residues are important for promoting cell death in
the glr1 trr2 mutant. For these experiments, mutant versions
of Trx3 were expressed in a glr1 trr2 trx3 mutant and the
growth was examined using plate assays. The glr1 trr2 trx3
mutant containing an empty vector grew well under respi-
ratory conditions and on media containing acetic acid,
whereas, expression of wild-type TRX3 prevented the growth
of this mutant in agreement with the finding that Trx3 pro-
motes cell death in the glr1 trr2 mutant (Fig. 4B). Mutations in
the Trx3 active site (trx3::C55S, trx3::C58S, trx3::C55S/C58S)
rescued growth under both respiratory and acetic acid stress
conditions. In contrast, mutations in the additional Cys resi-
dues (trx3::C70S, trx3::C92S) modestly improved the growth
redox mutants. (A) The wild-type glr1, trr2, trx3, glr1 trr2, and
glr1 trr2 trx3 mutant strains were grown to an exponential
phase in respiratory media (SGE) and the levels of total GSH
and oxidized GSH (GSSG) were determined in cytosolic and
mitochondrial fractions. Values shown are the means of three
independent determinations and are expressed as an nmol/mg
protein. There is no significant difference between the glr1 trr2
and glr1 trr2 trx3 mutants. (B) Redox Western blot analysis.
The wild-type, glr1 trr2 and glr1 trr2 trx3 mutant strains ex-
pressing cytosol-, IMS- or matrix rxYFP were grown to an
exponential phase in respiratory media (SGE). Cell extracts
were separated on nonreducing SDS-PAGE gels and Westen
blots probed with anti-GFP antibodies. Cells were treated with
DTT to fully reduce the redox probes as indicated. Oxidized
and reduced proteins are indicated. GSSG, oxidized glu-
tathione; GSH, glutathione; IMS, mitochondrial intermembrane
space; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel
electrophoresis; SGE, synthetic glycerol ethanol.
Regulation of GSH metabolism in mitochondrial
YEAST MITOCHONDRIAL THIOREDOXIN ACTIVATES CELL DEATH379
active site Cys residues. These data indicate that the redox
activity of Trx3 is important for promoting cell death.
Mitochondrial Prx1 regulates the redox state of Trx3
We made use of the Trx3 Cys mutants to search for possi-
ble Trx3 disulfide-bonded intermediates. Previous analyses
have indicated that disulfide-bonded thioredoxin interac-
the second Cys residue of the CXXC active site motif (Cys58
in Trx3). Analysis of Trx3 by nonreducing sodium dodecyl sul-
fate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed
intermediatesinaglr1 trr2 mutant(Fig.5A).MutationofCys55
(trx3::C55S/C58S) prevented the formation of these disulfides
suggesting that they represent physiological intermediates
formed with the firstCys residue oftheCXXC active site motif.
No disulfide-bonded interactions where stabilized in trx3 mu-
tants lacking the additional Cys80 or Cys92 residues (data not
shown). Additionally, no interactions were detected in cells
grown under fermentative conditions indicating that their for-
mation correlates with the oxidation of Trx3 under respiratory
growth conditions (Fig. 5A).
We have previously shown that the mitochondrial thior-
edoxin system can support Prx1 activity in an in vitro peroxi-
dase assay and suggested that Trx3 can directly reduce the
sulfenic acid form of the Prx1 peroxidatic Cys residue (13). We
therefore tested whether Prx1 accounts for one of the Trx3
disulfide-bonded intermediates detected in the glr1 trr2 mu-
tant. Western blot analysis of Prx1 indicated that a significant
proportion of Prx1 forms an intermediate with the trx3::C58S
mutant (Fig. 5B). This interaction was confirmed to be a Trx3-
conditions confirming that it is mediated by a disulfide bond.
Given the well-characterized roles of Prx’s in redox sig-
naling, we tested whether mitochondrial Prx1 might regulate
the redox state of Trx3. The cellular oxidation state was pre-
served by rapidly treating cells with trichloracetic acid (TCA),
the thiol-specific probe 4-acetamido-4¢maleimidyldystilbene-
2,2¢-disulfonic acid (AMS). AMS alkylates Cys residues in a
free-SH, but not in an oxidized state, increasing their relative
molecular mass which can be detected by SDS-PAGE and
Western blot analysis (39). The migration of all Trx3 from the
wild-type strain grown under respiratory conditions was
decreased following treatment with AMS indicating that the
vast majority is present in the reduced form (Fig. 5C). Si-
multaneous loss of TRR2 and GLR1 shifted the Trx3 redox
balance to an oxidized state in agreement with our previous
observations (40). Interestingly, deletion of PRX1 in the trr2
gl1 mutant restored the Trx3 redox balance to a more reduced
state indicating that Prx1 promotes Trx3 oxidation in a glr1
trr2 mutant (Fig. 5C). We reasoned that if Prx1 is responsible
for Trx3 oxidation in a glr1 trr2 mutant, then deletion of PRX1
should restore the growth of a glr1 trr2 mutant under respi-
ratory conditions comparable to deletion of TRX3. This was
indeed found to be the case indicating that Prx1 appears to act
as a signaling molecule that oxidizes Trx3 under respiratory
conditions, promoting cell death (Fig. 5D).
Loss of TRX3 increases resistance to hydrogen peroxide
(H2O2) stress. Our previous analysis of mitochondrial Trx3
showed that it is relatively resistant to oxidation by H2O2com-
pared with cytoplasmic thioredoxins (40). We therefore exam-
ined the redox state of Trx3 following a longer term exposure
and higher concentrations of peroxide in cells grown under re-
spiratory conditions. Analysis of the Trx3 redox state revealed
that it becomes progressively more oxidized following treat-
ments with increasing concentrations of H2O2for 4h (Fig. 6A).
Oxidation is detectable following exposure to 1mM H2O2, and
Trx3 appears predominantly oxidized following a treatment
H2O2treatments in a prx1 mutant (Fig. 6A). Less Trx3 was de-
tected in both the wild-type and prx1 mutant strains following
the 10mM treatment, suggesting that Trx3 may be subject to
degradation following exposures to higher concentrations of
H2O2. We examined the time course of oxidation following the
10mM treatment and found that Trx3 was partially oxidized
within 1h and fully oxidized by 4h (Fig. 6B). Oxidation of Trx3
in the trx3 mutant. A loading control was probed with an anti-
body that recognizes an elongation factor 1A (Tef1) and showed
type and trx3 mutant cells to 10mM H2O2(Fig. 6B).
If the oxidation state of mitochondrial Trx3 is an important
trigger for apoptosis, then one prediction would be that mu-
tants lacking TRX3 may be resistant to agents that oxidize
Trx3. We tested this idea by examining the resistance of a trx3
mutant to various concentrations of H2O2. Loss of TRX3 was
found to increase cell viability during H2O2exposure over a
concentration range up to 10mM (Fig. 6C).
Yeast mitochondrial thioredoxin reductase and the GSH
system play an overlapping role in maintaining the redox
to promote cell death. (A) Schematic of mitochondrial Trx3
showing the two redox active Cys residues (Cys55, Cys58)
within the Cys-Gly-Pro-Cys motif that are conserved among
thioredoxins from all species. Trx3 also contains two extra
Cys residues (Cys80, Cys92), which are not present in cyto-
plasmic Trx1 and Trx2. (B) A glr1 trr2 trx3 mutant containing
wild-type (TRX3) on a single copy vector (vector) or Cys
mutant versions of TRX3 (C55S, C58S, C80S, C92S) were
grown to an exponential phase and spotted onto fermenta-
tive synthetic dextrose (SD) or respiratory synthetic glycerol
ethanol (SGE) media containing acetic acid. Growth was
scored after 3 days. Cys, cysteine; SD, synthetic dextrose.
The active site Cys residues of Trx3 are required
380GREETHAM ET AL.
state of mitochondrial Trx3 (40). During normal fermentative
growth conditions, deletion of both TRR2 and GLR1 leads to
partial oxidation of Trx3, which is even more pronounced
during oxidative stress or respiratory growth conditions. In
this current study, we have shown that the accumulation of
oxidized Trx3 in the glr1 trr2 mutant during respiratory
growth conditions promotes apoptosis. ROS exposure and
the resulting oxidative stress have been well characterized
as an insult that induces a form of PCD in yeast (25). PCD is
thought to provide a mechanism for the selective death of a
subset of the yeast population, which can be beneficial in a
unicellular organism under certain stress conditions (10).
Many stress conditions that produce ROS, including H2O2
exposure and depletion of GSH, have been shown to pro-
mote yeast PCD (24, 33–34). Saccharomyces cerevisiae con-
tains a metacaspase (Yca1) that mediates cell death in
response to various stimuli, including oxidative stress,
caused by H2O2exposure and yca1 mutants are resistant to
H2O2consistent with apoptosis accounting for cell death
during ROS exposure (19, 25). Similarly, our current data
indicate that Yca1 is required to induce PCD mediated by
oxidized Trx3 in a glr1 trr2 mutant and trx3 mutants are
more resistant to H2O2.
Mitochondria are a main source of ROS in eukaryotic cells
and play well-established roles in cell death pathways (1). In
mammalian cells, the intrinsic cell death pathway is stimu-
lated by ROS exposure causing a complex cascade of events,
including permeabilization of the outer mitochondrial mem-
brane and release of cytochrome C (1, 7). Increasing evidence
suggests that redox regulatory mechanisms are important in
these processes. For example, GSH levels and redox state
are decreased in response to ROS-mediated apoptosis (7).
Mammalian mitochondrial Trx2 is required for normal de-
velopment of the mouse embryo, and lack of Trx2 results in
increased rates of apoptosis presumably due to ROS accu-
mulation (27). Furthermore, mitochondrial Trx2 is oxidized
cells grown under respiratory conditions. Western blot analysis of Trx3 is shown for the glr1 trr2 trx3 mutant containing
wild-type (TRX3) or Cys mutant versions of TRX3 (C55S, C58S, C55/58S) grown to an exponential phase under fermentative
(SD) or respiratory (SGE) growth conditions. Proteins were separated using nonreducing SDS-PAGE. (B) Prx1 can be
detected in a disulfide bonded form with Trx3::C58S. Western blot analysis of Prx1 is shown for the glr1 trr2 trx3 mutant
containing wild-type (TRX3) or Cys mutant of TRX3 (C58S) grown to an exponential phase under respiratory (SGE) growth
conditions. Proteins were separated using nonreducing (NR) or reducing (R) SDS-PAGE. The position of a Prx1-Trx3 dimer
is indicated. (C) Prx1 is required to oxidize Trx3 in a glr1 trr2 mutant. The wild-type glr1 trr2, glr1 trr2 prx1, and prx1 mutant
strains were grown to an exponential phase in SGE media. Proteins were precipitated with TCA and free thiols modified by
reaction with AMS. Samples were separated using SDS-PAGE and Trx3 detected by Western blot analysis. Oxidized and
reduced proteins are indicated. (D) Deletion of PRX1 restores the growth of a glr1 trr2 mutant under respiratory conditions.
The wild-type, glr1 trr2, glr1 trr2 prx1, and glr1 trr2 trx3 mutant strains were grown to a stationary phase and spotted onto
fermentative (YEPD) or respiratory (YEPGE) media. AMS, 4-acetamido-4¢maleimidyldystilbene-2,2¢-disulfonic acid; TCA,
Mitochondrial Prx1 regulates the redox state of Trx3. (A) Trx3::C58S is present in many disulfide-bonded forms in
YEAST MITOCHONDRIAL THIOREDOXIN ACTIVATES CELL DEATH 381
in mammalian cells undergoing apoptosis and oxidized
thioredoxin has been proposed to sensitize cells to ROS-
induced apoptotic cell death (6, 43). Mitochondrial Trx2 has
also been shown to prevent mitochondrial ROS-induced ap-
optosis following tumor necrosis factor -alpha exposure (15).
One key mechanism by which Trx2 influences apoptosis is
through modulation of the apoptosis signal-regulating kinase
1 (ASK1) (42). Inhibition and oxidation of mitochondrial
thioredoxin systems may therefore represent common events
in the activation of PCD in eukaryotic cells.
It was originally reported that Trx3 is the physiological
electron donor for Prx1 (31). This is unexpected since 1-Cys
Prx’s are not thought to form a disulfide that can act as a
substrate for thioredoxins. Our previous in vitro assays con-
firmed that mitochondrial Trx3 and Trr2 can support Prx1
activity with H2O2as a substrate (13). Since Trx3 therefore
appears to be able to reduce the sulfenic acid form of the Prx1
peroxidatic Cys residue, it is unclear why the mitochondrial
thioredoxin does not support Prx1 activity in vivo. Mamma-
lian Prx’s (Prx3, Prx5) have been proposed to function in mi-
tochondrial redox signaling (8). Kinetic analyses also suggests
that Prx’s are major targets of oxidation by mitochondrial
H2O2, which means that they will play a major role in deter-
mining the levels of mitochondrial H2O2available for sig-
naling redox sensitive proteins, such as thioredoxins.
Additionally, Prx’s can act as direct signal transducers by
oxidizing thiol proteins. We were able to trap Prx1 in a mixed
disulfide with the Trx3::C58S mutant. Based on the formation
of a Prx1-Trx3 reaction intermediate, we propose the mecha-
peroxidatic Cys residue of Prx1 is oxidized to the sulfenic acid
form. Reduced Prx1 can be regenerated in a reaction mecha-
nism requiring Trr2 and GSH as previously described (13).
Alternatively, oxidized Prx1 may react with Trx3 resulting in
the generation of oxidized Trx3, which can promote PCD. This
means that Prx1 normally functions as an antioxidant that de-
toxifies hydroperoxides. However, during an oxidative stress,
Prx1 can function as a signaling molecule that oxidizes Trx3
The wild-type and prx1 mutant strains were grown to an
exponential phase in YEPGE media and treated with
1mM, 3mM, or 10mM H2O2for 4h. Proteins were pre-
cipitated with TCA and free thiols modified by reaction
with AMS. Oxidized and reduced Trx3 are indicated. (B)
The wild-type and prx1 mutant strains were grown to an
exponential phase in YEPGE media, and the Trx3 redox
state analyzed following exposure to 10mM H2O2for 1, 2,
and 4h. Oxidized and reduced Trx3 are indicated. A
Western blot is shown probed with antibodies against the
elongation factor 1A (Tef1) as a loading control following
the same treatments. (C) A trx3 mutant shows increased
resistance to H2O2stress. The wild-type and trx3 mutant
strains were grown to an exponential phase in YEPGE
media and treated with the indicated concentrations of
H2O2for 4h. Percent survival is expressed relative to that
of untreated cultures. Experiments were repeated in trip-
licates and values shown are means. *p<0.05, **p<0.01,
***p<0.001 for comparisons between the trx3 mutant and
the wild-type strain. H2O2, hydrogen peroxide.
Prx1 oxidizes Trx3 in response to H2O2stress. (A)
PCD by Trx3. Reduction of H2O2by Prx1 results in oxidation
of the peroxidatic Cys residue (Cys91) of Prx1 to the sulfenic
acid form (Prx1-SOH). Reaction with GSH generates glu-
tathionylated Prx1 (Prx1-S-SG) (1), which is a substrate for
reduction by Trr2 in a reaction that requires GSH to regen-
erate reduced Prx1. Alternatively, Prx1-SOH reacts with the
catalytic Cys residue (Cys55) of Trx3 to form a disulfide-
bonded intermediate (2). This intermediate can be reduced by
the second Cys residue in Trx3 (Cys58) generating oxidized
Trx3 and reduced Prx1. Oxidized Trx3 can be reduced by Trr2
to prevent activation of PCD. PCD, programmed cell death.
Proposed reaction mechanism for regulation of
382GREETHAM ET AL.
and promotes apoptosis. This would mean that under condi-
tions where yeast cells cannot detoxify mitochondrial ROS,
they can induce PCD presumably to remove the affected cells.
is required to activate PCD indicates that Trx3 may directly
oxidize another substrate protein that forms part of the cell
death pathway. Thioredoxins are well-known oxidoreduc-
tases, which may therefore directly catalyze thiol/disulfide
exchange with an, as yet, unidentified protein. Mutation of
Cys55 would prevent oxidation of this unidentified protein
under conditions, where Trx3 normally becomes oxidized.
Similarly, mutation of the second Cys residue (Cys58) in Trx3
also prevented the activation of PCD. Presumably, this mu-
tant may trap substrate proteins in a mixed disulfide pre-
oxidation and reduction of the Trx3 catalytic residues (Cys55
and Cys58) may cause some conformational change in Trx3,
which triggers PCD. This could be analogous to the activation
of apoptosis by ASK1 in mammalian cells, which can be
regulated by redox-dependent binding of Trx2 (42). Thus,
direct oxidation–reduction of thioredoxin can alter its non-
covalent interaction with other proteins. Further work will be
required to elucidate the exact pathway by which Trx3 in-
Materials and Methods
Yeast strains and plasmids
The S. cerevisiae strains used in this study were isogenic
1 his3-11 can1-100). Strains deleted for the mitochondrial
thioredoxin (trx3::kanMX4), mitochondrial thioredoxin re-
and glutathione reductase (glr1::TRP1) have been described
previously (11, 13, 40). Strains deleted for YCA1 were con-
structed using a one-step polymerase chain reaction amplifi-
cation protocol that replaced its entire open reading frame
with the LEU2 gene (3). Standard yeast genetic techniques
were used to construct mutants lacking multiple genes (glr1
trr2, glr1 trr2 trx3, glr1 trr2 yca1, and glr1 trr2 prx1). Mutant
versions of Trx3 (trx3::C55S, trx3::C58S, trx3::C80S, and
trx3::92S) were made in TRX3 contained on plasmid pRS416
using the QuikChange method (Stratagene).
(YEPD) medium (2% w/v glucose, 2% w/v bactopeptone, 1%
w/v yeast extract) or a minimal synthetic dextrose (SD) me-
dium (0.17% w/v yeast nitrogen base withoutamino acids, 5%
w/v ammonium sulfate, 2% w/v glucose supplemented with
appropriate amino acids and bases). For growth on non-
fermentable carbon sources, synthetic glycerol ethanol (SGE)
3% (v/v) glycerol and 1% (v/v) ethanol. Media were solidified
by the addition of 2% (w/v) agar. Acetic acid sensitivity was
spotting onto agar plates containing 20mM acetic acid.
Protein extracts were electrophoresed under reducing or
nonreducing conditions on SDS-PAGE minigels and electro-
blotted onto polyvinylidene fluoride membrane (Amersham
Pharmacia Biotech). The bound antibody (aTrx3, aPrx1)
was visualized by chemiluminescence (ECL; Amersham
Pharmacia Biotech). The redox state of Trx3 was measured by
covalent modification with the thiol-reactive probe AMS
(Molecular Probes) as described previously (39). Glutathione
(GSH and GSSG) levels were determined as described previ-
ously (12). Intracellular redox states were measured using
probes (cytosolic rxYFP, matrix rxYFP, IMS rxYFP) as de-
scribed previously (17).
Cell death assays
Cell viability was determined by growing cells to an ex-
ponential phase and treating with H2O2for 4h. Aliquots of
cells were diluted into fresh YEPD media and plated in trip-
licate on YEPD plates to obtain viable counts after 3 days
growth. Apoptotic (Annexin V staining) versus necrotic (PI
staining) cells were detected using the ApoAlert?Annexin V
Kit (Clontech). DNA fragmentation was measured using
the TUNEL method (FragELTMDNA Fragmentation Kit;
Calbiochem). For scoring fluorescent cells, three or more in-
dependent views of at least 50 cells from biological replicates
were visualized using an Olympus widefield microscope and
MetaVue software (Bioimaging Facility, Faculty of Life Sci-
ence, University of Manchester). Results are expressed as
means–standard error. Significant differences were analyzed
using the Student’s t-test. Values of p<0.05 were considered
We thank Caryn Outten (University of South Carolina) for
the gift of the rxYFP plasmids used in this study. This work
was supported by funding from the Wellcome Trust and the
Biotechnology and Biological Sciences Research Council.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Prof. Chris M. Grant
Faculty of Life Sciences
The University of Manchester
The Michael Smith Building
Manchester M13 9PT
Date offirst submissiontoARS Central, March6,2012;dateof
final revised submission, July 3, 2012; date of acceptance, July
ASK1¼apoptosis signal-regulating kinase 1
IMS¼mitochondrial intermembrane space
PCD¼programmed cell death
ROS¼reactive oxygen species
SDS-PAGE¼sodium dodecyl sulfate–polyacrylamide
SGE¼synthetic glycerol ethanol
TUNEL¼terminal dUTP nick end-labeling
YEPD¼yeast extract, peptone dextrose
YEPGE¼yeast extract, peptone glycerol, ethanol
YEAST MITOCHONDRIAL THIOREDOXIN ACTIVATES CELL DEATH 385