Molecular basis of the regulation of iron homeostasis in fission and filamentous yeasts.

When iron load exceeds that needed by fission and filamentous yeasts, iron-regulatory GATA-type transcription factors repress genes encoding iron acquisition systems. In contrast, under iron starvation, optimization of cellular iron utilization is coordinated by a specialized regulatory subunit of the CCAAT-binding factor that fosters repression of genes encoding iron-using proteins. Despite these findings, there is still limited knowledge concerning the mechanisms by which these iron-responsive regulators respond to high- or low-iron availability. To provide a framework for understanding common and distinct properties of iron-dependent transcriptional regulators, a repertoire of their functional domains in different fungal species is presented here. In addition, discovery of interacting partners of these iron-responsive factors contributes to provide additional insight into their properties.

Shu1 is a cell-surface protein involved in iron acquisition from heme in Schizosaccharomyces pombe.

Iron is an essential metal cofactor that is required for many biological processes. Eukaryotic cells have consequently developed different strategies for its acquisition. Until now, Schizosaccharomyces pombe was known to use reductive iron uptake and siderophore-bound iron transport to scavenge iron from the environment. Here, we report the identification of a gene designated shu1(+) that encodes a protein that enables S. pombe to take up extracellular heme for cell growth. When iron levels are low, the transcription of shu1(+) is induced, although its expression is repressed when iron levels rise. The iron-dependent down-regulation of shu1(+) requires the GATA-type transcriptional repressor Fep1, which strongly associates with a proximal promoter region of shu1(+) in vivo in response to iron repletion. HA4-tagged Shu1 localizes to the plasma membrane in cells expressing a functional shu1(+)-HA4 allele. When heme biosynthesis is selectively blocked in mutated S. pombe cells, their ability to acquire exogenous hemin or the fluorescent heme analog zinc mesoporphyrin IX is dependent on the expression of Shu1. Further analysis by absorbance spectroscopy and hemin-agarose pulldown assays showed that Shu1 interacts with hemin, with a KD of ∼2.2 µm. Taken together, results reported here revealed that S. pombe possesses an unexpected pathway for heme assimilation, which may also serve as a source of iron for cell growth.

Fra2 is a co-regulator of Fep1 inhibition in response to iron starvation.

Iron is required for several metabolic functions involved in cellular growth. Although several players involved in iron transport have been identified, the mechanisms by which iron-responsive transcription factors are controlled are still poorly understood. In Schizosaccharomyces pombe, the Fep1 transcription factor represses genes involved in iron acquisition in response to high levels of iron. In contrast, when iron levels are low, Fep1 becomes inactive and loses its ability to associate with chromatin. Although the molecular basis by which Fep1 is inactivated under iron starvation remains unknown, this process requires the monothiol glutaredoxin Grx4. Here, we demonstrate that Fra2 plays a role in the negative regulation of Fep1 activity. Disruption of fra2+ (fra2Δ) led to a constitutive repression of the fio1+ gene transcription. Fep1 was consistently active and constitutively bound to its target gene promoters in cells lacking fra2+. A constitutive activation of Fep1 was also observed in a php4Δ fra2Δ double mutant strain in which the behavior of Fep1 is freed of its transcriptional regulation by Php4. Microscopic analyses of cells expressing a functional Fra2-Myc13 protein revealed that Fra2 localized throughout the cells with a significant proportion of Fra2 being observed within the nuclei. Further analysis by coimmunoprecipitation showed that Fra2, Fep1 and Grx4 are associated in a heteroprotein complex. Bimolecular fluorescence complementation experiments brought further evidence that an interaction between Fep1 and Fra2 occurs in the nucleus. Taken together, results reported here revealed that Fra2 plays a role in the Grx4-mediated pathway that inactivates Fep1 in response to iron deficiency.