Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1⌬aft2⌬ deletion was generated. Growth assays established that the single aft2⌬ strain exhibited no iron-dependent phenotype. However, the double-mutant aft1⌬aft2⌬ strain was more sensitive to lowiron growth conditions than the single-mutant aft1⌬ strain. A mutant allele of AFT2 (AFT2-1 up ), or overexpression of the wildtype AFT2 gene, led to partial complementation of the respiratorydeficient phenotype of the aft1⌬ strain. The AFT2-1 up allele also increased the uptake of 59 Fe in an aft1⌬ strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1 up -dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1 up allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles. metalloregulation ͉ iron homeostasis

The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and the YRO2 homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c and YRO2 appears to be direct. Induction of HAA1 using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.

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