BY4742 wild-type cells were grown to exponential phase in SC medium and challenged with 2 mM of FeSO₄. RNAs of Fe-treated and untreated cells were isolated and analyzed with DNA microarrays as detailed in . Genes up or downregulated were sorted into functional categories according to MIPS database. Downregulated genes comprised in the Ribosome biogenesis category (n = 48) were not included. A list of all the genes included in each functional category is shown in .

(A) Exponentially growing cells from wild-type (WT), yap5 mutant (yap5) and ccc1 mutant (ccc1) strains were harvested, serially diluted and spotted onto control SC plates or SC plates containing the designated FeSO₄ concentrations. (B) WT and yap5 strains were upshifted to high-Fe medium, by supplementation of SC medium with 2 mM of FeSO4, and harvested at the indicated time-points. The expression of CCC1 was assessed by qRT-PCR as described in . Values are the mean of biological triplicates ± s.d. (C) Schematic representation of the truncated promoter version of CCC1 gene (spCCC1). (D) ccc1 strain was transformed with a plasmid harboring spCCC1 gene (ccc1<spCCC1>), fpCCC1 gene (ccc1<fpCCC1>) or the plasmid alone (ccc1<vector>). Wild-type strain was transformed with the empty plasmid (WT<vector>). Exponentially growing cells were harvested, serially diluted and spotted onto control SD plates or SD plates containing 9 mM of FeSO₄.

BY4742 wild-type and yap5 mutant strains were grown to exponential phase in SC medium and challenged with 2 mM of FeSO₄ for 60 min. RNA of Fe-treated cells was isolated and analyzed with DNA microarrays as detailed in . Genes up- or downregulated were sorted into functional categories according to MIPS database. Dubious open reading frame unlikely to encode a protein (100) were not considered. A list of all the genes included in each functional category is shown in .

(A) Exponentially growing cells from wild-type (WT) and yap5 mutant (yap5) strains were upshifted to high-Fe medium, by supplementation of SC medium with the indicated FeSO₄ concentrations, and harvested at the indicated time-points. The expression of GRX4 was assessed by qRT-PCR as described in . Values are the mean of biological triplicates ± s.d. (B) WT and yap5 strains were transformed with a plasmid carrying GRX4-HA, treated with 5 and 15 mM of FeSO₄ for 20 min and analyzed by Western blot with an anti-HA antibody. Pgk1 protein levels were used as loading control. (C) Representation of the GRX4 constructs without (a) or with (b,c,d) mutations in the YREs. (D) Mutant grx4 cells expressing the constructs depicted in (C) were grown under Fe-adequate or Fe overload (5 mM FeSO₄) conditions, and the expression of GRX4 was assessed by qRT-PCR as described in . Values are the mean of biological triplicates ± s.d. (E) yap5 cells were transformed with HA-tagged Yap5 and grown in SD medium not supplemented (−Fe) or supplemented (+Fe) for 15 min with 2 mM of FeSO₄, before being processed for ChIP. ChIP analysis was performed using probes specific for GRX4. (F) ChIP analyses combined with qRT-PCR, were used to determine the fold enrichment of GRX4, SCR1 and ARN2. The sequence enrichment in the ChIP (i.e. IP/IN) was normalized using the ACT gene as a reference.

(A) Wild-type (WT) yap5 (yap5) grx4 (grx4) mutant strains were transformed with a high-copy AFT1-GFP plasmid. Exponentially growing cells in SD medium were untreated (SD) or treated with 2 mM of FeSO₄ for 30 min. GFP-Aft1 fusion protein was visualized by fluorescence microscopy. (B) Cells with nuclear fluorescence were counted as detailed in . Approximately 100 cells were analyzed per condition. Values are the mean of two biological duplicates (200 cells) ± s.d. (C) Exponentially growing cells from wild-type (WT) and yap5 mutant (yap5) strains were upshifted to high-Fe medium, by supplementation of SC medium with 2 mM of Fe SO4, and harvested at the indicated time-points. RNAs were isolated and the expression of FET3 was assessed by qRT-PCR as described in . Values are the mean of biological triplicates ± s.d. (D) WT and yap5 strains were transformed with the plasmid pCM64-CTH2-FeRE-CYC1-LacZ (CTH2:LacZ). Cells were grown exponentially in SD medium or in Sd medium supplemented with 2 mM of FeSO₄, for 30 min (+FeSO₄) and ß-galactosidase activity was assayed as detailed in . Values are the mean of biological decaplicates ± s.d. * P<0.01 **P<0.05.

Under Fe-adequate growth conditions Yap5 is active and the main regulator of CCC1 gene expression. The continuous Fe storage will end up with cytoslic Fe depletion and the consequent activation of Aft1 in order to re-establish Fe homeostasis. Under Fe overload, another factor is regulating CCC1 gene expression, bypassing Yap5 regulation. In addition Yap5 transactivation potential is higher and therefore it is able to induce GRX4 gene that together with Grx3 will inhibit Aft1 activity by promoting its nuclear export to the cytoplasm.