The budding yeast Saccharomyces cerevisiae responds to depletion of iron in the environment by activating Aft1p, the major iron-dependent transcription factor, and by transcribing systems involved in the uptake of iron. Here, we have studied the transcriptional response to iron deprivation and have identified new Aft1p target genes. We find that other metabolic pathways are regulated by iron: biotin uptake and biosynthesis, nitrogen assimilation, and purine biosynthesis. Two enzymes active in these pathways, biotin synthase and glutamate synthase, require an iron-sulfur cluster for activity. Iron deprivation activates transcription of the biotin importer and simultaneously represses transcription of the entire biotin biosynthetic pathway. Multiple genes involved in nitrogen assimilation and amino acid metabolism are induced by iron deprivation, whereas glutamate synthase, a key enzyme in nitrogen assimilation, is repressed. A CGG palindrome within the promoter of glutamate synthase confers iron-regulated expression, suggesting control by a transcription factor of the binuclear zinc cluster family. We provide evidence that yeast subjected to iron deprivation undergo a transcriptional remodeling, resulting in a shift from iron-dependent to parallel, but iron-independent, metabolic pathways.

In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1. cDNA microarrays were used to identify new iron and AFT1-regulated genes. Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters. These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease. Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxaminebound iron and growth on ferrioxamine as an iron source. The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments. Epitopetagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12. In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases. These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.

Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast. FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1 up , a constitutively active allele of AFT1, was expressed. Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60 -230-fold in response to iron deprivation in an Aft1p-dependent manner. Fit1p was localized exclusively to the cell wall by indirect immunofluorescence. Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin. FITdeletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake. Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources. Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains. Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space.