4554
`
`Research Article
`
`Glutaredoxins Grx3 and Grx4 regulate nuclear
`localisation of Aft1 and the oxidative stress response
`in Saccharomyces cerevisiae
`Nuria Pujol-Carrion1, Gemma Belli1, Enrique Herrero1, Antoni Nogues2 and Maria Angeles de la Torre-Ruiz1,*
`1Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida 25198, Spain
`2Servei d’analisis cliniques, Hospital Arnau de Vilanova, Lleida 25192, Spain
`*Author for correspondence (e-mail: madelatorre@cmb.udl.es)
`
`Accepted 23 August 2006
`Journal of Cell Science 119, 4554-4564 Published by The Company of Biologists 2006
`doi:10.1242/jcs.03229
`
`Summary
`two monothiol glutaredoxins of
`Grx3 and Grx4,
`regulate Aft1
`nuclear
`Saccharomyces
`cerevisiae,
`localisation. We provide evidence of a negative regulation
`of Aft1 activity by Grx3 and Grx4. The Grx domain of both
`proteins played an important role in Aft1 translocation to
`the cytoplasm. This function was not, however, dependent
`on the availability of iron. Here we demonstrate that Grx3,
`Grx4 and Aft1 interact each other both in vivo and in vitro,
`which suggests the existence of a functional protein
`complex.
`Interestingly,
`each
`interaction
`occurred
`independently on the third member of the complex. The
`absence of both Grx3 and Grx4 induced a clear enrichment
`of G1 cells in asynchronous cultures, a slow growth
`phenotype, the accumulation of intracellular iron and a
`constitutive activation of the genes regulated by Aft1.
`The grx3grx4 double mutant was highly sensitive to
`
`and
`peroxide
`hydrogen
`agents
`oxidising
`the
`t-butylhydroperoxide but not to diamide. The phenotypes
`of the double mutant grx3grx4 characterised in this study
`were mainly mediated by the Aft1 function, suggesting that
`grx3grx4 could be a suitable cellular model for studying
`endogenous oxidative stress induced by deregulation of the
`iron homeostasis. However, our results also suggest that
`Grx3 and Grx4 might play additional roles in the oxidative
`stress response through proteins other than Aft1.
`
`Supplementary material available online at
`http://jcs.biologists.org/cgi/content/full/119/21/4554/DC1
`
`Key words: Grx3, Grx4, Oxidative stress, Iron homeostasis, Aft1,
`Cell cycle
`
`Introduction
`Cells are exposed to a number of environmental changes and
`must therefore develop different strategies to respond and adapt
`to the various resulting stresses. Aerobic metabolism gives rise
`to reactive oxygen species (ROS), such as superoxide,
`hydrogen peroxide and hydroxyl ions (Cadenas, 1989), which
`provoke oxidative stress and cause damage to cells (Aruoma et
`al., 1991). As a consequence, cells need to develop a series of
`different mechanisms to protect themselves from the harmful
`reactive oxygen species (Poyton, 1999). Iron is an essential
`element for all organisms and appropriate iron homeostasis is
`required to prevent the impairment of cellular functions caused
`by excesses or deficiencies of this metal. Iron is also required
`in a number of essential proteins related to respiratory chain
`reactions, and it plays an essential role in at least one electron
`chain reaction (Arredondo and Núñez, 2005). An excess of iron
`can be very toxic for cells because it generates free radicals
`that may oxidise and damage DNA, lipids and proteins
`(Halliwell and Gutteridge, 1991). Iron deficiency, on the other
`hand, is responsible for several health problems including
`anaemia (Beard, 2001) and both neuronal (Ortiz et al., 2004)
`and immunological alterations. In Saccharomyces cerevisiae,
`the transcription factor Aft1 regulates a subset of genes defined
`as the high-affinity iron-uptake regulon (Yamaguchi-Iwai et al.,
`1995; Casas et al., 1997). This group comprises genes involved
`
`in the uptake, compartmentalisation and use of iron. Aft1 binds
`to specific promoter regions and induces expression of the iron
`regulon in conditions of iron depletion (Yamaguchi-Iwai et al.,
`1996). In a subsequent study, Yamaguchi-Iwai and co-workers
`(Yamaguchi-Iwai et al., 2002) reported that Aft1 responds to
`iron availability by changing its intracellular localisation. This
`means that under iron-replete conditions Aft1 localises to the
`cytoplasm, but under conditions of iron starvation Aft1
`translocates to the nucleus. Even so, the transcriptional activity
`of Aft1 is determined by its nuclear localisation regardless of
`the iron intracellular status.
`iron
`intracellular
`the
`Another physiological effect of
`accumulation mediated by Aft1 is cell-cycle arrest. Philpott and
`co-workers (Philpott et al., 1998) reported that the expression
`of an AFT1-up allele induces iron accumulation, and as a
`consequence, cells arrest in G1 at START. Constitutive
`activation of the iron-responsive regulon resulting from constant
`transcriptional induction driven by Aft1 therefore causes a
`reduced expression of the G1 cyclins, Cln1 and Cln2. One of
`the physiological effects derived from this accumulation of iron
`in cells affects cell-cycle progression in a similar way to that
`previously described for other environmental stresses, including
`heat shock and oxidative and nutritional stress (Cross, 1995;
`Lee et al., 1996).
`Aft2 is another transcription factor required for iron
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`

`homeostasis and resistance to oxidative stress in the absence
`of Aft1 function (Blaiseau et al., 2001). Aft2 also activates
`transcription of specific genes in response to low iron
`conditions (Rutherford et al., 2001; Rutherford et al., 2005). A
`recent study has demonstrated that Aft2 acts in the absence of
`Aft1 (Courel et al., 2005) and that the transcriptional function
`of both proteins is iron-dependent. DNA microarray clustering
`has revealed that both Aft1 and Aft2 share the regulation of a
`number of iron-responsive genes. However, there is a group of
`genes related to iron homeostasis whose regulation depends on
`Aft2 but not on Aft1.
`In proteins, cysteine residues are very susceptible to
`oxidation. Living cells contain regulatory proteins that are
`involved in maintaining the redox states of oxidised proteins
`(Rietsch and Beckwith, 1998; Carmel-Harel and Storz,
`2000; Grant, 2001). Monothiol glutaredoxins are
`thiol
`oxidoreductases, which
`require
`the
`reduced
`form of
`glutathione, GSH, as an electron donor to reduce protein-
`glutathione disulfides (Holmgren, 1989; Holmgren and
`Aslund, 1995; Grant, 2001; Herrero and Ros, 2002).
`In Saccharomyces cerevisiae, three different monothiol
`glutaredoxins, Grx3, Grx4 and Grx5 (Rodríguez-Manzaneque
`et al., 1999), have been described to date. Grx5 plays a role in
`the biogenesis of iron/sulphur clusters at the mitochondria and
`its function has been extensively characterised (Rodríguez-
`Manzaneque et al., 1999; Rodríguez-Manzaneque et al., 2002;
`Bellí et al., 2002). Recent reports have demonstrated that Grx3
`and Grx4 both localise to the nucleus (Lopreiato et al., 2004;
`Molina et al., 2004).
`In this study we describe a function for Grx3 and Grx4 in
`the cellular iron homeostasis through the regulation of the
`nuclear localisation of Aft1. At the time of submission of this
`manuscript, one study was accepted in press (Ojeda et al.,
`2006), which contained a number of similarities with respect
`to the present one. Both studies demonstrate the interaction
`between Aft1 and the monothiol glutaredoxins Grx3 and Grx4,
`and also that in the absence of both Grx3 and Grx4, the genes
`regulated by Aft1 are constitutively induced. Here we analyse
`the consequences of this regulation in the transcriptional
`response mediated by Aft1 and hypothesise a possible
`mechanism by which Grx3 and Grx4 might regulate Aft1
`translocation from the nucleus to the cytoplasm. In addition,
`we also demonstrate a physical nuclear interaction between
`Grx3, Grx4 and Aft1, which could reflect the functional
`specific regulation of Aft1 by both monothiol glutaredoxins.
`The simultaneous absence of both Grx3 and Grx4 proteins had
`a pronounced effect on cell-cycle progression, the rate of cell
`growth and sensitivity to oxidising agents. Hence, Grx3 and
`Grx4 might regulate the oxidative status of the cell by
`regulating iron homeostasis in iron-rich conditions.
`
`Functional characterisation of Grx3 and Grx4
`
`4555
`
`Results
`Grx3 and Grx4 are required for the cellular response to
`oxidative stress
`Since both Grx3 and Grx4 are monothiol glutaredoxins we
`wondered whether they each could play a role in reducing
`oxidised proteins and therefore in the oxidative stress response.
`In order to answer this question we assayed sensitivity to
`various oxidant agents in grx3, grx4, grx3grx4 and wild-type
`strains. We took aliquots from each of the cell cultures growing
`exponentially and spotted them on plates containing different
`concentrations of hydrogen peroxide, t-butylhydroperoxide
`and diamide. We obtained very encouraging results, although
`neither of the single mutants was substantially sensitive to the
`oxidising agents hydrogen peroxide and t-butylhydroperoxide,
`however, the double mutant turned out to be very sensitive
`to both agents compared with wild-type cells (Fig. 1).
`Interestingly, none of the mutants tested was sensitive to
`diamide. From this result we deduced that both Grx3 and Grx4
`are required for cells to respond to certain types of oxidative
`stress and that both glutaredoxins perform additive functions
`in protecting against oxidation.
`
`Both Grx3 and Grx4 interact in vivo and in vitro with Aft1
`in the nucleus
`In an attempt to further characterise the function of both Grx3
`and Grx4 glutaredoxins, we searched the SGD database and
`found a possible interaction between Grx3 and Aft1. This
`interaction turned out to be quite interesting for several
`reasons: (1) Aft1 is a transcription factor involved in the high
`affinity system for iron capture, and misregulation of iron
`inside cells is an important cause of oxidative stress (Gakh et
`al., 2006); (2) Glutaredoxins are molecules that detoxify
`oxidised residues; (3) it has recently been reported that Grx3
`and Grx4 localises in the nucleus and Aft1 operates in the
`nucleus by inducing the transcription of a subset of genes
`required for iron uptake. As a result, Aft1 proved a suitable
`candidate as a substrate for Grx3 and/or Grx4.
`We first constructed a number of plasmids to perform two-
`hybrid analysis between Grx3 and Aft1, Grx3 and Grx4, and
`Grx4 and Aft1. We obtained a clear result: the existence of
`strong in vivo interactions in the nucleus between Grx3 and
`Aft1, Grx3 and Grx4 and Aft1 and Grx4 (Fig. 2A). We
`wondered whether Grx3 and Grx4 were precluding the
`interaction of the other glutaredoxin with the transcriptional
`factor Aft1. In an attempt to gain a clearer picture of this
`interaction we therefore performed two-hybrid assays between
`Grx4 and Aft1 in grx3 background, between Grx3 and Aft1 in
`grx4 mutant cells and between Grx3 and Grx4 in aft1
`background (Fig. 2C). We subsequently observed that: (1) in
`the absence of Grx3, Grx4 still interacted with Aft, (2) in the
`
`Fig. 1. Grx3 and Grx4 are required for survival
`upon treatment with hydrogen peroxide and t-
`butylhydroperoxide. Exponentially growing cells
`from wild-type, grx3, grx4 and grx3grx4 strains
`were harvested, serially diluted and spotted onto
`control SD plates or on SD plates containing 1
`mM H2O2, 1 mM t-butylhidroperoxide or 0.75
`mM diamide. Plates were incubated at 30°C for 3
`days.
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`

`4556
`
`Journal of Cell Science 119 (21)
`
`Fig. 2. In vivo and in vitro interaction of Grx3 and Aft1, Grx4 and Aft1 and Grx3 and Grx4. (A) Two-hybrid analyses for the following
`interactions: Grx3 with Aft1, Grx4 with Aft1 and Grx3 with Grx4. Values for interaction between SNF4 and SNF1 were used as a strong
`positive control for nuclear interaction (+). Values obtained from the nuclear interaction between YAK1 and GRX5 were used as a positive
`control for weak nuclear interaction (–). e.v, empty vector. (B) Pull-down assays between Grx3 and Aft1, Grx4 and Aft1, Grx3 and Grx4. To
`detect these interactions, total protein extracts were obtained and subsequently bound to GST beads. In this first step we isolate either Grx3 or
`Grx4. To detect the second protein component of the complex, we tagged either Aft1 or Grx4 with the HA epitope and detected its presence by
`western blot with anti-HA antibody. As a loading control, we used anti-GST or anti-HA antibodies in aliquots taken from the same protein
`extracts. (C) Two-hybrid assay between: Grx3 and Aft1 in the grx4 mutant MML406; Grx4 and Aft1 in the grx3 strain MML405; Grx3 and
`Grx4 in the aft1 mutant CML126. +, positive control; –, negative control; e.v, empty vector. We performed a control for each of the
`backgrounds assayed, but for simplicity and because the three values were almost identical, the average values are shown. (D) Pull-down assays
`between Aft1 and Grx3 in the grx4 mutant; between Aft1 and Grx4 in the grx3 mutant; and Grx3 and Grx4 in the aft1 background.
`
`absence of Grx4, Grx3 interacted with Aft1; and (3) Grx3 and
`Grx4 also interacted in the absence of Aft1 (Fig. 2C). These
`results suggested that Grx3 and Grx4 both regulate Aft1 and
`also that Grx3, Grx4 and Aft1 form a complex and each of the
`three proteins interact with each other independently.
`These interactions were confirmed by pull-down assays in
`all the mutants detailed above (Fig. 2B,D). We used ferrocene
`to sequester iron because in iron-limiting conditions Aft1
`translocates to the nucleus. The addition of ferrocene produced
`a higher degree of isolation of the Aft1-Grx3 and Aft1-Grx4
`complexes, but the complex formed between Grx3 and Grx4
`did not vary. This indicated that Grx3 and Grx4 interact with
`Aft1 in the nucleus and that the greater the presence of Aft1 in
`
`the nucleus the greater the interaction with Grx3 and Grx4.
`This led us to conclude that the interaction between Grx3 and
`Grx4 with Aft1 was only limited by Aft1 cellular localisation.
`Another relevant finding was that the physical binding between
`Grx3 and Grx4 in the nucleus was not dependent on either the
`availability of iron or the presence of Aft1.
`
`Grx3 and Grx4 negatively regulate the transcriptional
`function of Aft1 in the nucleus
`In view of the previously mentioned results we decided to
`investigate the functional significance of the interaction
`between Grx3, Grx4 and Aft1. Since Aft1 regulates the
`transcription of the high-affinity iron uptake genes, we decided
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`

`Functional characterisation of Grx3 and Grx4
`
`4557
`
`to select two of the genes whose transcriptional control is
`tightly regulated by Aft1: FIT3 and FET3. FIT3 encodes a
`mannoprotein involved in the retention of siderophore iron in
`the cell wall whose transcription is regulated by Aft1 (Philpott
`et al., 2002). FET3 is a ferro-O2-oxidoreductase required for
`high-affinity iron uptake and is located in the plasma
`membrane (De Silva et al., 1995; Rutherford et al., 2003).
`Under conditions in which there is an excess of iron in the
`culture medium, neither of the two genes is transcriptionally
`induced but when iron concentration is a limiting factor in the
`external medium, Aft1 induces the expression of these genes.
`We used ferrocene, an iron-chelating agent, in order to mimic
`an environmental situation in which there was an iron
`deficiency. We then performed northern blot analysis in
`different backgrounds: grx3, grx4, grx3grx4 and wild-type
`cells and used FIT3 and FET3 as probes. In Fig. 3 we observed
`how the addition of ferrocene to the culture medium gradually
`induced a very pronounced expression of these genes in wild-
`type, grx3 and grx4 backgrounds with respect to the basal level.
`In both single mutants: grx3 and grx4, the constitutive mRNA
`levels of both FIT3 and FET3 genes were higher than those
`determined in wild-type cells; this indicated a negative
`regulation of each Grx3 and Grx4 on the Aft1 transcriptional
`function. However, the most revealing result was that obtained
`with the double mutant grx3grx4: both FIT3 and FET3 genes
`were constitutively induced with similar levels of expression
`in untreated cells and in cells treated with ferrocene (Fig. 3).
`These observations were in line with the previously mentioned
`results in which we observed that the glutaredoxins interacted
`and regulated Aft1 independently of each other, but that the
`two acted together in the regulation of Aft1 function in the
`nucleus. To ascertain whether the transcriptional upregulation
`detected in the grx3grx4 double mutant was specifically Aft1
`dependent, we constructed the grx3grx4aft1 triple mutant and
`observed that in this background neither the FET3 or FIT3
`transcriptional level was detectable at time 0 (exponentially
`growing cultures not treated with ferrocene): this was similar
`to that observed in the case of the aft1 null mutant (Fig. 3).
`This led us to conclude that the very high constitutive
`expression levels of FIT3 and FET3 observed in the grx3grx4
`double mutant were due to Aft1 gene regulation and that
`Grx3 and Grx4 consequently negatively regulate the Aft1
`transcriptional function regardless of iron availability.
`
`The existence of a low-affinity system for iron uptake,
`regulated mainly by Aft2 has already been widely documented.
`In the absence of Aft1, Aft2 induces the expression of the iron
`regulon of genes under conditions of iron limitation. Moreover,
`the expression levels of FET3 and FIT3 remarkably increased
`upon ferrocene treatment in both aft1 and grx3grx4aft1 strains,
`in a similar fashion as in wild-type cells (Fig. 3). These results
`are in accordance with a model in which Aft2 drives the
`expression of the iron regulon when iron constitutes a limiting
`factor in the culture medium and when Aft1 is not functional.
`It also suggests that Grx3 and Grx4 do not regulate the Aft2
`function, but further studies are required to validate this model.
`
`The absence of Grx3 and Grx4 do not influence the
`mRNA levels of Aft1
`One possible interpretation of the results presented above is
`that Grx3 and Grx4 could have regulated the transcriptional
`levels of AFT1: this would lead to the increase in Aft1
`protein levels and consequently
`to
`the
`transcriptional
`induction of the genes regulated by Aft1. We decided
`to perform northern blot analysis using samples from
`the wild-type, grx3grx4,
`aft1, grx3grx4tetO7Grx3HA,
`grx3grx4tetO7Grx4HA,
`grx3grx4tetO7Grx3HA+pGSTGrx4
`and tetO7Grx4HA+pGSTGrx3 strains and using the probe
`AFT1. We observed (Fig. 4) that the mRNA levels in AFT1
`were independent of the presence or absence of Grx3 and Grx4.
`We therefore conclude that neither Grx3 nor Grx4 regulated
`the expression of AFT1.
`
`The absence of Grx3 and Grx4 causes a growth and
`cell-cycle defect as a consequence of Aft1 upregulation
`In the course of this study we observed that the double mutant
`grx3grx4 presented a marked growth defect. This consisted of
`a much longer generation time (135 minutes) than that
`observed in wild-type cells (90 minutes in SD medium growing
`at 30°C) and also a curious accumulation of G1 cells in
`exponentially growing cells (Fig. 5). Since Aft1 overexpression
`also induces a delay in the G1 phase of the cell cycle (see Fig.
`5) and we have demonstrated that both proteins negatively
`regulate Aft1 function, we wondered whether the cell-cycle
`phenotype observed in the grx3grx4 double mutant was
`another consequence of Aft1 misregulation. When we
`measured the growth rate in the triple mutant grx3grx4aft1 we
`
`Fig. 3. Grx3 and Grx4 negatively regulate the expression of FIT3 and
`FET3 in a manner dependent on Aft1 activity. Cells from the
`following strains: wild type, grx3, grx4, grx3grx4, aft1 and
`grx3grx4aft1, were exponentially grown in SD medium plus amino
`acids, at 30°C, then treated with 2 mM ferrocene. Samples were
`taken after 4 and 8 hours as indicated. Samples were taken for
`mRNA isolation and northern blot using FIT3 and FET3 as probes,
`U2 was detected as a loading control.
`
`Fig. 4. AFT1 RNA levels are not regulated by Grx3 nor Grx4.
`Northern blot analysis of AFT1 expression levels in the wild type,
`grx3grx4 and aft1 mutants and under conditions of overexpression of
`Grx3, Grx4, or both. Overexpression of Grx3 and Grx4 was driven
`by the tetO7 or by the ADH1 promoter (pGSTGrx3 or pGSTGrx4) as
`stated. To regulate gene expression under the tetO7 promoter, we
`added (+) or not (–) 20 ␮g/ml doxycycline to the culture media.
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`

`4558
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`Journal of Cell Science 119 (21)
`
`Fig. 6. Total iron accumulates in the cell in the absence of Grx3 and
`Grx4. Total iron concentration was spectrophotometrically
`determined as described in the Materials and Methods in
`exponentially growing cultures of wild-type, grx3, grx4, grx3grx4
`and grx3grx4aft1 strains. Numerical values represented in the
`histograms of the figure are averages from three experiments. In all
`cases standard errors were lower than 10, therefore no error bars are
`distinguished.
`
`accumulation observed in the absence of both Grx3 and Grx4
`is a consequence of Aft1 activity. Therefore, we conclude that
`one cellular consequence of Aft1 upregulation in the grx3grx4
`mutant is the accumulation of intracellular iron.
`It has already been demonstrated that the accumulation of
`iron inside cells provokes oxidative stress through the Fenton
`reaction, which releases hydroxyl radical to the cytoplasm.
`Bearing this in mind, we decided to investigate whether the
`greatest sensitivity to oxidant agents observed in the grx3grx4
`double mutant was the consequence of the upregulation of the
`genes governing iron uptake driven by Aft1. To determine this,
`we first tested sensitivity to various oxidant agents in the
`following strains: wild type, grx3grx4, aft1 and grx3grx4aft1,
`and observed that the very high sensitivity of the grx3grx4
`double mutant to hydrogen peroxide and t-butyl hydroperoxide
`was only partly recovered to wild type levels upon aft1 deletion
`(Fig. 7A). In order to further characterise whether the
`abrogation of sensitivity in the grx3grx4aft1 triple mutant was
`due to iron accumulation, we decided to treat cells with
`hydrogen peroxide in the presence of ferrocene. The presence
`of the iron chelator abrogated most of the sensitivity to the
`oxidising agent observed in the grx3grx4 double mutant (Fig.
`7B). These data indicated that the phenotype of oxidative stress
`sensitivity characteristic of grx3grx4 is at least in part, due to
`the accumulation of high levels of iron inside cells as a
`consequence of Aft1 upregulation. However, other specific
`functions not mediated by Aft1 must also be regulated by Grx3
`and Grx4 within the oxidative stress response.
`
`Grx3 and Grx4 regulate Aft1 compartmentalisation
`Some authors have reported that the Aft1 function was
`determined by its nuclear localisation because the iron regulon
`of genes dependent on Aft1 was constitutively induced in a
`mutant of the protein permanently located in the nucleus
`(Yamaguchi-Iwai et al., 2002). We therefore wondered whether
`the upregulation of FET3 and FIT3 detected in the grx3grx4
`strain was also the consequence of Aft1 translocation to the
`nucleus. To check this, we constructed a GFP-Aft1 fusion
`protein in a multicopy plasmid to monitor, in vivo, the cellular
`localisation of the protein by fluorescence microscopy. In wild-
`
`Fig. 5. The simultaneous absence of Grx3 and Grx4 induces
`accumulation of cells in G1. FACS profiles of different strains
`growing exponentially in SD medium plus amino acids. The strains
`are: wild type, grx3, grx4, grx3grx4, wild type overexpressing Aft1,
`grx3grx4aft1, wild type overexpressing Grx3 and wild type
`overexpressing Grx4. All the proteins tested were overexpressed
`under the tetO7 promoter.
`
`observed that it was similar to that in wild-type cells (90
`minutes of generation time in SD medium growing at 30°C).
`We also observed that the FACS profile of grx3grx4aft1 was
`similar to that of wild-type cells in which the 2N DNA content
`of the population had been enriched in comparison with the 1N
`content. This was characteristic of the wild-type background
`used in this study. As expected, the absence of only Grx3 or
`Grx4 proteins did not affect cell-cycle progression nor did the
`overexpression of each of both proteins (Fig. 5). These results
`suggest that both the growth defects and cell-cycle defects
`observed in the grx3grx4 double mutant were mediated by Aft1
`activity.
`
`Grx3 and Grx4 are involved in the regulation of iron
`homeostasis through Aft1
`We then decided to investigate whether the Aft1 upregulation
`observed in this study in the absence of Grx3 and Grx4 would
`affect the concentration of intracellular iron. We measured total
`iron concentration in wild-type, grx3, grx4, grx3grx4 and
`aft1grx3grx4 strains. As shown in Fig. 6, in both grx3 and grx4
`single mutants, total iron concentration significantly increased
`with respect to that recorded in wild-type cells, but it was in
`the double mutant that the highest intracellular iron levels were
`detected. Interestingly, in the triple aft1grx3grx4 mutant the
`intracellular iron concentration was equivalent to that detected
`in wild-type cells, which clearly indicates that the iron
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`

`Functional characterisation of Grx3 and Grx4
`
`4559
`
`Fig. 7. The sensitivity to oxidising agents observed in the grx3grx4 double mutant
`is partly mediated by Aft1. (A) Cells from exponentially growing cultures of wild-
`type, grx3grx4, aft1 and grx3grx4aft1 strains were serial diluted and spotted onto
`SD plates either containing or not oxidising agents as in Fig. 1. (B) Wild-type and
`grx3grx4 strains growing exponentially were treated with 2 mM ferrocene for 6
`hours, after which cells were washed and placed in fresh medium to be
`subsequently serial diluted and plated on SD plates containing 1 mM hydrogen
`peroxide or 1 mM hydrogen peroxide plus 150 ␮M ferrocene (F).
`
`type cells, Aft1 was dispersed throughout the cell (Fig. 8A,
`panel 1) whereas
`in
`the grx3grx4 mutant, Aft1 was
`permanently located in the nucleus, as demonstrated by DAPI
`staining (Fig. 8B, panel 2). This finding is in accordance with
`the results shown in this study and explains the constitutive
`upregulation of FIT3 and FET3 detected in the grx3grx4
`double mutant.
`The next question to investigate was whether Grx3 and Grx4
`regulated
`the nucleus-cytoplasm Aft1
`localisation. We
`designed an in vivo experiment that allowed us to determine
`how the two monothiol proteins regulated Aft1 nuclear
`localisation. To do this, we constructed two double conditional
`mutants. In the first, Grx3 was regulated by the tetO7 promoter,
`so that the addition of doxycycline would inhibit Grx3
`expression and the release of doxycycline would induce Grx3
`overexpression. The second double mutant was similar to the
`first, but with the difference that the regulated protein under
`the tetO7 promoter was Grx4. When we checked the
`transcriptional regulation of FET3 we observed that there was
`a very high expression of this gene in samples taken from both
`mutants when they were exponentially grown in the presence
`of doxycycline. This was similar to what we observed in
`samples taken from the double mutant grx3grx4 (Fig. 8B, lines
`2, 5 and 7). Furthermore, under these conditions, Aft1 was
`located in the nucleus in all three cell populations (not shown).
`Upon removal of doxycycline, Grx3 protein was overexpressed
`and in this context Aft1 was partly translocated to the
`cytoplasm (Fig. 8A, panel 3) whereas expression of FET3
`diminished (Fig. 8B, line 6). We repeated this assay with the
`second strain, in which the regulatable protein was Grx4, and
`obtained qualitatively identical results (Fig. 8A, panel 4 and
`Fig. 8B, line 8). This result indicated that expression of either
`Grx3 or Grx4 was sufficient to allow the Aft1 translocation
`from the nucleus to the cytoplasm. We reasoned that if both
`Grx3 and Grx4 inhibited the importation of Aft1 to the nucleus,
`the treatment with ferrocene (which induces Aft1 nuclear
`translocation) in conditions of Grx3 and Grx4 overexpression,
`should impair Aft1 translocation to the nucleus. To test this
`
`hypothesis we simultaneously overexpressed Grx4 and Grx3 in
`a grx3grx4 strain. Upon the addition of ferrocene to the culture
`medium, Aft1 clearly accumulated in the nucleus after 6 hours
`of treatment (Fig. 9A) correlated to the induction of FIT3 (Fig.
`9B, line 9) and FET3 (not shown) transcripts, in a similar
`manner to that observed for wild-type cells (Fig. 9B, line 3).
`In a grx3grx4 double mutant, the addition of ferrocene did not
`produce variations in either patterns of gene expression or Aft1
`localisation (as shown in Fig. 9A,B). Moreover, Grx3 and Grx4
`protein levels were not affected by ferrocene, as indicated
`in Fig. 9C, which effectively rules out any possible
`posttranslational regulation of the two proteins resulting from
`iron depletion.
`These results suggest the possibility that neither Grx3 nor
`Grx4 regulates Aft1 nuclear import. Our hypothesis, however,
`is that both proteins negatively regulate Aft1 activity by
`promoting its nuclear exportation from the nucleus to the
`cytoplasm. This is further supported by the observation of a
`more-pronounced inhibition of FET3 transcription when we
`simultaneously overexpressed both Grx3 and Grx4 in the
`grx3grx4 background (Fig. 8B, line 9).
`
`Aft1 interacts with both the glutaredoxin and thioredoxin
`domains
`Since both Grx3 and Grx4 present two functional domains,
`namely Grx, a glutaredoxin-like sequence and Trx, the
`thioredoxin domain, we decided to investigate the role of these
`domains in the regulation of Aft1 function. We first performed
`two-hybrid assays between each of the Grx and Trx domains
`of Grx3 and Grx4 and Aft1. We found that both Grx and Trx
`domains from Grx3 or Grx4 physically interacted with Aft1 in
`the nucleus (Fig. 10A). However, we observed that the full-
`length Grx3 protein interacted more strongly with Aft1 than
`each of its single Grx or Trx domains, and that Grx4 increased
`the binding efficiency between Grx3 and Aft1 (see Fig. 2A,C
`and Fig. 10A). We conclude not only that Grx3 and Grx4 can
`independently interact with Aft1 but also that Grx and Trx
`domains of Grx3 and Grx4 are able to independently bind Aft1
`
`Journal of Cell Science
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`

`4560
`
`Journal of Cell Science 119 (21)
`
`in the nucleus. We next wondered which of those
`domains was responsible for the regulation of the Aft1
`transcriptional function and according to our model,
`for the translocation of Aft1 from the nucleus to the
`cytoplasm. To answer this question we overexpressed
`each of the pGrx and pTrx domains of Grx3 and Grx4,
`respectively, in a grx3grx4 double mutant strain and
`analysed the levels of FIT3 and FET3 mRNAs. We
`observed that the overexpression of each of the Grx
`domains from Grx3 and Grx4, significantly diminished
`the levels of expression of FIT3 and FET3 compared
`with the grx3grx4 double mutant (Fig. 10B). Following
`this observation and to investigate which domain was
`involved in Aft1 translocation from the nucleus to the
`cytoplasm, we decided to express each of the four
`domains in the nucleus. For this, we used the following
`plasmids: pTP17, pTP19, pTP20 and pTP21, which
`contain a nuclear localisation domain and a strong
`promoter such as ADH1. The results we obtained
`clearly indicated that high expression of the Grx
`domains of both Grx3 and Grx4 in the nucleus induced
`Aft1 translocation from the nucleus to the cytoplasm
`in a grx3grx4 double mutant bearing an Aft1GFP
`fusion protein (Fig. 10C). However, the overexpression
`of each of the Trx domains did not cause any variation
`in the levels of FIT3 and FET3 expression compared
`with that determined in the empty grx3grx4 double
`mutant (Fig. 10B) and under these conditions, Aft1
`remained localised in the nucleus. We conclude from
`all these results that both Grx domains from Grx3 and
`Grx4 are responsible for Aft1 transcriptional function
`by regulating its translocation from the nucleus to the
`cytoplasm.
`
`Discussion
`In this study we demonstrate that Grx3 and Grx4
`regulate the subcellular localisation of Aft1 under
`conditions in which iron is not a limiting factor. In the
`absence of both proteins Aft1 localisation is nuclear in
`iron-rich medium. As discussed below, this function is
`directly related to the oxidative stress response. Grx3 and Grx4
`negatively regulate Aft1 activity. The two proteins have
`overlapping additive functions, although the absence of either
`does not significantly affect cell viability under normal
`conditions or in response to oxidative treatment. Yamaguchi-
`Iwai et al. (Yamaguchi-Iwai et al., 2002) reported that Aft1
`responds to iron concentration by regulating changes in the
`subcellular localisation of Aft1. Here, we demonstrate that both
`Grx3 and Grx4 regulate the cellular localisation of Aft1 and
`that both proteins are required
`to permit cytoplasmic
`localisation of Aft1 under iron-rich conditions.
`subcellular
`If
`iron were
`to
`signal Aft1
`for
`compartmentalisation