Abstract
In response to excess iron, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in iron uptake through the Fep1 transcription factor. Fep1 mediates this control by interacting with the consensus sequence 5'-(A/T)GATAA-3', found in iron-dependent promoters. In this report, we show that Fep1 localizes to the nucleus under both iron-replete and iron-starved conditions. The Fep1 DNA binding domain (amino acids 1-241) contains two GATA-type zinc finger motifs. Although we determine that the Fep1 C-terminal zinc finger (ZF2) is essential for DNA binding, we show that the N-terminal zinc finger (ZF1) enhances DNA binding affinity approximately 5-fold. Between the two zinc finger motifs of Fep1 resides an invariant amino acid sequence, denoted the Cys-rich region (amino acids 68-94), in which four highly conserved Cys residues are found. Cells harboring mutant alleles in which two or more of the conserved Cys residues were substituted by alanine exhibited elevated fio1(+) mRNA levels. We determine that the dissociation constant for the resulting complex between each of the Cys mutants and the sequence 5'-(A/T)GATAA-3' reflects a much lower affinity that correlates with failure to repress fio1(+) gene expression. Deletion analysis identified two heptad repeats (amino acids 522-536) within the C-terminal region of Fep1 that are necessary and sufficient to mediate Fep1 dimerization. Moreover, mutations that impair dimerization also negatively affect transcriptional repression. Together these findings reveal several novel features of Fep1, a non-canonical GATA factor required for iron homeostasis.
Highlights
The transition metal iron is both essential and, at high levels, toxic to cells [1,2,3,4]
Fep1 is a member of the GATA factor family that binds to DNA sequences containing 5Ј-(A/T)GATAA-3Ј [18, 19]
Regulators with similar sequences and functions to Fep1 have been found in other fungi, e.g. U. maydis, N. crassa, A. nidulans, and C. albicans, but not in S. cerevisiae [30]
Summary
The transition metal iron is both essential and, at high levels, toxic to cells [1,2,3,4]. Fep1⌬ cells harboring the Cys mutant proteins exhibited increased activity of the cell surface reductase Frp1, as a consequence of lack of transcriptional repression of frp1ϩ.2 To gain insight how the mutated Cys residues alter regulation of fio1ϩ by iron, experiments were carried out using purified Cys-mutant MBP-2Fep1241 fusion proteins (Fig. 4D).
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