Characterization of iron uptake repressor Fep1 in Schizosaccharomyces pombe

Abstract

Iron is an important cofactor for a wide variety of proteins involved in the major life processes, such as respiration and tricarboxylic acid cycle, DNA replication and repair, and nitrogen fixation. The essential redox-active property of iron that enables facile switch between ferric (Fe3+) and ferrous iron (Fe2+) also renders toxicity via generating reactive oxygen species. Therefore, most organisms are equipped with regulated mechanisms to maintain optimal intracellular levels of iron. In the fission yeast Schizosaccharomyces pombe, two repressors Php4 and Fep1 regulate iron-dependent expression of genes for iron usage/storage and acquisition, respectively. The iron-sensing depends on the CGFS-type monothiol glutaredoxin Grx4 that binds Fe-S cluster in a homodimer or in a heterodimer with a BolA-type protein Fra2. Under iron-rich condition, Grx4 binding to Php4 causes its cytosolic sequestration, resulting in the induction of iron usage/storage genes. Grx4 also binds Fep1 regardless of iron availability. Under iron-starved condition, Grx4 and Fra2 inhibit Fep1 repressor activity, resulting in derepression of iron acquisition genes. Fep1, a GATA-family transcription factor, binds to the promoter regions of genes for iron acquisition, such as reductive iron import (fio1+, frp1+), siderophore transport (str1+, str2+, str3+), and vacuolar transport (abc3+), to avoid iron overload under iron-rich conditions. The N-terminal DNA-binding domain of Fep1 contains four conserved cysteines located between the two zinc finger motifs. It has been demonstrated that the N-terminal 241 aa residues of Fep1 can bind to the target promoters in vivo under iron-replete condition. Through this N-terminal domain, Fep1 is known to interact with the monothiol glutaredoxin domain of Grx4 under iron-starved condition. Under iron-starved condition, Fep1 is released from binding to its target sites, inducing iron uptake genes. Fep1 orthologs are well conserved, especially in the DNA binding domain, across filamentous fungi, such as Ustilago maydis (Urbs1), Neurospora crassa (SRE), Histoplasma capsulatum (Sre1), Aspergillus spp. (SREA), Candida albicans (Sfu1), and Cryptococcus neoformans (Cir1). Even though the function and interaction partners of Fep1 have been elucidated extensively in S. pombe, the molecular basis by which Fep1 is inactivated under iron starvation remains unknown. In this study, to elucidate the mechanism behind iron-sensing by Fep1, I pursued biochemical and spectroscopic analyses of Fep1, in full length and truncated forms, as isolated or reconstituted proteins, with the wild type or substituted cysteine mutations. Evidences are presented that Fep1 binds iron, in the form of Fe-S cluster. Spectroscopic and biochemical analyses of as isolated and reconstituted Fep1 suggest that the dimeric Fep1 binds Fe-S clusters. Iron to acid-labile sulfide stoichiometry of purified Fep1-N238 was roughly 1 : 1 in both before and after chemical reconstitution. Furthermore, the reconstitution of the His-tagged Fep1-N238 requires not only iron donor but also sulfide donor, indicating that Fep1 binds [Fe-S] clusters. The mutation study revealed that the cluster-binding depended on the conserved cysteines located between the two zinc fingers in the DNA binding domain. EPR analyses revealed [Fe-S]-specific peaks indicative of mixed presence of [2Fe-2S], [3Fe-4S], or [4Fe-4S]. Overall, the finding that Fep1 is an Fe-S protein fits nicely with the model that the Fe-S-trafficking Grx4 senses intracellular iron environment and modulates the activity of Fep1.