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First published online October 30, 2006
doi: 10.1242/10.1242/jcs.03229

Journal of Cell Science 119, 4554-4564 (2006)
Published by The Company of Biologists 2006
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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,*

1 Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, Lleida 25198, Spain
2 Servei d'analisis cliniques, Hospital Arnau de Vilanova, Lleida 25192, Spain


Figure 1
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  		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.

 

Figure 2
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  		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.

 

Figure 3
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  		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.

 

Figure 4
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  		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 µg/ml doxycycline to the culture media.

 

Figure 5
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  		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.

 

Figure 6
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  		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.

 

Figure 7
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  		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 µM ferrocene (F).

 

Figure 8
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  		Fig. 8. Aft1 nuclear localisation is regulated by both Grx3 and Grx4. (A) GFP-Aft1 fusion protein was visualised by fluorescence microscopy and recorded in different backgrounds. 1. wild type. 2. grx3grx4 double mutant. 3. grx3grx4 double mutant overexpressing GRX3 under the tetO7 promoter. 4. grx3grx4 double mutant overexpressing GRX4 under the tetO7 promoter. (B) Northern blot of FET3 from mRNA samples collected from the same experiment described in A. To regulate gene expression under the tetO7 promoter, we added (+) or not (–) 20 µg/ml doxycycline to the culture media.

 

Figure 9
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  		Fig. 9. Aft1 nuclear translocation mediated by iron deficit is independent of the Aft1 nuclear localisation mediated by both Grx3 and Grx4. (A) GFPAft1 localisation in the different strains: wild type, grx3grx4 and grx3grx4tetO7GRX4+pADH1Grx3, upon the addition of 2 mM ferrocene to exponentially growing cells at 30°C in SD medium. (B) Northern blot of FIT3 in mRNA samples extracted from aliquots collected from wild type, grx3grx4 and grx3grx4tetO7GRX4+pGSTGrx3 upon the addition of 2 mM ferrocene to exponentially growing cells at 30°C in SD medium. (C) Western blot of total protein extracts prepared from the previous experiment. We used anti-HA antibody to detect Grx4 protein and anti-GST antibody to detect Grx3 protein.

 

Figure 10
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  		Fig. 10. Aft1 interacts with both Grx and Trx domains of Grx3 and Grx4, but the Grx domains of Grx3 or Grx4 are responsible for Aft1 transcriptional function and cellular compartmentalisation. (A) Two-hybrid analysis for the following interactions: Aft1 with the Grx domain of Grx3, Aft1 with the Trx domain of Grx3, Aft1 with the Grx domain of Grx4 and Aft1 with the Trx domain of Grx4, determined as in Fig. 2A and Fig. 2C. (B) Northern blot analysis of FET3 and FIT3 expression in samples taken from cultures of the following strains: wild type, grx3grx4, grx3grx4+pMM491(pDomGrx of Grx3) (Dom, domain), grx3grx4+pMM486 (pDomTrx of Grx3), grx3grx4+ pMM506 (pDomGrx of Grx4) and grxgrx4+pMM502 (pDomTrx of Grx4). Cells were exponentially grown in SD at 30°C. (C) GFPAft1 localisation in the grx3grx4 strain co-transformed with pTP20 (pDomGrx, identical results were observed with pTP19) or with pTP17 (pDomTrx, identical results were observed with pTP21) in cultures growing exponentially at 30°C in SD medium.

 

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© The Company of Biologists Ltd 2006