The Role of the Yap5 Transcription Factor in Remodeling Gene Expression in Response to Fe Bioavailability

Abstract

The budding yeast Saccharomyces cerevisiae has developed several mechanisms to avoid either the drastic consequences of iron deprivation or the toxic effects of iron excess. In this work, we analysed the global gene expression changes occurring in yeast cells undergoing iron overload. Several genes directly or indirectly involved in iron homeostasis showed altered expression and the relevance of these changes are discussed. Microarray analyses were also performed to identify new targets of the iron responsive factor Yap5. Besides the iron vacuolar transporter CCC1, Yap5 also controls the expression of glutaredoxin GRX4, previously known to be involved in the regulation of Aft1 nuclear localization. Consistently, we show that in the absence of Yap5 Aft1 nuclear exclusion is slightly impaired. These studies provide further evidence that cells control iron homeostasis by using multiple pathways.

Repression of the Low Affinity Iron Transporter Gene FET4

“…The MUTp-ROX1 plasmid was generated using as template p-ROX1, and the primers depicted in Table 2 were used in a PCR-directed mutagenesis reaction to mutate the YRE 2 site located at Ϫ414 bp, as detailed in Ref. 31. A similar and sequential strategy was used to construct MUTp-FET4-HA using the primers listed in Table 2.…”

“…Yeast strains were grown in synthetic medium (SC) or medium lacking specific requirements (SD), as previously described (31). Phenotypic growth assays were carried out by spotting 5 l of cultures in early exponential phase (A 600 ϭ 0.4 -0.5) sequentially diluted (ϳ5 ϫ 10 3 to 50 cells) in medium containing the indicated concentrations of CdCl 2 and supplemented or not with FeSO 4 .…”

“…Relative ␤-galactosidase activity was monitored as described in Ref. 31. Enzymatic activity was assayed by following the degradation of the colorimetric substrate O-nitrophenyl-␤-D-galactopyranoside at A 420 and normalizing against A 600 .…”

“…Total proteins were extracted from cell cultures as described in Ref. 31. Proteins were resolved in a 10% SDS-PAGE and immunoblotted with horseradish peroxidase-bound anti-HA IgG (Roche).…”

“…ChIP Analysis-ChIP assays were carried out as previously described (31). Cells transformed with a c-Myc-tagged version of Yap1 (27) and treated with cadmium were harvested at A 600 0.6 and fixed with 1% formaldehyde.…”