EUKARYOTIC CELL, Dec. 2008, p. 2160–2167
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 7, No. 12
Thiol-Independent Action of Mitochondrial Thioredoxin To Support
the Urea Cycle of Arginine Biosynthesis in
Ji-Yoon Song, Kyoung-Dong Kim, and Jung-Hye Roe*
Laboratory of Molecular Microbiology, School of Biological Sciences and Institute of Microbiology,
Seoul National University, Seoul 151-742, Korea
Received 24 March 2008/Accepted 30 September 2008
Thioredoxins usually perform a role as a thiol-disulfide oxidoreductase using their active-site cysteines. The
fission yeast Schizosaccharomyces pombe contains two thioredoxins: Trx1 for general stress protection and Trx2
for mitochondrial functions. The ?trx2 mutant grows as well as the wild type on complex media containing
glucose. However, on nonfermentable carbon source such as glycerol, the mutant did not grow, indicating a
defect in mitochondrial function. The mutant also exhibited auxotrophy for arginine and cysteine on minimal
medium. In order to find the reason for the unexpected arginine auxotrophy, we searched for multicopy
suppressors and found that the arg3?gene encoding ornithine carbamoyltransferase (OCTase) in the urea
cycle of the arginine biosynthetic pathway rescued the arginine auxotrophy. The levels of arg3?transcript, Arg3
protein, and OCTase activity were all decreased in ?trx2. Through immunocoprecipitation, we observed a
direct interaction between Trx2 and Arg3 in cell extracts. The mutant forms of Trx2 lacking either one or both
of the active site cysteines through substitution to serines also rescued the arginine auxotrophy and restored
the decreased OCTase activity. They also rescued the growth defect of ?trx2 on glycerol medium. This contrasts
with the thiol-dependent action of overproduced Trx2 in complementing glutathione reductase. Therefore, Trx2
serves multiple functions in mitochondria, protecting mitochondrial components against thiol-oxidative dam-
age as a thiol-disulfide oxidoreductase, and supporting urea cycle and respiration in mitochondria in a manner
independent of active site thiols.
In various organisms, glutathione (GSH) and peptide thiols
in thioredoxin (Trx) and glutaredoxin (Grx) provides antioxi-
dative environment by reducing disulfide bonds (13, 30, 55).
Thioredoxins, initially isolated as a hydrogen donor for ri-
bonucleotide reductase (15), are small proteins with two
conserved active cysteines. They efficiently reduce disulfide
bonds in a wide variety of proteins and are reduced by
thioredoxin reductase using NADPH (14, 30, 55). Thiore-
doxins function as an antioxidative agent not only by reduc-
ing disulfide bonds in oxidized substrates but also by pro-
viding electrons to thioredoxin-dependent peroxidases.
They also serve as electron donors for several enzymes such
as methionine sulfoxide reductase and 3?-phosphoadenosyl-
5?-phosphosulfate (PAPS) reductase (30). The eukaryotic
signal transduction pathway is modulated by thioredoxins, as
observed in regulating the activities of NF-?B (13, 36) and
AP-1 family transcription factors (8, 19, 23) and in antiapop-
totic regulation (40).
In addition to its redox reaction, thioredoxin is also known to
play a key role in promoting growth and assembly of viruses in
Escherichia coli such as M13 and T7 (16, 29, 38). In T7 it
participates as an accessory protein of the phage-encoded
DNA polymerase complex (18, 46). Trx stabilizes the complex
between T7 DNA polymerase and DNA and confers proces-
sivity on the polymerizing reaction. Surprisingly, the oxi-
doreductase activity is not required for the function, and sub-
stitution of both cysteines in Trx did not significantly affect the
maximum polymerase activity (17).
Mitochondria are well known as the primary energy-gener-
ating system in eukaryotic cells. Besides its central task of ATP
generation, mitochondria play multiple roles to support bio-
chemical pathways for carbon and nitrogen metabolism. Var-
ious amino acid biosynthetic enzymes and metabolic pathways
are localized in mitochondria, and the tricarboxylic acid cycle
links both carbon and nitrogen metabolism by oxidizing or-
ganic acids from glycolysis and providing ?-ketoglutarate as a
carbon skeleton for amino acid synthesis (48). In addition,
mitochondria participate in iron-sulfur cluster assembly, fatty
acid oxidation, calcium signaling, and apoptosis (3, 6, 27).
As by-products of aerobic respiration, reactive oxygen spe-
cies (ROS) are generated in mitochondria and cause damages
in various components (5, 45, 50). Mitochondrial defense sys-
tem against ROS includes a number of antioxidant enzymes
such as Mn-superoxide dismutase, glutathione peroxidase, and
thioredoxin peroxidase (34, 50). Trx, Grx, and GSH maintain
the redox homeostasis not only in the cytosol but also in mi-
tochondria. It has been reported that the mitochondrial Trx
gene, TRX2, is an essential gene in chicken and that Trx2-
deficient cells undergo apoptosis with accumulation of intra-
cellular ROS (47). Overexpression of human mitochondrial
Trx causes increased membrane potential (7) and inhibits mi-
tochondrial ASK1-mediated apoptosis (57). Lack of mitochon-
drial Trx2 also causes early embryonic lethality in mice (35). In
contrast to the critical requirement of mitochondrial thiore-
* Corresponding author. Mailing address: School of Biological Sci-
ences, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul
151-742, Korea. Phone: 82-2-880-6706. Fax: 82-2-888-4911. E-mail:
?Published ahead of print on 10 October 2008.
doxin in avian and mammalian cell survival, its counterpart in
the yeast Saccharomyces cerevisiae (Trx3) is dispensable for cell
survival in complex, minimal, or respiratory media and even for
survival under oxidative stress conditions (37, 49).
The fission yeast S. pombe relies heavily on mitochondria for
growth under most laboratory conditions as partly reflected by
its petit-negative physiology (41). In this respect, it can serve as
a good model system to study genes that affect various aspects
of mitochondrial function. Maintenance of oxidation-labile
iron-sulfur cluster assembly system in mitochondria, for exam-
ple, is critical for aerobic growth of S. pombe even under
nutrient-rich conditions. Glutathione reductase (GR) supports
this function (25), and we previously found that thioredoxin
Trx2, when overproduced, can replace GR (43). In the present
study we examine the role of mitochondrial thioredoxin in
further detail and report an unexpected novel finding that it is
required for the urea cycle of arginine biosynthesis as well for
efficient energy generation on glycerol, both of which do not
involve the thiol-dependent oxidoreductase activity of thiore-
MATERIALS AND METHODS
Yeast strains and culture media. S. pombe strains used in the present study are
ED668 (h?ade6-M216 leu1-32 ura4-D18), ED665 (h?ade6-M210 leu1-32 ura4-
D18), JL38 (ura4?in ED665 background), JL36 (ura4??pgr1 nmt-pgr1 in
ED665), and JY31b (trx2::ura4?in ED668). Growth and maintenance of all of
the strains were done as described previously (2, 33). Cells were grown in YES
(0.5% yeast extract, 3% dextrose) medium or Edinburgh minimal medium
(EMM) with appropriate supplements as described by Alfa et al. (2). For respi-
ratory growth, glycerol medium (0.5% yeast extract, 0.1% dextrose, 3% glycerol,
and 250 mg of supplements/liter) was used.
Construction of ?trx2 mutants. To disrupt the trx2?gene, the BglII fragment
of the trx2 open reading frame cloned in pTZ18R vector was replaced with 1.8-kb
ura4?gene cassette. The 3.2-kb HindIII fragment containing the recombinant
construct was introduced to ED665/ED668 diploid cells for direct homologous
recombination at the trx2?locus. The transformants were selected by the ura?
marker, and the expected disruption was confirmed by both colony PCR and
genomic Southern hybridization. A haploid ?trx2 strain was isolated through
Construction of mutants and recombinants. Construction of pREP1-trx2?and
pREP42-trx2-EGFP-C was described previously (43). Substitution mutagenesis
of active-site cysteines to serines in Trx2 was done by using mutagenic primers;
T2-mut1 (5? GCGGTCCTTCGAAATACCTCAAACC 3?, the BstBI site is un-
derlined) and T2-mut2 (antisense of T2-mut1) for Cys50 substitution or T2-mut3
(5? GACTGGTCCGGACC TTCGAAATACCTC 3?, the BspEI and BstBI sites
are underlined) and T2-mut4 (antisense of T2-mut3) for substitution of both
Cys47 and Cys50. The arg3?gene fragment was amplified with the primer pair
ARG3-F (5? GATTTACTGCAGTTGGTAGAAGGC 3?, the PstI site is under-
lined) and ARG3-R (5? GCATTGTTTGCAGGATCCCCAGAT 3?, the BamHI
site is underlined). The 2.3-kb amplified DNA was subcloned into PstI/BamHI
site of pREP1. For C-terminal Myc tagging, the 9? His-human rhinovirus 3C
protease-9xMyc (HPM) cassette originated from pJS-HPM53H (a modified
TAP-tagging vector kindly provided by J.-H. Seol, Seoul National University)
was cloned into the SmaI site of pREP41, generating pREP41-HPM vector. To
construct chimeric Arg3-Myc protein with C-terminally fused 9? Myc tag, PCR
products with the primers Arg3-N (5? CAGTTTGCAATTGCATATGTCTTT C
3?, the NdeI site is underlined) and Arg3-HPM (5? ATTAAGGATTCCCGGG
TAGGCTGAG 3?, the SmaI site is underlined) were generated, cut with NdeI
and SmaI, and cloned into pREP41-HPM. To construct pREP41-Gld-Myc, a
1.4-kb DNA fragment containing the open reading frame of a putative glycerol
dehydrogenase gene (gld1; SPAC13F5.03c) was cloned in a similar way.
Northern hybridization. RNAs from exponentially grown cells in YES me-
dium were separated on an agarose gel containing formaldehyde, transferred
onto a Hybond-N?membrane (Amersham), and fixed by UV-cross-linker (43).
Hybridization was performed in Rapid-Hyb buffer (Amersham) with radioac-
tively labeled arg3?probes generated by PCR as recommended by the manu-
facturer. The signal was visualized by exposing the membrane to X-ray film, and
the radioactivity was quantified with a PhosphoImager (BAS-5000) and a Mul-
OCTase assay. Ornithine carbamoyltransferase (OCTase) activity was mea-
sured as described by Lee and Nussbaum (24) with some modification. Crude cell
extracts were added to 700 ?l of reaction mixture composed of 5 mM ornithine,
15 mM carbamoyl phosphate, and 270 mM triethanolamine (pH 7.7), followed by
incubation at 37°C for 30 min. The reaction was stopped by adding 250 ?l of 3:1
phosphoric acid-sulfuric acid (vol/vol). Citrulline production was determined by
measuring the absorbance at 490 nm, after the addition of 50 ?l of 3% 2,3-
butanedione monoxime and further incubation in the dark at 95 to 100°C for 15 min.
Western immunoblot analysis. Crude cell extracts (100 ?g of total protein)
prepared from cells grown to an optical density at 595 nm (OD595) of ?1.5 in
YES medium were separated by sodium dodecyl sulfate–10% polyacrylamide gel
electrophoresis (SDS–10% PAGE) and subjected to Western analysis using
anti-Arg3 polyclonal antibody raised in mice. The secondary antibody (goat
anti-mouse IgGAM; Cappell) conjugated with horseradish peroxidase was used
at 10?5dilution, and the light development was detected by using an ECL system
(Amersham). The signal was visualized by the LAS-3000 imaging system (Fuji)
and quantified with a MultiGauge (Fuji).
Coimmunoprecipitation. Crude cell extracts containing 1 mg of total protein
were mixed with 10 ?l of agarose beads conjugated with monoclonal antibodies
against c-Myc or green fluorescent protein (GFP; Santa Cruz Biotech). Each
sample was incubated at 4°C with rocking for 2 h, followed by washing with buffer
A (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, and 100 mg of
sodium azide/liter). Pellets were resuspended in 1? SDS sample buffer and
separated by SDS-PAGE. They were subjected to Western analysis with mono-
clonal antibodies against GFP or c-Myc (Santa Cruz Biotech).
The ?trx2 mutant shows auxotrophy for arginine and cys-
teine. The S. pombe mutant devoid of mitochondrial thiore-
doxin gene (?trx2) was constructed and examined for growth
on different media. On complex YES medium, the ?trx2 mu-
tant grew as well as the wild type. However, on a minimal
medium plate (EMM) it did not grow unless supplemented
with Casamino Acids (Fig. 1A). Among amino acids, arginine
alone partially restored growth, whereas additional supple-
ment with cysteine resumed full growth. In contrast to the
?trx1 (cytosolic thioredoxin) mutant that shows cysteine auxo-
trophy (44), the growth defect of ?trx2 was hardly reversed by
supplementing with cysteine alone, nor by dithiothreitol. In
liquid minimal medium, the growth stimulatory effect of argi-
nine was more pronounced in the mutant, reaching an OD595
near 6.0, albeit with a slightly slower growth rate than that of
the wild type (Fig. 1B). Supplementation with both arginine
and cysteine completely restored the fast growth of ?trx2 in
liquid minimal medium. The cysteine synthesis has been re-
lated to thioredoxin, since one of its biosynthetic enzymes
(PAPS reductase) is known to require thioredoxin for its ac-
tivity. The arginine auxotrophy, however, has not been associ-
ated with thioredoxin in previous studies.
The gene for OCTase (arg3?) in the urea cycle of arginine
biosynthetic pathway suppresses ?trx2 phenotype. Arginine is
synthesized from glutamate via ornithine and the urea cycle, as
summarized in Fig. 2A. Among the enzymes in the arginine
biosynthetic pathway, six (acetylglutamate synthase, N-acetyl-
glutamate kinase, N-acetyl-?-glutamyl phosphate reductase,
acetylornithine aminotransferase, ornithine acetyltransferase,
and OCTase; presented in italics in Fig. 2A) were reported to
reside in mitochondria (31, 54). We suspected that one or
more of these mitochondrial enzymes could have been com-
promised by the loss of Trx2. To test this hypothesis, we cloned
each of these genes with its own promoter on multicopy vector
(pREP1) and introduced it to ?trx2 mutant. The transformants
VOL. 7, 2008MITOCHONDRIAL THIOREDOXIN NECESSARY FOR UREA CYCLE 2161
that grew on minimal EMM plates were selected. We found
that the arg3?gene encoding OCTase partially and fully re-
covered growth on EMM and EMM?cysteine plates, respec-
tively (Fig. 2B). This suggests that OCTase, which converts
ornithine to citrulline through carbamoylation in the urea cy-
cle, could be the target affected by trx2 disruption. In contrast
to arginine biosynthetic enzymes, most cysteine biosynthetic
enzymes are predicted to reside in the cytosol, with the excep-
tion of mitochondrial cysteine synthase encoded by cys 11?and
cyc12?(31). Overexpression of Cys12, however, did not alle-
viate the cysteine requirement of the trx2 disruptant (data not
Loss of Trx2 causes a decrease in OCTase. To confirm the
relationship between Trx2 and Arg3, OCTase activity was mea-
sured in the wild-type, ?trx2, and ?arg3 cells. In exponentially
growing cells, the OCTase activity was reduced in the ?trx2
mutant to ca. 60% of the wild-type level (Fig. 3A). OCTase
activity in ?arg3 mutant was ca. 8% of the wild-type level,
confirming that Arg3 provides the major activity for OCTase.
When the same set of cells were introduced with arg3?gene
with its own promoter on pREP-based vector, the specific
enzyme activities all increased, confirming the OCTase activity
of the arg3?gene product. However, even with the provision of
FIG. 1. Requirement of arginine and cysteine for the growth of
?trx2. (A) Growth on plates. Wild-type (JL38) and ?trx2 (JY31b) cells
were streaked on complex (YES) and minimal (EMM) media either
not supplemented or supplemented with 1 mM dithiothreitol, arginine,
or arginine plus cysteine. The photos were obtained after 4 days of
incubation at 30°C. (B) Growth in liquid media. The wild-type (open
symbols connected with dotted lines) and ?trx2 (filled symbols con-
nected with solid lines) cells were inoculated from overnight seed
culture to an OD595of 0.01 in EMM (diamonds), EMM plus arginine
(R, squares), EMM plus cysteine (C, triangles), and EMM plus R and
C (circles). Cell growth was monitored by measuring the OD595. WT,
FIG. 2. Introduction of arg3?gene encoding OCTase overcomes ar-
ginine auxotrophy of the ?trx2 mutant. (A) Biosynthetic pathway for
arginine from glutamate. The enzymes that are reported to be mitochon-
drial proteins are presented in italics. The standard names for encoding
genes as they appear in S. pombe database (www.genedb.org/pombe) are
indicated in parentheses (arg6?, SPBC725.14; arg11?, SPAC4G9.09c;
arg1?, SPCC777.09c; arg3?, and SPAC4G9.10; arg12?, SPBC428.05c;
arg7?, SPBC1773.14; car1?, SPBP26C9.02c). (B) Complementation by
or arg3?genes containing their own promoters in pREP1-based vector
(V) and incubated on EMM plates with supplements at 30°C for 4 days.
2162 SONG ET AL.EUKARYOT. CELL
multicopy arg3?genes, the OCTase activity was much lower in
?trx2 cells than in the wild-type and in ?arg3 mutant back-
grounds, indicating that Trx2 is necessary for proper level of
Arg3 enzyme activity.
To unravel how the loss of Trx2 caused a decrease in
OCTase activity, we examined the expression level of the arg3?
gene. The Western blot against the Arg3 protein demonstrated
that in the exponentially growing ?trx2 cells, Arg3 protein level
was reduced to ca. 50% of its wild-type level (Fig. 3B). This
result coincides with the decrease in OCTase activity in ?trx2
cells. When the level of arg3?mRNA was examined by North-
ern analysis, we found that it was also lowered to ca. 30% level
in ?trx2 (Fig. 3C). Reductions in the level of protein and
transcripts were consistently observed in ?trx2 cells provided
with pREP-arg3?plasmids compared to the wild-type and
?arg3 mutant backgrounds. We observed that the stability of
FIG. 3. Levels of OCTase, Arg3 protein, and arg3?transcripts in ?trx2. The wild-type (ED668), ?trx2 (JY31b), and ?arg3 cells with or without
pREP-arg3?plasmid were harvested at exponential phase (OD595?1.5 in YES medium) to prepare cell crude extract and RNA. The amount of
OCTase and Arg3 protein in cell extracts was measured by determining the enzyme activity (A) and by Western blotting (B). (C) The arg3?
transcripts were detected by Northern analysis with rRNAs as loading controls. The relative amounts of OCTase, Arg3 protein, and arg3?-specific
RNA compared to the wild-type level (set as 100) are also presented. In panel A, the relative enzyme activities in strains with pREP-arg3?plasmid
are presented compared to the wild-type background. The specific enzyme activities are shown at the top. Average values with standard deviations
from four (A and C) or three (B) independent experiments are given. WT, wild type.
VOL. 7, 2008 MITOCHONDRIAL THIOREDOXIN NECESSARY FOR UREA CYCLE2163
arg3?mRNA did not change in ?trx2 (data not shown). The
decreased mRNA level was restored by introducing trx2?gene
on pREP-based plasmid (data not shown). When the protein
half-life of Arg3 was examined by Western blotting after cy-
cloheximide treatment, no significant difference was observed
between wild-type and ?trx2 cells (data not shown). Therefore,
it seems most likely that Trx2 affects arg3?gene expression at
the level of transcription, which in turn affects the protein level,
and hence the activity.
The active-site cysteines of Trx2 is not required to maintain
OCTase activity. Trx2 shares many conserved residues with
other known thioredoxins, including the highly conserved ac-
tive-site sequence of Trp-Cys-Gly-Pro-Cys, which is essential
for oxidoreductase activity. To investigate whether the thiol-
dependent oxidoreductase activity of Trx2 is necessary to
maintain OCTase, one (C50) or both (C47 and C50) of cys-
teine residues in the active site were mutated to serines, re-
sulting in T2-CS and T2-SS mutants, respectively. The wild-
type and mutant trx2 genes were introduced into ?trx2 cells on
pREP-based multicopy plasmid retaining their own promoters.
To our surprise, both the T2-CS and the T2-SS variants al-
lowed ?trx2 cells to overcome arginine auxotrophy (Fig. 4A).
The OCTase activity also increased in cell extracts expressing
either wild-type or mutant Trxs, a finding consistent with
growth promotion on minimal medium (Fig. 4B).
Direct interaction of Trx2 with Arg3 protein. We examined
whether Trx2 directly interacts with Arg3 protein in vivo by
coimmunoprecipitation analysis. Arg3 and Trx2 were C-termi-
nally tagged with 9?Myc and GFP, respectively, and intro-
duced into the wild-type cells through pREP41- and pREP42-
based expression system, respectively. Cell extracts were
immunoprecipitated with anti-Myc antibody against Arg3-Myc,
followed by Western blotting with anti-GFP antibody against
Trx2-GFP. The results in Fig. 5 demonstrate that Trx2 was co-
precipitated with Arg3. The specificity of coprecipitation was ex-
amined by probing interaction of Trx2 with another mitochon-
drial enzyme (a putative glycerol dehydrogenase; Gld1) that is
similarly tagged at C terminus with Myc. The negative result with
Gld1 demonstrates that the interaction between Trx2 and Arg3 is
specific. Interaction with Trx2 mutants was examined likewise,
revealing that both mutant forms were able to interact with Arg3
as the wild type. When immunocoprecipitation was performed
with anti-GFP antibody against Trx2, followed by Western with
anti-Myc against Arg3, Arg3 was detected only in cells with Trx2
or their variants (data not shown). Therefore, it is evident that
Trx2 interacts with Arg3 protein in vivo, and this interaction is not
affected by the loss of active-site cysteines.
Trx2 and its cysteine mutants support growth on glycerol, a
nonfermentable carbon source. On complex media with glyc-
erol as a carbon source, S. pombe cells rely heavily on aerobic
respiration for growth. We found that ?trx2 cells were unable
to grow on glycerol medium (Fig. 6A). The respiration rate of
?trx2 was drastically reduced in both YES and glycerol media,
suggesting a role for Trx2 in maintaining proper mitochondrial
function including respiration (data not shown). Introduction
of trx2?gene on pREP-based vector restored growth on glyc-
erol (Fig. 6A). Interestingly, the T2-CS and T2-SS variants of
Trx2 also restored growth on glycerol plate. Considering that
YES or glycerol medium does not lack arginine, it is evident
that the role of Trx2 to maintain proper mitochondrial func-
FIG. 4. Thiol-independent complementation of arginine auxotro-
phy by mutant Trx2. (A) The ?trx2 (JY31b) mutant was transformed
with pREP1 (V) or pREP-based recombinant plasmids with cloned
arg3?, trx2?, or mutated trx2 with C50S (T2-CS) or C47/50S (T2-SS)
substitutions. The cloned genes contained their own promoters to
allow expression. Cells were streaked onto EMM plates supplemented
with cysteine or with cysteine plus arginine, followed by incubation at
30°C for 4 days. (B) OCTase activity of wild-type (ED668) or ?trx2
cells complemented with various trx2 constructs. Freshly grown cells at
the early exponential phase (OD595?0.4) were used to prepare crude
cell extracts. Error bars indicate the standard deviations from three
independent experiments. WT, wild type.
FIG. 5. Interaction of Trx2 with Arg3 monitored by coimmunopre-
cipitation. Cell extracts prepared from ED665 containing pREP41-
Arg3-Myc and pREP42-Trx2-GFP were subjected to immunoprecipi-
tation with anti-Myc antibodies conjugated on agarose beads. Total
crude extract and the immunoprecipitates were resolved on SDS-
PAGE, followed by Western blotting with anti-GFP antibodies. As a
negative control, parallel immunoprecipitation was done with cells
expressing Trx2-GFP alone and cells containing pREP41-Gld1 (glyc-
erol dehydrogenase)-Myc instead of pREP-Arg3-Myc. Trx2 variants
(T2-CS and T2-SS) were also fused with GFP tag and examined in the
2164SONG ET AL.EUKARYOT. CELL
tion goes beyond amino acid biosynthesis and does not require
the thiol-dependent oxidoreductase activity of Trx2.
Suppression of GR deficiency requires active site cysteines
in Trx2. In our previous work, the trx2?gene was found as a
multicopy suppressor of GR deficiency, which hinders cell
growth under all conditions examined. The restoration of
growth by overproduced Trx2 coincided with the restoration of
oxidant-labile Fe-S enzymes that were compromised in GR-
deficient mutants. We examined whether the role of overpro-
duced Trx2 as a substitute for GR is also independent of the
thiol oxidoreductase activity. Using a thiamine-suppressible
nmt1-pgr1?strain that lacks GR and therefore cannot grow on
plates containing thiamine (25), we examined the compensa-
tory activity of Trx2 variants. The results in Fig. 6B demon-
strate that the compensating effect of Trx2 for GR is depen-
dent on its thiol-oxidoreductase activity, since active site
cysteine mutants did not restore growth. Therefore, the com-
pensatory role of Trx2 to replace GR is mediated through
providing thiol-disulfide redox activity, whereas other roles to
support arginine biosynthesis and mitochondrial respiratory
function are independent of thiol-redox function.
S. pombe possesses two thioredoxins: Trx1 for general stress
protection (44) and Trx2 for mitochondrial functions (43). A
recently annotated thioredoxinlike protein (Txl1 or Trx3) con-
tains an N-terminal thioredoxinlike domain and C-terminal
domain of unknown function, but its function is not well char-
acterized except for defense against alkylhydropeorxide (20) or
other oxidants (21). We present evidence here that Trx2, the
mitochondrial thioredoxin in S. pombe, contributes to maintain
proper mitochondrial functions that include aerobic respira-
tion and biosynthesis of arginine. Trx2 served to maintain
proper level of OCTase (Arg3) in particular by regulating its
synthesis. It even interacted directly with it. Arginine is one of
the most versatile amino acids that serve as a precursor for
making not only proteins but also nitric oxide, urea, polyamine,
proline, glutamate, creatine, and agmatine (10). It is dispens-
able for healthy adult humans but is essential for young, grow-
ing animals. Interestingly, most of the enzymes in the arginine
biosynthetic pathway (from glutamate to citrulline) are local-
ized in mitochondria. It has been reported that some of these
mitochondrial arginine biosynthetic enzymes are associated
with mitochondrial nucleoids in S. cerevisiae (Arg5/6 [11, 22]),
as well as in humans (Arg4; carbamoyl phosphate synthetase
). In S. cerevisiae Arg5/6 has been shown to associate with
specific nuclear loci and regulate nuclear gene expression.
Since the arginine pathway is tightly interlinked with carbon
and nitrogen metabolism, it seems logical that this pathway
could be at a crossroad to connect nutrient signals with mito-
chondrial function, as well as with nuclear gene expression.
The fact that loss of Trx2 contributed to decreasing arg3?
transcription could be interpreted as a phenomenon reflecting
communication between mitochondria and the nucleus. Re-
cent findings on mitochondrial stress signaling accumulate to
indicate that mitochondrial dysfunction due to mitochondrial
DNA loss or altered membrane potential triggers retrograde
response that involves multiple factors in the signaling pathway
to regulate nuclear target gene expression (28, 39). Concen-
tration changes in metabolites originating from mitochondria
such as oxaloacetate, ?-ketoglutarate, glutamate, ammonia,
and [Ca2?] are known as signaling molecules to trigger this
pathway. Although the RTG-dependent pathway in S. cerevi-
siae has been investigated in most detail, the composition and
the mechanism of the signaling pathway appear to be diverse
among organisms (4). It is tempting to speculate from our
study that a kind of retrograde signaling that involves Trx2 may
exist in S. pombe to connect mitochondrial status with nuclear
gene expression. In this case, mitochondrial dysfunction result-
ing from the lack of Trx2 somehow downregulates gene ex-
pression for Arg3, shutting off arginine synthesis and the orni-
thine cycle. There is no available information on retrograde
mitochondrial signaling in S. pombe, and it appears highly
intriguing to unravel the mechanism by which Trx2 affects
nuclear gene expression.
The observation that Trx2 supports efficient respiration and
Arg3 (OCTase) production independently of thiol oxidoreduc-
tase activity implies that these functions are mediated through
protein-protein interaction without involving thiol-disulfide re-
dox reaction. This hypothesis has been partly confirmed by
direct interaction between Arg3 and Trx2. How the interaction
of Trx2 with Arg3 in mitochondria is related to arg3?gene
expression in the nucleus is very intriguing. Considering pre-
vious reports that thioredoxins can stabilize proteins, enhance
enzyme activities, and assist proper folding, it is conceivable
FIG. 6. Thiol-independent function of Trx2 for growth on glycerol
versus thiol-dependent function for substituting GR. (A) Growth on a
modified YES medium that contain 3% glycerol instead of glucose.
The ?trx2 mutant was transformed with pREP1 (V), or pREP-trx2?,
pREP-T2-CS, or pREP-T2-SS and monitored for growth on plates
containing glycerol as a carbon source. (B) Complementation of GR
deficiency. A pgr1 mutant strain (JL36 ) that contain thiamine-
repressible pgr1?gene (Pnmt1-pgr1) in the chromosome was trans-
formed with pREP1 (V), pREP-trx2?, pREP-T2-CS, and pREP-T2-
SS. Thiamine represses expression from Pnmt-pgr1?without affecting
trx2 gene expression from its own promoter. The wild type (JL38) with
control vector (V) was also examined in parallel. The growth was
monitored on EMM plates containing 10 ?M thiamine after incuba-
tion at 30°C for 4 days. WT, wild type.
VOL. 7, 2008MITOCHONDRIAL THIOREDOXIN NECESSARY FOR UREA CYCLE2165
that it can somehow ensure optimal OCTase activity, main-
taining a proper balance of metabolites, which in turn signal
proper gene regulation. According to this scenario, the loss of
Trx2 would disturb this balance and decrease arg3?expression.
The validity of this scenario requires further systematic anal-
ysis of Trx2 function as well as search for additional interaction
partners. In our hands, Arg3 enzyme activity itself was not
affected by Trx2 in vitro (data not shown), suggesting that Trx2
may not modulate OCTase enzyme activity in vivo. How Trx2
is related to the expression of arg3?gene is an interesting
question for future investigations.
The observation that ?trx2 mutation in S. pombe caused a
severe decrease in mitochondrial functions contrasts the ab-
sence of such phenotype in S. cerevisiae mutant (?TRX3) that
lack mitochondrial thioredoxin (37, 49). Although OCTases
are present from bacteria to higher eukaryotes, the regulation
of its synthesis could be different. In contrast to S. pombe and
other higher eukaryotes, OCTase in S. cerevisiae is reported to
be a cytosolic enzyme (53, 54) and is regulated by protein-
protein interaction with arginase (1). In higher eukaryotes,
mitochondrial TRX2 is an essential gene as reported for cell
viability in chickens and for embryonic development in mice
(35, 47). Previously, the reason for the essentiality of mito-
chondrial Trx for mammalian cell survival was proposed to be
due to the inhibition of apoptosis and cell death (35, 47, 57).
On the basis of present study, we present another possible
reason for Trx2 requirement for cell survival, namely, to main-
tain OCTase activity and to ensure proper mitochondrial func-
tion. In mammals, OCTase deficiency causes urea cycle disor-
der, resulting in the accumulation of ammonia and excess or
deficiency of other metabolites that lead to hyperammonemia,
encephalopathy, and respiratory alkalosis (32). In humans, in-
herited OCTase deficiency leads to an increase in blood am-
monia and glutamate/glutamine level and a decrease in citrul-
line and arginine concentration. Clinically severe symptoms
start at a younger age even within several days after birth (9).
Therefore, it is worthwhile to examine whether mitochondrial
Trx regulates OCTase in other eukaryotes including humans.
Our study in the S. pombe system has demonstrated that
mitochondrial thioredoxin as a thiol oxidoreductase can re-
place the function of GR when overproduced (43) and serve as
an electron donor for other oxidoreductases such as Gpx1, a
thioredoxin peroxidase (26). We presented additional novel
roles of mitochondrial thioredoxin to support optimal mito-
chondrial function and to ensure the proper level of Arg3, both
of which do not involve thiol-disulfide redox function. The
present findings imply that Trx2 participates in communication
between mitochondria and the nucleus to adjust nuclear gene
expression in response to changes in mitochondrial function.
This study was supported by a National Research Laboratory grant
(2004-02397) from the Ministry of Science and Technology to
J.-H.R. J.-Y.S. was supported by the second stage of BK21 program for
Biological Sciences at Seoul National University.
1. Alami, M., E. Dubois, Y. Oudjama, C. Tricot, J. Wouters, V. Stalon, and F.
Messenguy. 2003. Yeast epiarginase regulation, an enzyme-enzyme activity
control. J. Biol. Chem. 278:21550–21558.
2. Alfa, C., P. Fantes, J. Hyams, M. Mcleod, and E. Warbrick. 1993. Experi-
ments with fission yeast: a laboratory course manual. Cold Spring Harbor
Laboratory Press, New York, NY.
3. Borutaite, V., and G. Brown. 2003. Mitochondria in apoptosis of ischemic
heart. FEBS Lett. 24:1–5.
4. Butow, R., and N. Avadhani. 2004. Mitochondrial signaling: the retrograde
response. Mol. Cell 14:1–15.
5. Cahill, A., X. Wang, and J. B. Hoek. 1997. Increased oxidative damage to
mitochondrial DNA following chronic ethanol consumption. Biochem. Bio-
phys. Res. Commun. 235:286–290.
6. Chan, D. 2006. Mitochondria: dynamic organelles in disease, aging, and
development. Cell 125:1241–1252.
7. Damdimopoulos, A. E., A. Miranda-Vizuete, M. Pelto-Huikko, J.-Å. Gustafs-
son, and G. Spyrou. 2002. Human mitochondrial thioredoxin. Involvement in
mitochondrial membrane potential and cell death. J. Biol. Chem. 277:33249–
8. Delaunay, A., A. Isnard, and M. B. Toledano. 2000. H2O2sensing through
oxidation of the Yap1 transcription factor. EMBO J. 19:5157–5166.
9. Endo, F., T. Matsuura, K. Yanagita, and I. Matsuda. 2004. Clinical mani-
festations of inborn errors of the urea cycle and related metabolic disorders
during childhood. J. Nutr. 134(Suppl. 6):1605S–1609S.
10. Guoyao, W. U., and S. M. Morris. 1998. Arginine metabolism: nitric oxide
and beyond. Biochem. J. 336:1–17.
11. Hall, D. A., H. Zhu, X. Zhu, T. Royce, M. Gerstein, and M. Snyder. 2004.
Regulation of gene expression by a metabolic enzyme. Science 306:482–484.
12. Reference deleted.
13. Herrero, E., and J. Ros. 2002. Glutaredoxins and oxidative stress defense in
yeast. Methods Enzymol. 348:136–146.
14. Hirota, K., M. Murata, Y. Sachi, H. Nakamura, J. Takeuchi, K. Mori, and
J. Yodoi. 1999. Distinct roles of thioredoxin in the cytoplasm and in the
nucleus: a two-sept mechanism of redox regulation of transcription factor
NF-?B. J. Biol. Chem. 274:27891–27897.
15. Holmgren, A. 1984. Enzymatic reduction-oxidation of protein disulfides by
thioredoxin. Methods Enzymol. 107:295–300.
16. Holmgren, A. 1985. Thioredoxin. Annu. Rev. Biochem. 54:237–271.
17. Holmgren, A. 1989. Thioredoxin and glutaredoxin systems. J. Biol. Chem.
18. Huber, H. E., M. Russel, P. Model, and C. C. Richardson. 1986. Interaction
of mutant thioredoxins of Escherichia coli with the gene 5 protein of phage
T7. J. Biol. Chem. 261:15006–15012.
19. Huber, H. E., S. Tabor, and C. C. Richardson. 1987. Escherichia coli thiore-
doxin stabilizes complex of bacteriophage T7 DNA polymerase and primed
template. J. Biol. Chem. 262:16224–16232.
20. Izawa, S., K. Maeda, K. Sugiyama, J. Mano, and Y. Inoue. 1999. Thioredoxin
deficiency causes the constitutive activation of Yap1, an AP-1-like transcrip-
tion factor in Saccharomyces cerevisiae. J. Biol. Chem. 274:28459–28465.
21. Jime ´nez, A., L. Mateos, J. R. Pedrajas, A. Miranda-Vizuete, and J. L. Re-
vuelta. 2007. The txl1?gene from Schizosaccharomyces pombe encodes a
new thioredoxin-like 1 protein that participates in the antioxidant defense
against tert-butyl hydroperoxide. Yeast 24:481–490.
22. Kim, S. J., E. M. Jung, H. J. Jung, Y. S. Song, E. H. Park, and C. J. Lim.
2007. Cellular functions and transcriptional regulation of a third thioredoxin
from Schizosaccharomyces pombe. Can. J. Microbiol. 53:775–783.
23. Kucej, M., and R. A. Butow. 2007. Evolutionary tinkering with mitochondrial
nucleoids. Trends. Cell. Biol. 17:586–592.
24. Kuge, S., M. Arita, A. Murayama, K. Maeta, S. Izawa, Y. Inoue, and A.
Nomoto. 2001. Regulation of the yeast Yap1p nuclear export signal is me-
diated by redox signal-induced reversible disulfide bond formation. Mol.
Cell. Biol. 21:6139–6150.
25. Lee, J. T., and R. L. Nussbaum. 1989. An arginine to glutamine mutation in
residue 109 of human ornithine transcarbamylase completely abolishes en-
zymatic activity in Cos1 cells. J. Clin. Investig. 84:1762–1766.
26. Lee, J., I. Dawes, and J. H. Roe. 1997. Isolation, expression, and regulation
of the pgr1?gene encoding glutathione reductase absolutely required for the
aerobic growth of Schizosaccharomyces pombe. J. Biol. Chem. 272:23042–
27. Lee, S.-Y., J.-Y. Song, E.-S. Kwon, and J.-H. Roe. 2008. Gpx1 is a stationary
phase-specific thioredoxin peroxidase in fission yeast. Biochem. Biophys.
Res. Commun. 367:67–71.
28. Lill, R., and U. Mu ¨hlenhoff. 2006. Iron-sulfur protein biogenesis in
eukaryote: components and mechanisms. Annu. Rev. Cell Dev. Biol. 22:457–
29. Liu, Z., and R. Butow. 2006. Mitochondrial retrograde signaling. Annu. Rev.
30. Mark, D. F., and C. C. Richardson. 1976. Escherichia coli thioredoxin: a
subunit of bacteriophage T7 DNA polymerase. Proc. Natl. Acad. Sci. USA
31. Masutani, H., and J. Yodoi.2002. Thioredoxin. Methods Enzymol. 347:279–
32. Matsuyama, A., R. Arai, Y. Yashiroda, A. Shirai, A. Kamata, S. Sekido, Y.
Kobayashi, A. Hashimoto, M. Hamamoto, Y. Hiraoka, S. Horinouchi, and
M. Yoshida. 2006. ORFeome cloning and global analysis of protein local-
2166SONG ET AL.EUKARYOT. CELL
ization in the fission yeast Schizosaccharomyces pombe. Nat. Biotechnol.
33. Mian, A., and B. Lee. 2002. Urea-cycle disorders as a paradigm for inborn
errors of hepatocyte metabolism. Trends Mol. Med. 8:583–589.
34. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fission
yeast Schizosaccharomyces pombe. Methods Enzymol. 194:795–823.
35. Netto, L. E. S., A. J. Kowaltowski, R. F. Castilho, and A. E. Vercesi. 2002.
Thiol enzymes protecting mitochondria against oxidative damage. Methods
36. Nonn, L., R. R. Williams, R. P. Erickson, and G. Powis. 2003. The Absence
of mitochondrial thioredoxin 2 causes massive apoptosis, exencephaly, and
early embryonic lethality in homozygous mice. Mol. Cell. Biol. 23:916–922.
37. Okamoto, T., K. Asamitsu, and T. Tetsuka. 2002. Thioredoxin and mecha-
nism of inflammatory response. Methods Enzymol. 347:349–360.
38. Pedrajas, J. R., E. Kosmidou, A. Miranda-Vizuete, J. A. Gustafsson, A. P.
Wright, and G. Spyrou. 1999. Identification and functional characterization
of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae.
J. Biol. Chem. 274:6366–6373.
39. Russel, M., and P. Model. 1986. The role of thioredoxin in filamentous phage
assembly. J. Biol. Chem. 261:14997–15005.
40. Ryan, M. T., and N. J. Hoogenraad. 2007. Mitochondrial-nuclear communi-
cations. Annu. Rev. Biochem. 76:701–722.
41. Saitoh, M., H. Nishitoh, M. Fujii, K. Takeda, K. Tobiume, Y. Sawada, M.
Kawabata, K. Miyazono, and H. Ichijo. 1998. Mammalian thioredoxin is a
direct inhibitor of apoptosis signal-regulating kinase (ASK) 1. EMBO J.
42. Scha ¨fer, B. 2003. Genetic conservation versus variability in mitochondria:
the architecture of the mitochondrial genome in the petite-negative yeast
Schizosaccharomyces pombe. Curr. Genet. 43:311–326.
43. Schmitt, M. E., T. A. Brown, and B. L. Trumpower. 1990. A rapid and simple
method for preparation of RNA from Saccharomyces cerevisiae. Nucleic
Acids Res. 18:3091–3092.
44. Song, J.-Y., J. Cha, J. Lee, and J.-H. Roe. 2006. Glutathione reductase and
a mitochondrial thioredoxin play an overlapping role for maintaining iron-
sulfur enzymes in fission yeast. Eukaryot. Cell 5:1857–1865.
45. Song, J.-Y., and J.-H. Roe. 2008. The role and regulation of Trx1, a cytosolic
thioredoxin in Schizosaccharomyces pombe. J. Microbiol. 46:408–414.
46. Stadtman, E. R. 1993. Oxidation of free amino acids and amino acid residues
in proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Bio-
47. Tabor, S., H. E. Huber, and C. C. Richardson. 1987. Escherichia coli thiore-
doxin confers processivity on the DNA polymerase activity of the gene 5
protein of bacteriophage T7. J. Biol. Chem. 262:16212–16223.
48. Tanaka, T., F. Hosoi, Y. Yamaguchi-Iwai, H. Nakamura, H. Masutani, S.
Ueda, A. Nishiyama, S. Takeda, H. Wada, G. Spyrou, and J. Yodoi. 2002.
Thioredoxin-2 (TRX-2) is an essential gene regulating mitochondria-depen-
dent apoptosis. EMBO J. 21:1695–1703.
49. Taylor, N. L., D. A. Day, and A. H. Millar. 2004. Targets of stress-induced
oxidative damage in plant mitochondria and their impact on cell carbon/
nitrogen metabolism. J. Exp. Bot. 55:1–10.
50. Trotter, E. W., and C. M. Grant. 2005. Overlapping roles of the cytoplasmic
and mitochondrial redox regulatory systems in the yeast Saccharomyces cer-
evisiae. Eukaryot. Cell 4:392–400.
51. Turrens, J. F. 2003. Mitochondrial formation of reactive oxygen species.
J. Physiol. 552:335–344.
52. Reference deleted.
53. Urrestarazu, L., S. Vissers, and J. Wiame. 1977. Change in location of
ornithine carbamoyltransferase and carbamoylphosphate synthetase among
yeasts in relation to the arginase/ornithine carbamoyltransferase regulatory
complex and the energy status of the cells. Eur. J. Biochem. 79:473–481.
54. Van Huffel, C., E. Dubois, and F. Messenguy. 1992. Cloning and sequencing
of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA
fragment: heterologous expression and mitochondrial targeting of their
translation products. Eur. J. Biochem. 205:33–43.
55. Vlamis-Gardikas, A., and A. Holmgren. 2002. Thioredoxin and glutaredoxin
isoforms. Methods Enzymol. 347:286–296.
56. Wang, Y., and D. F. Bogenhagen. 2006. Human mitochondrial DNA nucle-
oids are linked to protein folding machinery and metabolic enzymes at the
mitochondrial inner membrane. J. Biol. Chem. 281:25791–25802.
57. Zhang, R., R. Al-Lamki, L. Bai, J. W. Streb, J. M. Miano, J. Bradley, and
W. Min. 2004. Thioredoxin-2 inhibits mitochondria-located ASK1-medi-
ated apoptosis in a JNK-independent manner. Circ. Res. 94:1483–1491.
VOL. 7, 2008MITOCHONDRIAL THIOREDOXIN NECESSARY FOR UREA CYCLE 2167