Dataset Information

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.

SUBMITTER: Mercier A

PROVIDER: S-EPMC2740451 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Both Php4 function and subcellular localization are regulated by iron via a multistep mechanism involving the glutaredoxin Grx4 and the exportin Crm1.

Mercier Alexandre A Labbé Simon S

The Journal of biological chemistry 20090605 30

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 ...[more]

PMID: 19502236

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Project description:The acquisition of iron and the maintenance of iron homeostasis are important aspects of virulence for the pathogenic fungus Cryptococcus neoformans In this study, we characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis and virulence in C. neoformans Monothiol glutaredoxins are important regulators of iron homeostasis because of their conserved roles in [2Fe-2S] cluster sensing and trafficking. We initially identified Grx4 as a binding partner of Cir1, a master regulator of iron-responsive genes and virulence factor elaboration in C. neoformans We confirmed that Grx4 binds Cir1 and demonstrated that iron repletion promotes the relocalization of Grx4 from the nucleus to the cytoplasm. We also found that a grx4 mutant lacking the GRX domain displayed iron-related phenotypes similar to those of a cir1Δ mutant, including poor growth upon iron deprivation. Importantly, the grx4 mutant was avirulent in mice, a phenotype consistent with observed defects in the key virulence determinants, capsule and melanin, and poor growth at 37°C. A comparative transcriptome analysis of the grx4 mutant and the WT strain under low-iron and iron-replete conditions confirmed a central role for Grx4 in iron homeostasis. Dysregulation of iron-related metabolism was consistent with grx4 mutant phenotypes related to oxidative stress, mitochondrial function, and DNA repair. Overall, the phenotypes of the grx4 mutant lacking the GRX domain and the transcriptome sequencing (RNA-Seq) analysis of the mutant support the hypothesis that Grx4 functions as an iron sensor, in part through an interaction with Cir1, to extensively regulate iron homeostasis.IMPORTANCE Fungal pathogens cause life-threatening diseases in humans, particularly in immunocompromised people, and there is a tremendous need for a greater understanding of pathogenesis to support new therapies. One prominent fungal pathogen, Cryptococcus neoformans, causes meningitis in people suffering from HIV/AIDS. In the present study, we focused on characterizing mechanisms by which C. neoformans senses iron availability because iron is both a signal and a key nutrient for proliferation of the pathogen in vertebrate hosts. Specifically, we characterized a monothiol glutaredoxin protein, Grx4, that functions as a sensor of iron availability and interacts with regulatory factors to control the ability of C. neoformans to cause disease. Grx4 regulates key virulence factors, and a mutant is unable to cause disease in a mouse model of cryptococcosis. Overall, our study provides new insights into nutrient sensing and the role of iron in the pathogenesis of fungal diseases.

Project description:Due to iron's essential role in cellular metabolism, most organisms must maintain their homeostasis. In this regard, the fission yeast Schizosaccharomyces pombe (sp) uses two transcription factors to regulate intracellular iron levels: spFep1 under iron-rich conditions and spPhp4 under iron-deficient conditions, which are controlled by spGrx4. However, bioinformatics analysis to understand the role of the spGrx4/spFep1/spPhp4 axis in maintaining iron homeostasis in S. pombe is still lacking. Our study aimed to perform bioinformatics analysis on S. pombe proteins and their sequence homologs in Aspergillus flavus (af), Saccharomyces cerevisiae (sc), and Homo sapiens (hs) to understand the role of spGrx4, spFep1, and spPhp4 in maintaining iron homeostasis. The three genes' expression patterns were also examined at various iron concentrations. A multiple sequence alignment analysis of spGrx4 and its sequence homologs revealed a conserved cysteine residue in each PF00085 domain. Blast results showed that hsGLRX3 is most similar to spGrx4. In addition, spFep1 is most closely related in sequence to scDal80, whereas scHap4 is most similar to spFep1. We also found two highly conserved motifs in spFep1 and its sequence homologs that are significant for iron transport systems because they contain residues involved in iron homeostasis. The scHap4 is most similar to spPhp4. Using STRING to analyze protein-protein interactions, we found that spGrx4 interacts strongly with spPhp4 and spFep1. Furthermore, spGrx4, spPhp4, and spFep1 interact with spPhp2, spPhp3, and spPhp5, indicating that the three proteins play cooperative roles in iron homeostasis. At the highest level of Fe, spgrx4 had the highest expression, followed by spfep1, while spphp4 had the lowest expression; a contrast occurred at the lowest level of Fe, where spgrx4 expression remained constant. Our findings support the notion that organisms develop diverse strategies to maintain iron homeostasis.

Dataset Information

Both Php4 function and subcellular localization are regulated by iron via a multistep mechanism involving the glutaredoxin Grx4 and the exportin Crm1.

Publications

Both Php4 function and subcellular localization are regulated by iron via a multistep mechanism involving the glutaredoxin Grx4 and the exportin Crm1.

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