The txl1+ gene from Schizosaccharomyces pombe encodes a new
thioredoxin-like 1 protein that participates in the antioxidant defense against
tert-butyl hydroperoxide
Alberto Jiménez1, Laura Mateos1, José R. Pedrajas2, Antonio Miranda-Vizuete3,
José L. Revuelta1,4
1Grupo
de Ingeniería Metabólica, Instituto de Microbiología Bioquímica y
Departamento de Microbiología y Genética, CSIC/Universidad de Salamanca,
Campus Miguel de Unamuno, 37007 Salamanca, Spain.
2Grupo
de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad
Asociada al CSIC (EEZ), Área de Bioquímica y Biología Molecular, Universidad
de Jaén, Spain
3Centro
Andaluz de Biología del Desarrollo (CABD-CSIC), Departamento de
Fisiología, Anatomía y Biología Celular, Universidad Pablo de Olavide, 41013
Sevilla, Spain.
4To
whom correspondence should be addressed:
revuelta@usal.es
Phone: +34 923 294671
Fax: +34 923 224876
Short title: Thioredoxin-like 1 from Schizosaccharomyces pombe
Abbreviations: DTT, dithiothretitol; GFP, green fluorescent protein; GST,
glutathione-S-transferase; Trx, thioredoxin; TrxR, thioredoxin reductase; Txl,
thioredoxin-like.
Abstract
Yeasts are equipped with several putative single-domain thioredoxins located in
different subcellular compartments. However, additional proteins containing
thioredoxin domains are also encoded by the yeast genomes as described for
mammals and other eukaryotic organisms. We report here the characterization of
the fission yeast orthologue thioredoxin-like 1 (txl1+), which has been previously
identified in mammals. Similarly to the human protein, the fission yeast Txl1 is a
two-domain protein comprising an N-terminal thioredoxin-like domain and a Cterminal domain of unknown function. Many other yeasts and fungi species
contain homologues of txl1+, however there is no evidence of txl1+ orthologues
either in Saccharomyces cerevisiae or plants. Txl1 is found in both the nucleus
and the cytoplasm of S. pombe cells and exhibits a strong reducing activity
coupled to thioredoxin reductase. In humans, TXL1 expression is induced by
glucose deprivation and overexpression of TXL1 confers resistance against this
stress. In contrast, a S. pombe txl1 mutant was not affected in the response
against glucose starvation but the txl1 mutant strain showed a clear
hypersensitivity to alkyl hydroperoxide. The mRNA levels of txl1+ in a h20 strain
did not change in response to any oxidative insult (hydrogen peroxide or alkyl
hydroperoxide) and the overexpression of an integrated copy of the wild-type txl1+
gene did not confer a significant increased resistance against alkyl hydroperoxide.
Overall, these results indicate that the Txl1 role in the cellular detoxification of
alkyl hydroperoxide is exerted through a constitutive transcription of txl1+.
Introduction
Thioredoxins (Trx) are redox proteins that function as general protein disulphide
oxidoreductases, maintaining the reduced cellular environment (Hirota, et al.,
2002; Nakamura, 2005). The redox activity of thioredoxins is based on the ability
of the cysteines of their active site (Cys-Gly-Pro-Cys) to undergo reversible
oxidation from a dithiol to a disulphide form. Thioredoxins activity is coupled to the
flavoenzyme thioredoxin reductase (TrxR), which maintains thioredoxins in their
reduced active form using NADPH as the electron donor (Holmgren, 2000). It has
been described that alterations in the thioredoxin system can lead to several
pathological processes (Burke-Gaffney, et al., 2005).
All organisms investigated so far, from prokaryotes to humans, contain different
forms of thioredoxins (Hirota, et al., 2002; Nakamura, 2005). Saccharomyces
cerevisiae contains two thioredoxin systems: one in the cytosol composed by two
thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1), and other in
the mitochondria formed by a thioredoxin (Trx3) and a thioredoxin reductase
(Trr2) (Gan, 1991; Pedrajas, et al., 1999). In contrast, the genome of
Schizosaccharomyces pombe encodes for only one cytosolic thioredoxin (Trx1),
one mitochondrial thioredoxin (Trx2) and one thioredoxin reductase (Trr1), which
can be localized both in the cytosol and the mitochondria (Casso and Beach,
1996; Cho, et al., 2001; Lee, et al., 2002).
In fission yeast, the expression of the cytosolic Trx1 is moderately induced by
hydrogen peroxide and other environmental stresses, while the mitochondrial Trx2
is not. Consequently, trx1+ is considered as a part of the “Core Environmental
Stress Response” (CESR) in S. pombe (Chen, et al., 2003). Additionally, the
expression of thioredoxin reductase is strongly induced in response to both
hydrogen peroxide and methyl methane sulfonate (MMS) (Chen, et al., 2003;
Weeks, et al., 2006).
The structure of thioredoxins is globular and consists of a central core of sheets surrounded by -helixes with the active site situated in a protrusion
emerging from the protein surface (Jeng, et al., 1994). While all thioredoxins
reported from yeast species comprise only one thioredoxin domain, some
mammalian members of the thioredoxin family are composed of additional
domains with known or unknown counterparts in the databases (Cunnea, et al.,
2003; Lee, et al., 1998; Miranda-Vizuete, et al., 1998; Miranda-Vizuete, et al.,
2001; Sadek, et al., 2001; Sadek, et al., 2003).
In a recent work, we have characterized the human thioredoxin-like 1 (TXL1),
which is a two-domain protein composed of a N-terminal thioredoxin domain
followed by a C-terminal domain of unknown function with no homology to any
other protein in the databases (Jimenez, et al., 2006). Human TXL1 is a cytosolic
thioredoxin that can also translocate to the nucleus, it is predominantly expressed
in the central nervous system and other organs with an elevated metabolic rate
and it is involved in the cellular response to glucose deprivation (Jimenez, et al.,
2006).
We
have
identified
a
TXL1
orthologue
in
a
S.
pombe
database
(http://www.sanger.ac.uk/Projects/S_pombe/) and here we show that the S.
pombe Txl1 is a novel thioredoxin-like, which participates in the cellular protection
against oxidative stress induced by alkyl hydroperoxide.
Materials and Methods
Strains, Growth Conditions and Chemicals
All the S. pombe strains used in this work are listed in Table 1. S. pombe cells
were routinely grown at 28ºC in YES rich medium or minimal EMM medium with
the required supplements (Moreno, et al., 1991). Growth was monitored
spectrophotometrically at 595nm and standard genetic manipulations were used
(Moreno, et al., 1991). S. pombe transformations were carried out as described
elsewhere (Ito, et al., 1983).
Amino acids, insulin, NADPH, DTT, geneticin (G418), H2O2 and tert-butyl
hydroperoxide (t-BOOH) were purchased from Sigma (Steinheim, Germany).
S. pombe txl1+ protein expression and purification
The ORF encoding the S. pombe Txl1 was PCR-amplified from cDNA prepared
using the SuperScripTM II RT enzyme (Invitrogen, Carlsbad, USA) and S. pombe
total RNA as template. The ORF was cloned into the BamHI-EcoRI sites of the
pGEX-4T-1 expression vector (Amsershan Biosciences, Madrid, Spain), verified
by sequencing and used to transform E. coli HMS174(DE3). Induction and
purification of the recombinant protein was achieved as previously reported
(Miranda-Vizuete, et al., 1998). An overnight incubation with thrombin (5 units/mg
of fusion protein) was used to remove the Txl1 recombinant protein from the
glutathione S-transferase domain. S. pombe Txl1 was eluted and protein
concentration was determined with the Bio-Rad protein assay kit (Bio-Rad,
Madrid, Spain) using BSA as a standard.
Enzymatic Thioredoxin Activity Assays
Enzymatic activity of recombinant Txl1 was performed using two different
assays. In the DTT assay, DTT is used as reducing agent and the assay was
carried out as previously described (Wollman, et al., 1988) In the thioredoxin
reductase assay, recombinant Txl1 activity was determined by its capability to
reduce insulin disulfide bonds using NADPH as electron donor in the presence of
the mitochondrial thioredoxin reductase (Trr2) from S. cerevisiae (Pedrajas, et al.,
1999). The activity assay was performed essentially as described elsewhere
(Spyrou, et al., 1997) but monitoring insulin precipitation at 595nm. In both cases,
the mitochondrial thioredoxin (Trx3) from S. cerevisiae (Pedrajas, et al., 1999)
was used as a positive control.
Gene Deletion, Overexpression and GFP-tagging
The entire txl1+ ORF was replaced in a h20 strain of S. pombe with a kanMX6
cassette, which confers resistance to G418, following the method described by
Bahler et al. (Bahler, et al., 1998). The deletion cassette was constructed by PCR
means using the pFA6kanMX6 vector (Bahler, et al., 1998) as template and 90-95
nucleotides long primers (Supplementary Data Table 1).
For the overexpression of the S. pombe txl1+, we used PCR techniques to
replace the native txl1+ promoter by the inducible strong nmt1+ promoter. A
pFA6kanMX6-P3nmt1 vector was used as PCR template (Bahler, et al., 1998)
using appropriate primers (Supplementary Data Table 1). The nmt1+ promoter
allows expression at a relatively high level in culture media lacking thiamine. For
overexpression experiments using the nmt1+ promoter, cells were grown in EMM
medium (without thiamine).
Gene deletion and integration of the nmt1+ promoter in S. pombe was confirmed
by Southern-blotting and expression analysis was verified by northern-blotting.
GFP C-terminal tagging was achieved using also PCR techniques, employing a
pFA6-GFP(S65T)-kanMX6 (Bahler, et al., 1998) and using the primers listed in
Supplementary Data Table 1. A fusion fragment containing the txl1+ ORF in frame
to the GFP coding region was constructed by direct genome integration. The
fluorescence of the Txl1-GFP(S65T) fusion protein was monitored in living cells as
previously described (Niedenthal, et al., 1996). Micrographs were acquired using
a Photometrics Sensys CCD camera coupled to a Leica DMXRA microscope
equipped with Nomarski optics and epifluorescence.
Northern blot analysis
Cells grown under different conditions were harvested and total RNA was
prepared as previously described (Percival-Smith and Segall, 1984). For northern
blot analyses, 10 g of each RNA sample was used. A 580 bp HindIII fragment
from the S. pombe txl1+ ORF was labeled with [-32P] dCTP (Rediprime random
primer labeling kit; Amersham Pharmacia-Biotech) and used as radioactive probe.
The blots were also hybridized with a 1.3 kb SacII-HindII fragment of the S.
pombe -
For quantitative analysis, the blots were scanned
and quantified with a BAS1500-Mac image analyzer (Fuji Film Co).
Glucose deprivation and oxidative treatments
For the glucose deprivation treatment, cells were grown in YES medium with 2%
glucose, 0.5% glucose or 2% glycerol (without glucose). Cultures were carried out
in microtiter 96-well plates using 180 l of culture media per well. The h20 and
txl1 strains were replicated from a saturated master 96-well plate where both
strains had been previously grown in YES medium.
Additionally, 10 ml EMM cultures were achieved with h20, txl1 and P3nmt1txl1+ strains using 2% glucose (EMM) or 0.5% glucose (EMM-L). In this case,
cultures were initiated from saturated precultures using 10-50 l.
For the treatments with hydrogen peroxide and t-BOOH, cells were grown in
EMM medium (lacking thiamine) to allow the overexpression of txl1+ in the
P3nmt1-txl1+ strain.
Results
Cloning of a novel thioredoxin-like protein from S. pombe
A BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) using the human TXL1
ORF as a bait identified several entries including a S. pombe orthologue (ORF
No. SPBC577.08c), which showed a 38% of identity at the protein level when
compared with that of the human TXL1. The information related to the
SPBC577.08c
ORF
in
the
S.
pombe
Database
(http://www.genedb.org/genedb/pombe/index.jsp) considered the ORF product as
a member of the thioredoxin family, inferring its role from homology comparisons.
The SPBC577.08c gene comprises 925 bp, it maps at the chromosome 2 of S.
pombe genome and it is organized into two exons with a spliced length of 873 bp.
Surprisingly, there is no apparent orthologue in the S. cerevisiae genome
although the gene is present all along the evolution from lower eukaryotes to
humans (see Supplementary Data Fig. 1 in (Jimenez, et al., 2006)), indicating that
the protein could have an important cellular role. Therefore, we designed specific
primers (Supplementary Data Table 1) to amplify the SPBC577.08c ORF by PCR
from a S. pombe cDNA library.
Structurally, the SPBC577.08c protein of S. pombe (290 residues) comprises a
N-terminal thioredoxin-like domain and a C-terminal domain of unknown function
(DUF1000) as described for the human TXL1 (Jimenez, et al., 2006) and other
orthologues (Fig. 1). The predicted chemical properties of the SPBC577.08c
encoding protein are also the same as described for other orthologues (Jimenez,
et al., 2006) with no subcellular localization signals within the protein sequence
either. Therefore, we decided to consider SPBC577.08c as the corresponding
thioredoxin-like 1 (txl1+) gene in S. pombe.
S. pombe Txl1 shows reducing thioredoxin activity
We wanted to confirm whether the Txl1 from S. pombe was able to catalyze the
reduction of disulphide bonds, thus having thioredoxin activity. Consequently, we
expressed a recombinant GST-Txl1 fusion protein in E. coli and purified a
thrombin-cleaved Txl1 (see Materials and Methods).
We next checked the enzymatic activity of the recombinant S. pombe Txl1 using
DTT or thioredoxin reductase coupled to NADPH as reductants, and we used the
mitochondrial thioredoxin (Trx3) from S. cerevisiae as a positive control (Pedrajas,
et al., 1999). As shown in figure 2, the recombinant Txl1 from S. pombe is able to
reduce the insulin disulphide bonds either with DTT or thioredoxin reductase.
Remarkably, the kinetics of S. cerevisiae Trx3 and S. pombe Txl1 were quite
similar and the specific thioredoxin activity of both protein preparations was also
comparable. Conversely, we have previously reported that human TXL1
thioredoxin activity shows a long latency phase and much lower reducing activity
than that of human TRX1 (Jimenez, et al., 2006). This discrepancy between the
human and S. pombe Txl1 thioredoxin activities might reflect other functional
divergences.
Subcellular localization of the S. pombe Txl1
In order to investigate the subcellular localization of Txl1, we carried out a Cterminal GFP tagging of the S. pombe Txl1 protein. Fluorescence could be seen
both in the nucleus and the cytoplasm of cells expressing the Txl1-GFP fusion
(Fig. 3), indicating that Txl1 can translocate into the nucleus even though a
nuclear localization signal is lacking in its sequence. The same localization pattern
has been previously reported for the human TXL1 (Jimenez, et al., 2006) and,
more recently, a comprehensive global analysis of the S. pombe ORFome has
also
confirmed
these
localizations
(Matsuyama,
et al.,
2006).
Nuclear
translocation has also been reported for other thioredoxins lacking nuclear
targeting signals (Hirota, et al., 1999).
S. pombe Txl1 is not involved in the cellular response to glucose
starvation.
Our previous results indicate that human TXL1 could play a protective role
against sugar starvation stress (Jimenez, et al., 2006). Thus, we decided to check
whether S. pombe Txl1 could be functioning in a similar way as human TXL1
regarding the response against glucose deprivation.
In a first experiment we looked for the S. pombe txl1+ transcription pattern in h20
cells cultured under glucose starvation stress. A northern-blot analysis revealed
that the txl1+ mRNA levels did not change along the treatment when glucose or
glycerol were used as carbon sources (Fig. 4A). Hence, our results seem to
indicate that txl1+ transcription is not affected by glucose deprivation.
To confirm this assumption, we decided to investigate whether the absence of
TXL1 might influence the growth pattern of S. pombe under glucose starvation
stress. Thus, we constructed a txl1 null mutant by replacing the TXL1 gene with
the kanMX6 dominant marker, which confers resistance to G418 (see Materials
and methods for details). Gene deletion was verified by Southern-blot and
northern-blot (Fig. 5).
The txl1 strain did not show any apparent morphologic defect and the growth
pattern in rich medium (2% glucose) was identical to the h20 parental strain. As
shown in figures 4B and 6A, there were no differences among the txl1 and the
h20 strains with low (0.5%) glucose concentrations in the culture media. Glycerol
did not support the growth of either the txl1 or the h20 strains (Fig. 4B), and the
addition of exogenous glucose after 108 hours immediately induced the same
growth enhancement on both strains. Therefore, we can conclude that Txl1 is not
involved in the glucose starvation response in S. pombe.
The txl1 strain of S. pombe shows hypersensitivity to an alkyl
hydroperoxide
The results shown above indicate that human and S. pombe txl1+ orthologues
might accomplish different tasks in the cell. S. pombe Txl1 holds a high disulphide
reducing activity, which might reflect a possible role for Txl1 in the cellular defense
against oxidants. Therefore, to further characterize the txl1 strain we decided to
explore the effect of two prooxidants, hydrogen peroxide (H2O2) and tert-butyl
hydroperoxide (t-BOOH), on the growth of the txl-1 strains on liquid cultures. The
h20 and txl1 strains were grown in minimal media with 2mM H2O2 or 0.5mM tBOOH during 40 hours. Our results showed that hydrogen peroxide does not
affect the growth of the txl1 strain when compared with the h20 strain (Fig. 6A).
However, t-BOOH did induce a marked delay in the growth of the txl1 strain (Fig.
6A), suggesting that Txl1 might be involved in the detoxification of t-BOOH and
probably other alkyl hydroperoxides.
We assumed that Txl1 protection against alkyl hydroperoxides might be
mediated by an induction of the txl1+ transcription. However, when we tested the
effect of the two prooxidants previously used on the levels of the txl1+ transcripts
in a h20 strain, we found that neither t-BOOH nor H2O2 were able to induce an
increase on the mRNA levels of txl1+ (Fig. 6B). To confirm this result, we
expressed an integrated copy of txl1+ under the control of the strong promoter
nmt1+ (Fig. 5) and checked the growth rate of the txl1+ overexpressing strain in
the presence of t-BOOH or H2O2. As shown in figure 6A, no significant increase in
the resistance to t-BOOH was achieved through the overexpression of txl1+,
indicating that non-transcriptional mechanisms might underlie the protective effect
of Txl1 against t-BOOH.
Discussion
Redox balance is a main issue in the cellular physiology and is maintained by
means of a steady state situation between oxidants and antioxidants (Kondo, et
al., 2006; Nakamura, 2005). However, it has been proposed that oxidative stress
may be considered as a disruption of redox signalling and control, and that
specific redox mechanisms determine discrete signalling and control events
(Jones, 2006).
Thioredoxin like-1 was first identified and characterized in humans (Lee, et al.,
1998; Miranda-Vizuete, et al., 1998). More recently, we proposed that human
TXL1 participates in the cellular response against glucose deprivation-mediated
stress in a TRX1 independent manner (Jimenez, et al., 2006). In this work, we
have identified the fission yeast orthologue of the human TXL1 and we have
characterized it in order to complement our data with the human TXL1.
We have demonstrated that the thioredoxin-like 1 protein from S. pombe has a
strong reducing activity coupled to thioredoxin reductase, differing significantly
from that of the human TXL1, which shows a much lower activity. A possible
explanation for this difference may be the fact that the active site of the fission
yeast protein is preceded by a tryptophan residue (WCGPC), as occurs in most
thioredoxins (Holmgren and Björnstedt, 1995). In humans and other mammals,
the tryptophan residue is substituted in the TXL1 protein by a glycine residue
(GCGPC) (Jimenez, et al., 2006). Indeed, the presence of the tryptophan in the
thioredoxins active site has been proposed to regulate their catalytic activity
(Krause and Holmgren, 1991).
The domain organization of the S. pombe thioredoxin-like 1 protein is identical to
the other reported orthologues, having a thioredoxin-like N-terminal domain and a
C-terminal domain (DUF100), which has been suggested to be a regulatory
domain (Jin, et al., 2002). The subcellular localization of the S. pombe Txl1
protein also coincides with the nuclear and cytosolic localization of the human
TXL1. Furthermore, neither S. pombe Txl1 nor human TXL1 have any nuclear
translocation signal in their respective sequences, so the translocation into this
organella must be mediated by cotransport with other proteins rather than passive
diffusion given its size (Jimenez, et al., 2006).
The transcription of txl1+ remained unaltered in response to the stress induced
by glucose starvation or hydroperoxides. This feature constitutes a significant
difference between the human and the fission yeast orthologues. Indeed, a
mutant txl1 strain did not show any growth defect when cultured in media lacking
glucose, with low glucose concentrations or in the presence of hydrogen peroxide.
However, the deletant txl1 displayed a notorious growth defect when an alkyl
hydroperoxide (t-BOOH) was present in the culture medium but, surprisingly, a
mutant strain overexpressing txl1+ did not exhibit higher resistance to t-BOOH
compared to that of the S. pombe h20 strain, indicating that induction of txl1+
transcription is not responsible for the Txl1 protection against t-BOOH. A genome
wide analysis of S. cerevisiae viable deletion strains showed that most genes
involved in the resistance against oxidative stress are not transcriptionally induced
(Thorpe, et al., 2004). Besides, it has been shown that stress-mediated induction
in many genes is evolutionarily conserved in S. cerevisiae and S. pombe; and the
same work has reported that only eight genes with annotated antioxidant function
are included in the Core Environmental Stress Response (CESR) in S. pombe
(Chen, et al., 2003). Indeed, most of the genes that are induced in response to
hydrogen peroxide in S. pombe do not code for antioxidant enzymes (Chen, et al.,
2003). Therefore, we can consider that S. pombe is equipped with a set of
constitutively expressed genes, which participate in the antioxidant housekeeping
defense like txl1+ in response to t-BOOH or grx2+ against paraquat (Chung, et al.,
2004). Additionally, S. pombe has the CESR set of genes that are transcriptionally
induced by oxidative stress as described for the trx1+ and trr1+ genes (Chen, et
al., 2003). A recent report has evidenced that posttranslational processing
contribute to the function of the cytosolic thioredoxin 1 (Haendeler, 2006), and we
cannot exclude the possibility that Txl1 might suffer some posttranslational
modifications to regulate its function.
A S. cerevisiae ahp1 mutant lacking an alkyl hydroperoxide reductase has been
described to be hypersensitive to t-BOOH (Lee, et al., 1999). The fission yeast
database at Sanger Institute (http://www.genedb.org/genedb/pombe/index.jsp)
displays the entry number SPCC330.06c as a sequence similar to the S.
cerevisiae AHP1, although the percent of identity at protein level is only a 25%.
The SPCC330.06c gene product is annotated as a thioredoxin peroxidase and the
subcellular localization has been shown to be both cytosolic and nuclear
(Matsuyama, et al., 2006). Whether SPCC330.06c may be involved in the
resistance against alkyl hydroperoxides is still unknown, but considering that
SPCC330.06c and Txl1 colocalize within the same subcellular compartments in S.
pombe, and that the proposed SPCC330.06c orthologue in S. cerevisiae functions
as an alkyl hydroperoxide reductase, we might speculate that SPCC330.06c and
Txl1
could
participate
in
the
same
specific
mechanism
against
alkyl
hydroperoxides.
A comparison of proteins among S. pombe and S. cerevisiae revealed that gene
duplication had occurred more in S. cerevisiae than in S. pombe, thus accounting
for the high number of extra proteins found in S. cerevisiae (Wood, et al., 2002).
However, there is a number of S. pombe genes (17%) with no homologues in S.
cerevisiae (Wood, et al., 2002). The budding yeast genome does not encode for a
txl1+ orthologue. Nevertheless, other yeast species and higher eukaryotic
organisms retain a gene encoding a thioredoxin-like protein. Neofunctionalization
mechanisms in which a duplicated gene may loose or retain functions, or acquire
novel functions (He and Zhang, 2005), might explain the functional differences
between the human and S. pombe txl1+ orthologues. Such functional divergence
among eukaryotic species has been reported in other families of proteins as the
exporting-5 orthologues (Shibata, et al., 2006). In the thioredoxin-like 1 family,
mutations in the sequence of the active site leading to the substitution of a
tryptophan residue (present in lower eukaryotes) by a glycine (present in
mammals) may strongly affect the reducing thioredoxin activity. In this regard, the
human TXL1 gene might have acquired the ability to participate in the cellular
response against glucose deprivation; a function that is not accomplished by the
S. pombe thioredoxin-like 1 protein.
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Acknowledgements
This work was supported by grants GEN2001-4707-C08-01 and AGL2005-07245C03-03 from the Ministerio de Educación y Ciencia, Spain. A. J. is a recipient of a
postdoctoral contract (Programa Juán de la Cierva) from the Spanish Ministerio
de Educación y Ciencia, L. M. was supported by a predoctoral fellowship from the
Universidad de Salamanca, Spain. A. M.-V. is supported by a research contract
under the Ramón y Cajal Programme of the Spanish Ministerio de Educación y
Ciencia. We thank M. D. Sánchez for excellent technical help, and N. Skinner for
correcting the manuscript.
Figure lengends
Figure 1. Schematic domain organization of thioredoxin-like 1 proteins from
human and different yeast species. N. crassa, Neurospora crassa; C.
neoformans, Cryptococcus neoformans; Y. lipolytica, Yarrowia lipolytica; C.
albicans, Candida albicans; S. pombe, Schizosaccharomyces pombe.
Figure 2. Thioredoxin activity assay of Txl1 from S. pombe. A) Purified
recombinant Txl1 (2M) from S. pombe was assayed for its ability to reduce
insulin disulfide bonds in the presence of NADPH and DTT. B) Thioredoxin activity
assay of S. pombe Txl1 (2M) using NADPH and S. cerevisiae thioredoxin
reductase (Trr2, 0.5M). Trx3 (2M) from S. cerevisiae was used as positive
control. In the TR control, the assay was performed without any thioredoxin.
Identical results were obtained from three independent experiments.
Figure 3. Txl1 from S. pombe localizes both into the nucleus and cytosol. S.
pombe cells expressing an integrated txl1+-GFP(S65T) fusion were monitored
under epifluorescence microscopy. Left image corresponds to the differential
interference contrast (DIC) picture and right image represents the GFP-channel of
the same field.
Figure 4. S. pombe Txl1 is not involved in the cellular response against glucose
deprivation. A) h20 cells growing in YES medium (O.D.595nm = 0.4) were washed
and transferred to fresh media containing different carbon sources. Aliquots were
taken during 8 hours and RNA levels of txl1+ were determined by northern-
blotting. B) h20 and txl1 strains were grown in YES media containing the
indicated carbon sources. Cultures were made in microtitter 96-well plates and
growth was monitored at 595nm during 108 hours. Exogenous glucose (2%) was
added to the glycerol cultures after 108h.
Figure 5. Construction of mutant txl1 knock-out and overexpressing P3nmt1-txl1
strains. A) Schematic representation of the wild type txl1+, txl1::kanMX6 and
txl1::P3nmt1-txl1 loci. The genomic fragment used as radioactive probe in the
Southern-blot analysis is indicated. B) Southern-blot analysis of EcoRI digested
genomic DNA from the h20, txl1 and P3nmt1- txl1+ strains. C) Northern-blot
analysis of total RNA (10g) from the h20, txl1 and P3nmt1- txl1+ strains grown
in EMM (without thiamine) and YES (with thiamine) media.
Figure 6. S. pombe Txl1 is required for the cellular resistance against t-BOOH. A)
h20, txl1 and P3nmt1-txl1+ strains were grown on minimal EMM media with 2%
glucose (EMM), 0.5% glucose (EMM-L) or supplemented with H2O2 (2mM) or tBOOH (0.5mM). B) h20 cells growing in YES medium (O.D.595nm = 0.4) were
washed and transferred to fresh media containing H2O2 (2mM) or t-BOOH
(0.5mM). Aliquots were taken during 12 hours and RNA levels of txl1+ were
determined by northern-blotting.
Table 1. S. pombe strains used in this work
Strain
Genotype
Source
h20
h- leu1.32
S. Moreno*
SP-3
h- leu1.32, txl1-GFP
This work
SP-8
h- leu1.32, txl1::kanMX6
This work
SP-14
h- leu1.32, txl1::P3nmt1-txl1
This work
*Centro de Investigación del Cáncer, University of Salamanca, Spain.