Both Php4 Function and Subcellular Localization Are Regulated by Iron via a Multistep Mechanism Involving the Glutaredoxin Grx4 and the Exportin Crm1

Abstract

In Schizosaccharomyces pombe, the CCAAT-binding factor is a multisubunit complex that contains the proteins Php2, Php3, Php4, and Php5. Under low iron conditions, Php4 acts as a negative regulatory subunit of the CCAAT-binding factor and fosters repression of genes encoding iron-using proteins. Under conditions of iron excess, Php4 expression is turned off by the iron-dependent transcriptional repressor Fep1. In this study, we developed a biological system that allows us to unlink iron-dependent behavior of Php4 protein from its transcriptional regulation by Fep1. Microscopic analyses revealed that a functional GFP-Php4 protein accumulates in the nucleus under conditions of iron starvation. Conversely, in cells undergoing a transition from low to high iron, GFP-Php4 is exported from the nucleus to the cytoplasm. We mapped a leucine-rich nuclear export signal that is necessary for nuclear exclusion of Php4. This latter process was blocked by leptomycin B. By using coimmunoprecipitation analysis, we showed that Php4 and Crm1 physically interact with each other. Although we determined that nuclear retention of Php4 per se is not sufficient to cause a constitutive repression of iron-using genes, we found that deletion of the grx4 ؉ -encoded glutaredoxin-4 renders Php4 constitutively active and invariably localized in the nucleus. Further analysis by bimolecular fluorescence complementation assay and by two-hybrid assays showed that Php4 and Grx4 are physically associated in vivo. Taken together, our findings indicate that Grx4 and Crm1 are novel components involved in the mechanism by which Php4 is inactivated by iron in a Fep1-independent manner.

Cells Lacking Pfh1, a Fission Yeast Homolog of Mammalian Frataxin Protein, Display Constitutive Activation of the Iron Starvation Response

“…Thus, upon low iron, Php4 accumulates at the nucleus and represses transcription of genes coding for iron storage proteins or iron-consuming proteins (Fig. 4A) (46). First of all, we confirmed by Northern blot that not only protein levels but also mRNA levels for some Php4-dependent genes, such as pcl1 and isa1, are constitutively low in ⌬pfh1 cells (Fig.…”

“…Downstream of Grx4, the inactivation of Fep1 and activation of Php4 transcriptional repressors mediate the cellular response to iron deficiency (44). Briefly, when iron is experimentally depleted by the use of chelators, Fep1 is released from promoters of genes involved in iron uptake (45), whereas Php4 accumulates at the nucleus and represses transcription of genes coding for iron storage or iron-consuming proteins (46). Importantly enough, Grx4 is the real sensor of iron deprivation, probably through its ISC.…”

Coming into View: Eukaryotic Iron Chaperones and Intracellular Iron Delivery

“…Recent studies in fission yeast indicate that a monothiol glutaredoxin, Grx4, is required for iron sensing by its iron-dependent transcription factors, Fep1 and Php4 (39,40). Although these transcription factors have no similarity to Aft1, Grx4 can be found in a complex with each transcription factor in this fungal species, but it is not known whether direct transfer of an iron cofactor occurs.…”

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…Despite the fact that Aft1 is restricted to a few species, similar iron regulation systems might exist in other organisms including plants. For instance, in Schizosaccharomyces pombe, Fep1 and Php4, two transcriptional regulators of iron homeostasis, are regulated by a Grx4 ortholog (55,56). Whether a BolA is required in this case is not yet elucidated.…”