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. Author manuscript; available in PMC: 2012 Oct 20.
Published in final edited form as: FEBS Lett. 2011 Sep 29;585(20):3342–3347. doi: 10.1016/j.febslet.2011.09.025

Iron influences the abundance of the iron regulatory protein Cir1 in the fungal pathogen Cryptococcus neoformans

Won Hee Jung 1, James W Kronstad 2,*
PMCID: PMC3200532  NIHMSID: NIHMS328841  PMID: 21963719

Abstract

The GATA-type, zinc-finger protein Cir1 regulates iron uptake, iron homeostasis and virulence factor expression in the fungal pathogen Cryptococcus neoformans. The mechanisms by which Cir1 senses iron availability, although as yet undefined, are important for understanding the proliferation of the fungus in mammalian hosts. We investigated the influence of iron availability on Cir1 and found that the abundance of the protein decreases upon iron deprivation. This destabilization was influenced by reducing conditions and by inhibition of proteasome function. The combined data suggest a post-translational mechanism for the control of Cir1 abundance in response to iron and redox status.

Keywords: Cryptococcus neoformans, Iron, Cir1, Protein stability, Redox, Proteasome

1. Introduction

Iron plays an important role in metabolism during infection and in regulating virulence factor expression for many pathogenic microbes [1–2]. The fungal pathogen Cryptococcus neoformans can utilize iron from transferrin and heme within mammalian hosts and Barluzzi et al. showed that iron overload exacerbates Cryptococcal disease [3–5]. This fungus is the frequent cause of meningoencephalitis in immunocompromised people such as AIDS patients [6]. We previously found that C. neoformans utilizes iron from transferrin via the high-affinity, reductive iron transport pathway composed of the iron permease Cft1 and the ferroxidase Cfo1 [4–5]. These genes are required for full virulence in a mouse model of cryptococcosis and for proliferation in brain tissue. We also identified regulatory proteins that control the expression of genes for iron utilization, including the GATA-type, zinc-finger transcription factor Cir1 and a bZIP transcription factor HapX [7–8].

Cir1 is particularly important because it regulates the expression of genes for iron uptake functions, such as CFT1 and CFO1, as well as all of the known, major virulence factors of C. neoformans [7]. Related GATA-type, iron-regulatory transcription factors have been identified in other fungi. These factors include Fep1 in Schizosaccharomyces pombe, Sre in Neurospora crassa, SREB in Blastomyces dermatidis, SreA in Aspergillus nidulans and A. fumigatus, Sre1 in Histoplasma capsulatum, Sfu1 in Candida albicans and Urbs1 in Ustilago maydis [9–17]. For Sre1, Sre and Fep1, a conserved cysteine (cys)-rich region between the two zinc finger domains has been implicated in iron and DNA binding [18]. The influence of iron on expression and activity has been characterized for some of these proteins. For example, iron levels influence the expression of the SreA protein in A. nidulans, and Fep1 is regulated transcriptionally by iron availability in a Php4-dependent manner, but the stability and localization of the protein were not affected by iron status [19]. In contrast, iron and oxygen are key post-translational regulators of the mammalian iron-regulatory protein Irp2 [20]. The level of the protein is reduced in iron-replete cells via the ubiquitin-proteasomal pathway while mRNA stability and protein synthesis are unaffected.

We previously found that CIR1 is transcriptionally regulated by the Php4-related protein HapX in C. neoformans [8], as well as by the Gat201 transcription factor [8,20]. However, mRNA levels for CIR1 remained constant regardless of iron availability [7–8]. Here we investigated the abundance of the Cir1 protein upon growth of cells in low- and high-iron conditions.

2. Materials and Methods

2.1. Strains and media

C. neoformans var. neoformans strain B3501A was used in this study [21]. S. cerevisiae strain YPH499 (MATa, ura3-52, lys2-801, ade2-101, his3-Δ200, trp1-Δ63, leu2-Δ1) and a derivative lacking PDR5 were used for heterologous expression of Cir1 and testing MG132 inhibition [22]. Strains were maintained in yeast extract-Bacto peptone medium with 2.0% dextrose (YPD; Difco) or yeast nitrogen base (YNB; Difco) with 2.0% glucose. Bathophenanthrolinedisulfonate (BPS; Sigma) was added to YNB medium at 100 μM to achieve a low-iron condition and this medium is termed LIYNB. For the iron-replete conditions, FeCl3 was added to LIYNB at the final concentrations indicated in the text. Cells were pre-cultured in LIYNB at 30°C overnight to deplete intracellular iron before challenge with iron. S. cerevisiae cells were grown in YNB medium without uracil and with 2% glucose at 30°C overnight and washed with phosphate buffered saline (PBS). To induce and maintain the expression of Cir1, the cells were then transferred to YNB medium without uracil and with 2% galactose and 2% raffinose. These cells were employed for all experiments to test the influence of iron.

2.2. Preparation of cell extracts and Western blot analysis

Cells of C. neoformans or S. cerevisiae were collected by centrifugation and resuspended in cold protein extraction buffer containing 50 mM HEPES KOH pH7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycolate, 1 mM PMSF and protease inhibitor cocktail (Sigma). Cells were lysed by bead-beating, and the protein concentration in the lysates was measured by the Bradford assay [23]. Equal amounts of protein were resolved in a SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Sigma). Western blots were performed using an anti-Myc mouse monoclonal antibody or an anti-His mouse monoclonal antibody (Abgent) as a primary antibody, and a goat anti-mouse IgG horseradish peroxidase conjugate (Bio-Rad) as a secondary antibody, followed by visualizing by chemiluminescence. The methods for the expression of epitope-tagged Cir1 and the construction of amino acid substitution mutants can be found in Supplementary methods.

2.3. RNA isolation and analysis

Total RNA was prepared using an RNeasy mini-kit (Qiagen) and quantified using a NanoDrop spectrophotometer. A total of 10 μg of RNA per sample was used for Northern blot analysis as described [24]. The SacI/XhoI fragment from the plasmid pYES2.1/LacZ was used as a probe for the gene encoding β–galactosidase. A DNA fragment was amplified by PCR using primers Cir28-NF (5′-CCCGAAACGGGATCCTCCGCGCCTCAGGACAACGAGCATGAG-3′) and Cir28-NR (5′-GAGGAGAAGCTCGAGGAGCTTTGTCATCAAGAGGCGGTGCCAC-3′) for use as a probe for CIR1. Additional information on RNA analysis is provided in Supplementary methods.

3. Results

3.1. Iron-dependent regulation of Cir1 abundance

We previously observed that CIR1 mRNA levels are not influenced by iron although Cir1 mediates practically all of the transcriptional response to the metal in C. neoformans [7–8]. To investigate the mechanism of the Cir1 response to iron, we tested the hypothesis that the abundance of the protein may be regulated by iron availability. A cir1 deletion allele was replaced with a construct encoding a Cir1-13Myc fusion protein and the function of the epitope-tagged protein in C. neoformans was confirmed with a TTC overlay assay for Cir1-regulated cell surface reductase activity (Fig. 1A). The function of the Cir1-13Myc fusion protein was also confirmed by RT-PCR analysis showing that up-regulation of LAC1 expression in the cir1 mutant was restored by the fusion protein (Fig. 1B). To test whether the abundance of the tagged Cir1 protein was responsive to iron concentrations, cells were grown in low-iron media supplemented with various concentrations of iron and the levels of Cir1 were examined. This experiment revealed that higher levels of iron resulted in greater abundance of Cir1, relative to actin as a loading control (Fig. 1C). In particular, the protein was not detectable in the cells grown in medium containing less than 10 μM of iron thus suggesting that Cir1 abundance was positively influenced by iron concentration.

Figure 1.

Figure 1

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Epitope-tagged Cir1 is functional and its abundance is positively influenced by iron levels. (A) Wild-type (WT), the cir1 mutant (cir1Δ) and the mutant expressing the 13Myc epitope-tagged Cir1 protein (cir1Δ::Cir1-13Myc) were grown on YPD medium overnight and overlaid with TTC to detect cell surface reductase activity. (B) The abundance of LAC1 mRNA was evaluated by RT-PCR. cDNA was synthesized from total RNA and PCR products were withdrawn at cycles 24, 27 and 30 during the reactions and analyzed on an agarose gel. (C) Cells were grown in low-iron YNB medium (LIYNB) with various concentrations of FeCl3 at 30°C for 12 h and proteins were extracted as described in Materials and Methods. Equal amounts of protein (40 μg) were subjected to SDS-PAGE followed by Western blot analysis using anti-Myc13 (Cir1) or anti-actin (Act1) antibodies. Actin was used as a loading control and the tagged Cir1 protein is ~135 Kda.

Cir1 protein was also expressed in S. cerevisiae to further confirm the influence of iron in a heterologous host and to eliminate a possible transcriptional explanation for the observed influence of iron. For expression, the cDNA for CIR1 was fused with the inducible GAL1 promoter, a CYC1 terminator and the dual epitope tag V5-HIS in an S. cerevisiae expression vector. The levels of the Cir1 protein were analyzed in the presence of different concentrations of iron in the media under inducing conditions for the GAL1 promoter. As seen in C. neoformans, the abundance of Cir1 protein was also greater in cells grown in elevated iron versus low-iron medium in S. cerevisiae (Fig. 2A). Expression of β-galactosidase from the same expression vector was used as a control, and no influence of iron was observed for this protein. The Cir1 polypeptide is 952 amino acids in length with a cys-rich domain (amino acids 162–189) and a conserved zinc-finger domain (amino acids 307–359) in the N-terminal region. We hypothesized that the N-terminal region of the protein was responsible for the iron-dependent stability, and we tested this idea by expressing an epitope-tagged polypeptide from amino acids 1 to 408 in S. cerevisiae. This truncated protein also displayed the iron-dependent abundance suggesting that the N-terminal region contained a necessary domain for iron-dependent stability of Cir1, although a contribution of the C-terminal portion of the protein remains to be characterized (Fig. 2B). In a separate assay, pre-growing cells in high-iron medium with galactose and shifting them to low-iron medium resulted in a reduction in Cir1 abundance over time (Fig. 2C).

Figure 2.

Figure 2

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Cir1 is more abundant in S. cerevisiae cells grown in medium with elevated iron concentrations. (A) S. cerevisiae cells expressing the His epitope-tagged Cir1 protein under the GAL1 promoter and the CYC1 terminator were grown in LIYNB with various concentrations of FeCl3 at 30°C for 12 h. Proteins were extracted as described in Materials and Methods and equal amounts of protein (40 μg) were subjected to SDS-PAGE followed by Western blot analysis using anti-His (Cir1) or anti-actin (Act1) antibodies. β–galactosidase (β–gal) was used as a control protein that did not change in response to iron, and actin was used as a loading control. The tagged Cir1 protein is ~119 Kda. (B) S. cerevisiae cells expressing truncated Cir1 protein were grown in the presence of various concentrations of FeCl3 at 30°C for 12 h and were subjected to SDS-PAGE followed by Western blot analysis. Equal amounts of protein (40 μg) were loaded. The truncated Cir1 polypeptide is ~46 Kda. (C) The S. cerevisiae cells expressing the full-length Cir1 protein were grown in the presence of 100 μM FeCl3 and subsequently transferred to low-iron medium. Proteins were extracted at the times indicated and levels of Cir1 were accessed by Western blot analysis using anti-His (Cir1) or anti-actin (Act1) antibodies.

3.2. A cysteine-rich domain is not required for iron-dependent regulation of Cir1

The N-terminal region of Cir1 contains a cys-rich domain and a single zinc-finger domain, and is sufficient for the iron-dependent regulation of Cir1 abundance as shown above. Other fungal GATA-type, iron regulatory proteins also possess these conserved domains and the cys-rich region has been implicated in iron and DNA binding activity for some of these proteins (Fig. 3A) [14,18,25]. The role of the cys-rich region of Cir1 was tested in relation to iron-dependent abundance by replacing the Cys180 and Cys183 residues with Ala to generate the substitution mutant C180A/C183A (Fig. 3A). The construct containing the mutations C180A/C183A was transformed into S. cerevisiae, cells were grown in the presence of various concentrations of iron, and Cir1 protein level was analyzed. The protein containing C180A/C183A displayed a similar pattern to the wild-type protein (Fig. 3B, C180A/C183A), therefore indicating that the cys-rich region of the protein did not influence iron responsiveness. Within the zinc-finger region, Cys308 and Cys311 residues were replaced with Ala to generate the substitution mutant C308A/C311A, and Arg320 and Arg321 were replaced with Ala to construct the mutant R320A/R321A. The latter region has been implicated in iron binding in the Urbs1 GATA factor in U. maydis, although it may also function in DNA binding [10]. In contrast to the C180A/C183A mutation, the C308A/C311A and R320A/R321 mutations both reduced Cir1 abundance suggesting that the zinc-finger domain is critical for stability of the protein. Furthermore, the analysis revealed that the mutations described above affect the protein at the post-transcriptional and/or post-translational levels because Northern blot analysis revealed that CIR1 transcript levels are constant regardless of iron concentration (Fig. 3B).

Figure 3.

Figure 3

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Influence of amino acid substitutions on the abundance of Cir1. (A) Amino acid alignment of the N-terminal region of C. neoformans Cir1 with other GATA-type iron regulatory transcription factors including S. pombe Fep1 (AAM29187), C. albicans Sfu1 (AAM77345), H. capsulatum Sre1 (ABY66603), N. crassa Sre (AAC64946) and U. maydis Urbs1 (AAB05617). The domains containing the highly conserved cys-rich region (top) and the C-terminal zinc finger (bottom) are shown. Substituted amino acids are labeled at the bottom of each alignment and the A at the top of the alignments also indicates the alanine substitution. (B) S. cerevisiae cells expressing wild-type Cir1 and the mutant proteins containing substitutions were grown in LIYNB with various iron concentrations as indicated. Proteins were extracted and Western blot analysis was performed using anti-His (Cir1) or anti-actin (Act1) antibodies. β–galactosidase (β–gal) was used as a control protein that did not respond to iron and actin was used as a loading control. The abundance of Cir1 and Act1 mRNA in the cells grown under the same conditions was visualized by Northern blot analysis as described in Materials and Methods.

3.3. Reducing conditions influence the stability of Cir1

Redox regulation is known to influence the stability of some iron regulatory proteins and we hypothesized that stability of the C. neoformans Cir1 protein might be regulated in a similar fashion [26]. We first examined the influence of reducing conditions by expressing the protein in S. cerevisiae, followed by extraction using buffer containing iron with and without the reducing agent dithiothreitol (DTT). Both the full-length protein and the C-terminal truncated protein were tested and, as expected, higher concentrations of iron in the media increased the levels of both proteins (Fig. 4). Protein extraction using buffer containing iron did not significantly affect the level of the full-length protein, although slightly increased levels of the truncated version were observed. This result implied that C-terminal region of the protein may contribute to stability, perhaps by masking a domain in the N-terminal region that may interact with iron. Although the influence was not dramatic, maintaining reducing conditions with the inclusion of DTT resulted in increased levels of both the full-length and the truncated proteins thus indicating that the redox status of Cir1 may influence its stability (Fig. 4).

Figure 4.

Figure 4

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Reducing conditions increase the stability of Cir1. S. cerevisiae cells expressing the full length Cir1 (Full-length) or the C-terminal truncated protein (Truncated) were grown in LIYNB medium with or without 100 μM FeCl3 (Medium). Proteins were extracted using lysis buffer containing 100 mM DTT alone, or both 100 mM DTT and 100 μM FeCl3 (Buffer) and Western blot analysis were performed using anti-His (Cir1) or anti-actin (Act1) antibodies. Actin was used as a loading control.

3.4. Inhibition of proteasome function stabilizes Cir1

The possibility that the stability of Cir1 was influenced by proteasome activity was tested by treating S. cerevisiae cells expressing Cir1 with the peptide aldehyde MG132, a potent inhibitor of the 20S subunit of the proteasome. Western blot analysis showed that the levels of Cir1 were increased by MG132 treatment when cells were grown in the low-iron condition (Fig. 5A). The levels of β-galactosidase remained constant suggesting specificity of MG132 treatment for proteasomal degradation of Cir1. These experiments were performed in an S. cerevisiae strain that lacks the multidrug efflux protein Pdr5 because the wild-type strain does not efficiently accumulate MG132. We next tested whether proteasomal degradation of Cir1 takes place in C. neoformans in response to low iron. No variation in abundance was observed for the epitope-tagged Cir1 protein upon expression in C. neoformans in the presence of MG132, which was probably due to limited MG132 accumulation, similar to wild-type S. cerevisiae (data not shown). The assay was modified to overcome this problem by adding MG132 directly to the cell lysis buffer for protein extraction, incubating the cell lysates for different time periods, and analyzing Cir1 abundance. With this approach, an increased level of Cir1 protein in the iron-depleted condition was observed with the inhibitor suggesting a role for proteasome-dependent degradation of Cir1 in C. neoformans (Fig. 5B).

Figure 5.

Figure 5

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Proteasome-dependent degradation of Cir1. (A) S. cerevisiae pdr5 mutant cells expressing the full length Cir1 (Full-length, ~119 Kda) or the C-terminal truncated protein (Truncated, ~46 Kda) were grown in LIYNB medium containing various concentrations of FeCl3 with or without the proteasome inhibitor MG132 (20 μg/mL) at 30°C for 12 h. Proteins were extracted and Western blot analysis were performed using anti-His (Cir1), anti-actin (Act1) or anti-3-phosphoglycerate kinase (Pgk1) antibodies. β–galactosidase (β–gal) was used as a control protein that did not respond to iron, and Actin and Pgk1 were used as loading controls. (B) C. neoformans cells expressing Cir1-Myc13 were grown in LIYNB medium. Proteins were extracted using lysis buffer with or without MG132 (20 μg/mL) and were incubated for the indicated times followed by Western blot analysis using anti-Myc. Equal amounts of proteins (40 μg) were loaded.

4. Discussion

In this study, we demonstrated that lower iron levels correlate with reduced abundance of the Cir1 protein both in C. neoformans and upon heterologous expression in S. cerevisiae. Based on these findings, we propose a model in which the transcription and translation of Cir1 occur independent of iron levels, but iron directly or indirectly influences the stability of the protein (Fig. 6). The enhanced stability of Cir1 under iron-replete conditions is in contrast to the destabilization of Irp2 in response to elevated iron in mammalian cells [26]. Irp2 is stable in cells that are iron deficient and/or hypoxic, and degradation is mediated by the SKP1-CUL1-FBXL5 ubiquitin ligase complex, which catalyzes Irp2 ubiquitination and proteasomal degradation in response to increased iron and oxygen levels [27]. FBXL5 is stabilized by elevated iron and oxygen levels and this activates the ligase complex for Irp2 degradation. Other proteins involved in iron homeostasis and regulated through ubiquitination and proteasomal degradation include human frataxin, the divalent metal transporter Dmt1 in mammalian cells and the siderophore transporter Arn1 in S. cerevisiae [28–30]. In this context, Cir1 may also be targeted for ubiquitination under low-iron conditions, although candidate ubiquitin ligase(s) that are responsive to iron remain to be identified. Given our use of S. cerevisiae for heterologous expression of Cir1 and β-galactosidase, we note the possibility that iron levels could influence expression based on the finding of Shi et al. that iron limitation reduced GAL1 promoter activity during galactose induction in yeast [31]. However, we employed cells that were induced for expression by growth in galactose before incubation with different iron concentrations, and we also did not observe an influence of iron levels on the mRNAs for β-galactosidase or Cir1 under the growth conditions that we employed (Fig. 3B).

Figure 6.

Figure 6

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A model for the influence of iron on Cir1 stability. The model postulates that transcription and translation of CIR1 occur independent of intracellular iron status but that iron directly or indirectly causes stabilization of the Cir1 protein and, in turn, the protein acts as a repressor to negatively regulate transcription of target genes for iron acquisition. In contrast, low intracellular iron levels may result in a conformational change and increased susceptibility to oxidation such that the protein is more likely to be targeted for ubiquitin-dependent degradation. Oxidation of Cir1 may contribute to targeting the protein for degradation. It is presumed that Cir1 may also act as a transcriptional activator of target genes under the low-iron conditions since its dual functions (i.e., as both a repressor and an activator) have been demonstrated [7]. In this situation, reduced levels of Cir1 may influence promoter selection and/or interactions with partner proteins.

The influence of iron on protein abundance has not been observed for the iron regulatory GATA-type proteins in other fungi. The fungal GATA-type zinc-finger iron regulators are thought to be iron-binding proteins based on observations in N. crassa, H. capsulatum and S. pombe, although this property has not yet been established for Cir1. For example, Sre of N. crassa and Sre1 of H. capsulatum displayed an orange and orange-brown color, respectively, during purification, which is a typical characteristic of iron-binding proteins. Interestingly, the color of both proteins disappeared when a strong reducing reagent was added suggesting an influence of the redox status of iron on the proteins [14,18]. However, whether this redox status affects the stability of the proteins has not been established. As mentioned, the cys-rich domain in other fungal iron regulators from H. capsulatum, N. crassa and S. pombe is implicated in iron and DNA binding. For example, mutations in the cys-rich region influence the iron binding of Sre1 in H. capsulatum and proteins with substitutions for the cysteine residues had lower DNA binding affinity [14]. Our mutational analysis with Cir1 suggested that the conserved cysteine residues in the cys-rich region were not involved in the influence of iron on protein abundance. There are four additional cys residues in the zinc-finger domain. Substitution of two of these resulted in no protein accumulation, although this may be due to loss of zinc binding leading to destabilization.

The model fungus Saccharomyces cerevisiae has iron regulatory proteins, Aft1 and Aft2, which are unrelated to the GATA-type regulators from the other fungi. Aft1 localizes to the nucleus and induces the expression of genes required for iron uptake in iron-deficient conditions although the level of the protein does not change [32]. Aft1 activity is dependent on mitochondrial iron levels and is influenced by the nuclear monothiol glutaredoxins Grx3 and Grx4 [33–35]. A monothiol glutaredoxin Grx4 also regulates the GATA-type iron regulator Fep1 in S. pombe [36]. Our observed influence of reducing conditions on Cir1 abundance under low iron conditions suggests that redox responsive binding partners may also regulate the stability of the protein.

Supplementary Material

01

Highlights.

  • The fungus Cryptococcus neoformans is an important pathogen of AIDS patients.

  • The transcription factor Cir1 regulates iron uptake and virulence in C. neoformans.

  • We examined the influence of iron levels on the abundance of Cir1.

  • Iron deprivation results in reduced abundance of Cir1.

  • Cir1 stability is also regulated by redox conditions and proteasome activity.

Acknowledgments

The authors thank Joyce Wang and Jeongmi Kim for assistance, and Jan Stoepel, Shay Ben-Aroya, Philip Hieter and Thibault Mayor for S. cerevisiae reagents and helpful discussions. This work was supported by awards from the National Institutes of Health R01 AI053721 (JWK) and the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0005810) (WHJ). JWK is a Burroughs Wellcome Fund Scholar in Molecular Pathogenic Mycology.

List of abbreviations

Cir1

Cryptococcus iron regulator 1

LIYNB

low-iron yeast nitrogen base medium

BPS

Bathophenanthrolinedisulfonate

DTT

dithiothreitol

TTC

triphenyltetrazolium chloride

sec

second

min

minute

h

hour

Kda

kilodaltons

Footnotes

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