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.
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