Schizosaccharomyces pombe cells acquire iron under high affinity conditions through the action of a cell surface ferric reductase encoded by the frp1 ؉ gene and a two-component iron-transporting complex encoded by the fip1 ؉ and fio1 ؉ genes. When cells are grown in the presence of iron, transcription of all three genes is blocked. A conserved regulatory element, 5-(A/ T)GATAA-3, located upstream of the frp1 ؉ , fip1 ؉ , and fio1 ؉ genes, is necessary for iron repression. We have cloned a novel gene, termed fep1 ؉ , which encodes an iron-sensing transcription factor. Binding studies reveal that the putative DNA binding domain of Fep1 expressed as a fusion protein in Escherichia coli specifically interacts with the 5-(A/T)GATAA-3 sequence in an iron-dependent manner. In a fep1⌬ mutant strain, the fio1 ؉ gene is highly expressed and is unregulated by iron. Furthermore, the fep1⌬ mutation increases activity of the cell surface iron reductase and renders cells hypersensitive to the iron-dependent free radical generator phleomycin. Mutations in the transcriptional corepressors tup11 ؉ and tup12 ؉ are phenocopies to fep1 ؉ . Indeed, strains with both tup11⌬ and tup12⌬ deletions fail to sense iron. This suggests that in the presence of iron and Fep1, the Tup11 and Tup12 proteins may act as co-repressors for down-regulation of genes encoding components of the reductive iron transport machinery.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

We have identified genes encoding candidate proteins involved in iron storage (pcl1 ؉ ), the tricarboxylic acid cycle (sdh4 ؉ ), and iron-sulfur cluster assembly (isa1 ؉ ) that are negatively regulated in response to iron deprivation. Promoter deletion and site-directed mutagenesis permitted identification of a new cis-regulatory element in the promoter region of the pcl1 ؉ gene. This cis-acting regulatory sequence containing the pentanucleotide sequence CCAAT is responsible for transcriptional repression of pcl1 ؉ under low iron supply conditions. In Schizosaccharomyces pombe, the CCAAT-binding factor is a heteromeric DNA-binding complex that contains three subunits, designated Php2, Php3, and Php5. Inactivation of the php2 ؉ locus negatively affects the transcriptional competency of pcl1 ؉ . A fourth subunit, designated Php4, is not essential for the transcriptional activation of target genes under basal and iron-replete conditions. We demonstrate that, in response to iron-limiting conditions, Php4 is required for down-regulation of pcl1 ؉ , sdh4 ؉ , and isa1 ؉ mRNA levels. In vivo RNase protection studies reveal that the expression of php4 ؉ is negatively regulated by iron and that this regulated expression requires a functional fep1 ؉ gene. The results of these studies reveal that Fep1 represses php4؉ expression in response to iron. In contrast, when iron is scarce, Fep1 becomes inactive and php4؉ is expressed to act as a regulatory subunit of the CCAAT-binding factor that is required to block pcl1 ؉ , sdh4 ؉ , and isa1 ؉ gene transcription.

When iron repletes, Schizosaccharomyces pombe cells repress transcription of genes encoding components involved in the reductive iron transport system. Fep1 mediates this transcriptional control by interacting specifically with GATA-type cis-acting elements. To further investigate the role that Fep1 plays in iron homeostasis, we searched for additional Fep1-regulated genes. We found that str1+ is subject to negative transcriptional regulation, which is exerted through binding of Fep1 to a single GATA element in the str1+ promoter. Introduction of str1+ into a Saccharomyces cerevisiae fet3Delta arn1-4Delta strain led to assimilation of iron from ferrichrome, revealing that Str1 functions as a siderophore-iron transporter in S.pombe. We also identified two additional target genes of Fep1, named str2+ and str3+. We demonstrate that the str1+, str2+ and str3+ genes share a common promoter element, 5'-(A/T)GATAA-3'. We found that the N-terminal 241 residue segment of Fep1 expressed in Escherichia coli specifically interacts with the 5'-(A/T)GATAA-3' element present in each of these promoters. Consistent with this, constitutive high level str1+, str2+ and str3+ gene expression was observed in a fep1Delta mutant strain. Taken together, these results demonstrate that Fep1 occupies a central role in coordinating transcriptional regulation of genes encoding components of the reductive and non-reductive iron transport systems in fission yeast.