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. Author manuscript; available in PMC: 2009 Jun 1.
Published in final edited form as: Cell Metab. 2008 Jun;7(6):555–564. doi: 10.1016/j.cmet.2008.04.010

Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency

Sergi Puig 1,*,, Sandra V Vergara 2,, Dennis J Thiele 2,*
PMCID: PMC2459314  NIHMSID: NIHMS54026  PMID: 18522836

Summary

Iron (Fe) is an essential co-factor for a wide range of cellular processes. We have previously demonstrated that during Fe-deficiency yeast Cth2 is expressed and promotes degradation of a battery of mRNAs leading to reprogramming of Fe-dependent metabolism and Fe-storage. We report that the Cth2-homologous protein, Cth1, is transiently expressed during Fe-deprivation and participates in the response to Fe-deficiency through the degradation of mRNAs primarily involved in mitochondrially-localized activities including respiration and amino acid biosynthesis. In parallel, wild type but not cth1Δ cth2Δ cells accumulate mRNAs encoding proteins that function in glucose import and storage and store high levels of glycogen. In addition, Fe-deficiency leads to Snf1 phosphorylation, a member of the AMP-activated protein kinase family required for the cellular response to glucose starvation. These studies demonstrate a metabolic reprogramming as a consequence of Fe-starvation that is dependent on the coordinated activities of two mRNA-binding proteins.

Introduction

The ability of iron (Fe) to engage in redox reactions makes it is a widely utilized co-factor in many central biochemical processes including oxygen delivery and storage, mitochondrial oxidative phosphorylation, DNA replication and repair, lipid metabolism, and chromatin modification. Abnormal Fe accumulation, either in excess or insufficient levels, underlies several human diseases including hereditary hemochromatosis, Friedreich’s ataxia, aceruloplasminemia and Fe-deficiency anemia (Dunn et al., 2007; Hentze et al., 2004; Rouault, 2006). Indeed, Fe-deficiency represents the most common nutritional deficiency, estimated to affect more than two billion people worldwide (Baynes and Bothwell, 1990).

Given the importance of Fe in health and disease, Fe-homeostasis has been under intensive investigation aimed at elucidating the mechanisms of Fe acquisition, distribution and regulation (Escolar et al., 1999; Hentze et al., 2004; Kaplan et al., 2006; Rouault, 2006; Dunn et al., 2007; Philpott and Protchenko, 2008). One regulatory mechanism for Fe-homeostasis in mammals involves the iron-regulatory proteins IRP1/IRP2, which post-transcriptionally modulate the expression of specific mRNAs in response to intracellular Fe (Hentze et al., 2004; Rouault, 2006). When cellular Fe is low, IRP1 and IRP2 bind to stem-loop structures, known as iron-responsive elements (IREs), within the 5’-untranslated region (UTR) of transcripts including those encoding the iron storage protein ferritin, mitochondrial aconitase, the heme biosynthetic enzyme eALAS, and the Fe efflux transporter ferroportin, thereby inhibiting their translation. Concurrently, IRP1 and IRP2 also bind IRE sequences within the 3’-UTR of the transferrin receptor mRNA, stabilizing the transcript and thus increasing its translational efficiency. As a consequence, there is a decrease in sequestration of Fe by ferritin and an increase in the capacity to mobilize transferrin-bound Fe. Once Fe levels are adequate, IRP1 acquires a [4Fe–4S] cluster, inter-converting to a cytoplasmic aconitase, and IRP2 is degraded (Hentze et al., 2004; Rouault, 2006). A post-transcriptional mechanism that controls cellular Fe homeostasis has also been described in bacteria. During Fe-starvation a small non-coding RNA from Escherichia coli, RyhB, is synthesized and promotes the degradation of mRNAs encoding proteins involved in Fe homeostasis, Fe-dependent metabolism and Fe storage. Once Fe levels are adequate, expression of RyhB ceases due to its transcriptional repression by the ferric uptake regulator, Fur (reviewed in Massé et al., 2007).

Previous studies have demonstrated that under Fe-limiting conditions S. cerevisiae utilizes two Fe-responsive transcription factors, Aft1 and Aft2, to activate expression of genes collectively known as the Fe-regulon (Rutherford et al., 2003; Shakoury-Elizeh et al., 2004; Courel et al., 2005). Additional mechanisms of Fe induced gene transcription occur in an Aft1/2-independent manner (Shakoury-Elizeh et al., 2004; Li et al., 2007). Genes in the Fe-regulon encode proteins involved in Fe-uptake, redistribution of intracellular Fe-stores, Fe-S biogenesis, and heme-utilization.

Concomitant with transcription of the Fe-regulon under conditions of Fe deficiency, the steady state levels of mRNAs encoding enzymes involved in many Fe-dependent metabolic processes including the tricarboxylic acid (TCA) cycle, mitochondrial respiration, heme biosynthesis and fatty acid synthesis, as well as that coding for the Ccc1 Fe storage protein, are markedly reduced (Lesuisse et al., 2003; Shakoury-Elizeh et al., 2004; Puig et al., 2005). A member of the Fe-regulon, Cth2, belongs to a family of mRNA-binding proteins conserved from yeast to humans, that is characterized by the presence of two tandem zinc-fingers of the CX8CX5CX3H type (CCCH) that constitute an mRNA-binding domain (Blackshear, 2002). Members of this family of proteins promote the rapid degradation of select mRNA molecules, by recruiting RNA decay enzymes to AU-rich Element (ARE)-containing mRNAs (Lykke-Andersen and Wagner, 2005). Cth2 protein binds AREs within the 3’-untranslated region (3’-UTR) of many mRNAs encoding proteins involved in Fe homeostasis and Fe-dependent metabolic processes, thereby promoting mRNA degradation (Puig et al., 2005). Consistent with a role for Cth2 in orchestrating genome-wide changes in metabolism in response to Fe deficiency, cth2Δ cells exhibit a growth defect on low Fe medium. Furthermore, the growth defect of cth2Δ cells is exacerbated by a mutation in the CTH1 gene, encoding a putative homologue of Cth2, whose function has not been previously described.

Here we demonstrate that the CTH1 gene is a direct target of the Aft1/2 transcription factors that is activated rapidly and transiently in response to Fe deficiency. Microarray analyses revealed that, while Cth2 predominantly stimulates the degradation mRNAs encoding general Fe homeostasis and Fe utilizing proteins that function in metabolism, Cth1 preferentially targets mRNAs encoding components of the mitochondrial oxidative phosphorylation machinery and other known or predicted mitochondrial functions. Moreover, we demonstrate that Cth1 and Cth2-stimulated mRNA decay results in the elevated expression of genes that are involved in the transport, metabolism, and storage of glucose. Consistent with this regulation, Fe starvation results in elevated glycogen levels and activation of Snf1 protein kinase, a central regulator of cellular carbon metabolism. Taken together, the S. cerevisiae Cth1 and Cth2 mRNA binding proteins play critical roles in targeting specific functional classes of mRNAs for degradation in response to Fe deficiency. Moreover, the coordinated activities of these two proteins impart changes in cellular metabolism consistent with a shift away from oxidative phosphorylation and toward carbohydrate utilization.

Results

CTH1 function in response to Fe-deficiency

The yeast genome harbors a gene encoding a protein structurally related to Cth2, Cth1, which shares an overall 46% identity and 56% similarity with Cth2, as well as 79% identity within the tandem zinc finger (TZF) domains (Figure 1A). The TZF region of Cth2, and other TZF protein family members, is essential for binding to ARE-sequences within the 3’-UTR of their target mRNAs (Lai et al., 2000; Hudson et al. 2004; Puig et al., 2005) We previously demonstrated that cth2Δ cells grow poorly on Fe-limited media and exhibit defects in the degradation of a specific set of ARE-containing mRNAs (Puig et al., 2005). Furthermore, deletion of the CTH1 gene exacerbates the growth and mRNA degradation defects of cth2Δ cells under Fe-limiting conditions (Puig et al., 2005). To evaluate the function of Cth1 during Fe deficiency, Cth1 was expressed under conditions of Fe-deficiency by fusing the CTH2 promoter region to the CTH1 coding sequence (CTH2:CTH1). The CTH2:CTH1 plasmid conferred an Fe-regulated pattern of expression to CTH1 similar to CTH2 mRNA, as ascertained by RNA blotting experiments (data not shown). Cells (cth1Δ cth2Δ) harboring the empty vector, or containing the CTH1, CTH2 or CTH2:CTH1 plasmid were evaluated for cell growth under conditions of Fe limitation and for steady state levels of mRNAs known to be degraded in a Cth2-dependent manner. As shown in Figure 1B, the growth defect of cth1Δcth2Δ cells on Fe-chelated medium was only partially suppressed by native CTH1 expression levels, but more significantly rescued in cells harboring the CTH2, or CTH2:CTH1 plasmid. The steady-state levels of two Cth2 target mRNAs, SDH4 and CCP1, was examined by RNA blotting. Both SDH4 and CCP1 encode mitochondrial Fe-dependent enzymes – a subunit of the succinate dehydrogenase complex and cytochrome c peroxidase, respectively. As previously observed (Puig et al., 2005) and shown in Figure 1C, both CCP1 and SDH4 mRNA levels were greatly reduced as a consequence of Fe-limitation in wild-type cells, but not in cth1Δcth2Δ cells carrying an empty vector. In contrast, there is a significant decrease in both CCP1 and SDH4 mRNA steady state levels in cth1Δcth2Δ cells expressing CTH2, CTH1 or CTH2:CTH1. Taken together these results indicate that Cth1 can promote a decrease in steady state levels of two Cth2 mRNA targets under Fe deprivation conditions.

Figure 1. Over-expression of CTH1 partially rescues cth2Δ-phenotypes.

Figure 1

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A. Representation of the Cth1 and Cth2 proteins. The mutation within the tandem zinc-fingers (TZF) of Cth1 is indicated. B. Over-expression of CTH1 partially suppresses the growth defect of cth1Δcth2Δ cells on Fe-depleted medium. BY4741 cells transformed with pRS416 (Wild-type), and cth1Δcth2Δ cells transformed with pRS416 (vector), pRS416-CTH2, pRS416-CTH1, or pRS416-CTH2:CTH1 were spotted on SC-Ura or SC-Ura + 750 µM Ferrozine to induce Fe-deprived conditions. C. Over-expression of CTH1 promotes down-regulation of Cth2 mRNA targets. cth1Δcth2Δ cells transformed with pRS416, pRS416-CTH1, pRS416-CTH2, or pRS416-CTH2:CTH1 were grown in medium containing either 300 µM Ferrous Ammonium Sulfate (FAS; Fe +) or 100 µM Bathophenanthroline disulfonate (BPS; Fe −) and steady-state levels of SDH4 and CCP1 were analyzed by RNA blotting. ACT1 was used as a loading control. D. Cth1 and Cth2 promote down-regulation of the SDH4 transcript independently of Fe-levels. cth1Δcth2Δ cells transformed with p416TEF1, p416TEF1-CTH1, or p416TEF1-CTH2 were grown and assayed as in panel C.

Although both Cth1 and Cth2 negatively regulate SDH4 and CCP1 mRNA levels under conditions of Fe deficiency, it is not clear if Fe deficiency is needed for their function. We ascertained whether Cth1 or Cth2 could promote a decrease in SDH4 mRNA levels under Fe sufficiency conditions. The CTH1 and CTH2 genes were expressed independently in cth1Δcth2Δ cells using the constitutive yeast TEF1 promoter. Cells expressing either the TEF1:CTH1 or TEF1:CTH2 genes, but not vector alone, exhibited decreased steady-state levels of the SDH4 transcript, under Fe-deficiency or Fe-replete conditions (Figure 1D). These observations demonstrate that both Cth1 and Cth2, when constitutively expressed, are able to promote a decrease in SDH4 steady-state mRNA levels irrespective of Fe availability. These results suggest that their activity is not regulated post-translationally by Fe, but rather they are regulated predominantly at the level of gene expression.

Aft1 and Aft2 regulate CTH1 transcription

While Cth1 contributes to growth and SDH4 mRNA degradation under conditions of Fe deficiency, previous studies demonstrate that CTH2, but not CTH1 mRNA levels are elevated in response to long-term Fe-deficiency (Shakoury-Elizeh et al., 2004; Puig et al. 2005). Visual inspection of the CTH1 promoter region revealed two putative Aft1/Aft2-binding sites starting at nucleotide positions -286 (5’-GCACCCAA-3’) and -94 (5’-TCACCCAA-3’) from the CTH1 translation initiation codon (Figure 2A). Furthermore, a genome-wide localization analysis of transcription factors in the S. cerevisiae revealed occupancy of the CTH1 promoter by the Aft1 transcription factor (Lee et al., 2002). To ascertain if Aft1 directly activates CTH1, Aft1 recruitment to the CTH1 promoter was assessed by chromatin immuno-precipitation (ChIP) experiments using a strain harboring a functional TAP-tagged AFT1 allele. While there is a low level of Aft1-TAP occupancy on the FET3 and CTH2 promoters in cells grown under Fe-supplemented conditions, Aft1-TAP is enriched in cells grown under Fe-starvation conditions, as expected (Figure 2B). Moreover, we detect occupancy by Aft1-TAP on the CTH1 promoter under both Fe-supplemented and Fe-deprived conditions (Figure 2B). No Aft1-TAP occupancy was detected on the CMD1 promoter, a gene not responsive to Fe levels, under either condition (Figure 2B). To ascertain whether Aft1 recruitment to the CTH1 promoter results in transcriptional activation, the CTH1 promoter region was fused to the coding sequence of a lacZ reporter gene (Figure 2A, top) and β-galactosidase activity was measured. As shown in Figure 2C, the wild type CTH1 promoter strongly induced expression of the lacZ reporter gene under Fe-limitation conditions in wild-type cells. This activity was decreased by ~80% in the aft1Δ strain and the remaining β-galactosidase activity was further reduced in the aft1Δaft2Δ strain to levels approximating those detected under high Fe conditions in all three strains (Figure 2C). To ascertain the potential role of the putative Aft1/Aft2-binding sites within the CTH1 promoter on transcriptional activation, both Aft1/2 consensus sites within the CTH1:lacZ fusion were mutated at nucleotide residues previously demonstrated to be required for activation via Aft1/2 (Yamaguchi-Iwai et al., 1996; Puig et al. 2005) (Figure 2A, M2 mutant). The wild type and M2 reporter genes were assayed for β-galactosidase activity in response to Fe-deprivation in wild-type cells. As shown in Figure 2D, mutagenesis of both Aft1-Aft2 consensus promoter elements severely compromised activation of the CTH1:lacZ fusion gene in response to Fe-deprivation. Immunoblotting experiments from cells grown under Fe-limitation demonstrates that a Flag-Cth1 fusion protein is detectable within 0.5hr of the imposed Fe-limitation, reaching the highest steady state levels of expression by 2hr (Figure 2E). These data demonstrate that the CTH1 promoter is transcriptionally activated in response to Fe-deficiency in a manner that is dependent on the Fe-responsive transcription factors, Aft1 and Aft2. In addition, these data suggest that the expression of Cth1 occurs early and transiently in the response to Fe-limitation.

Figure 2. CTH1 is rapidly and transiently activated by Aft1 and Aft2 during Fe-limiting conditions.

Figure 2

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A. Representation of the promoter region of CTH1 and the two putative Aft1-Aft2 binding sites, and the CTH1:lacZ fusion used for β-galactosidase assays. B. Aft1 occupancy of the CTH1 promoter was tested by chromatin immunoprecipitation (ChIP) from cells grown in medium containing either 300 µM FAS (Fe +) or 100 µM BPS (Fe −) to exponential phase. ChIP performed as described under Experimental Procedures. CMD1 was used as a negative control, and FET3 and CTH2 as positive controls. C. Aft1 and Aft2 transcription factors are required for the CTH1:lacZ reporter activation upon Fe−deficiency. Wild-type, aft1Δ and aft1Δaft2Δ cells expressing a CTH1:lacZ fusion were grown under Fe+ or Fe− conditions and β-galactosidase activity was assayed. Results from four independent experiments are shown. D. Mutations in the putative Aft1-Aft2 binding sites in the CTH1 promoter region compromise Fe deficiency-dependent activation of the CTH1:lacZ reporter in wild-type cells. Wild-type cells expressing either CTH1:lacZ or M2-CTH1:lacZ fusion were assayed as in panel C. E. Expression of the Flag-Cth1 fusion protein. Described under Experimental Procedures.

Cth1 stimulates mRNA turnover

The mRNA destabilizing activity of Cth2 requires the integrity of the two CX8CX5CX3H tandem zinc fingers (TZFs), which constitute critical Zn-coordinating elements within the mRNA-binding domain, as well as the integrity of AU-rich elements (AREs) within the 3’-UTR of target mRNAs (Puig et al., 2005). Given that Cth1 over-expression can partially rescue the cth1Δcth2Δ-associated Fe-deficiency phenotypes, including the SDH4 mRNA turnover deficit, we investigated the mechanisms by which Cth1 mediates SDH4 mRNA down-regulation. We assayed the ability of Cth1 to interact with ARE-containing mRNAs using the yeast three-hybrid method (SenGupta et al., 1996). Wild type and mutated DNA fragments encoding ARE-containing RNA from the SDH4 and ACO1 3’-UTRs (Figure 3A) were fused to bacteriophage MS2 RNA (Puig et al., 2005) and co-expressed in a reporter yeast strain with a Cth1-Gal4 trans-activation domain fusion protein. Interactions between Cth1 and the fusion RNAs were monitored by growth on medium lacking histidine (-His) and by measuring reporter gene-driven β-galactosidase activity. As shown in Figure 3B (right panel), cells co-transformed with plasmids expressing the wild type SDH4 or ACO1 3’-UTR RNA fragments fused to MS2 RNA and the Cth1-Gal4 fusion protein can grow on medium lacking histidine, indicative of an interaction between Cth1 protein and the 3’-UTR of the SDH4 and ACO1 mRNAs. This interaction is not observed when SDH4 and ACO1 mRNAs are co-expressed with a mutant form of the Cth1-Gal4 fusion protein in which cysteine residue 225 within the TZF of Cth1 has been replaced with an arginine residue (C225R mutant, Figure 1A), suggesting that the integrity of the TZFs is required for Cth1 to interact with the mRNAs (Figure 3B). To further evaluate specific binding to SDH4 mRNA, we assayed Cth1 binding to SDH4 mutant alleles in which one patch (mt1) or all ARE consensus sequences (mt2) within the SDH4 3’-UTR mRNA were mutagenized (Figure 3A). As shown in Figure 3B, cells co-expressing the Cth1-Gal4 fusion protein and SDH4-mt1 fusion RNA grow poorly in the absence of histidine as compared to cell expressing wild-type SDH4 RNA. Furthermore, mutation of all AREs within the SDH4 3’-UTR (SDH4-mt2 allele) completely abrogates growth on medium lacking histidine, without altering growth on synthetic complete medium (Figure 3B). As an independent means to assess the Cth1-SDH4 ARE interaction, β-galactosidase activity driven by the Gal4-dependent reporter gene was measured (Figure 3C). Cells co-expressing the wild-type Cth1-Gal4 protein and SDH4 RNA fusions exhibited abundant β-galactosidase activity. However, this activity was decreased by ~40% in cells expressing the SDH4-mt1 fusion RNA and by ~90% in cells expressing the SDH4-mt2 fusion RNA (Figure 3C). Very low β-galactosidase activity was detected from cells co-expressing the wild-type SDH4 fusion RNA with the Cth1 TZF mutant (Cth1 C225R-Gal4) fusion protein. These results strongly suggest that, like Cth2 (Puig et al., 2005), Cth1 specifically binds, within the 3’-UTR of the SDH4 and ACO1 mRNAs in a manner that is dependent on the integrity of the AREs and the TZF RNA binding domain.

Figure 3. Cth1 accelerates the decay of SDH4 mRNA.

Figure 3

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A. Representation of the SDH4 transcript, the location of the AREs within its 3’-UTR, and the mutations introduced in the ARE clusters (mt1 and mt2; previously described in (Puig et al., 2005)). B. Yeast three-hybrid assay used to monitor in vivo interactions between Cth1 protein and the ARE-containing fragment of the SDH4 3’-UTR mRNA. L40-coat cells were co-transformed with (1) pIIIA/MS2-1 vector alone or containing the 3’-UTR of SDH4, SDH4-mt1, SDH4-mt2, ACO1, and the iron-responsive element (IRE) as positive control, and (2) pACT2 vector alone or fused to CTH1, CTH1-C225R, and the iron regulatory protein (IRP) as a positive control. Cells were grown on SC-Ura-Leu (+His) and SC-Ura-Leu-His (-His) plates for 7 to 10 days at 30°C. Positive interactions are indicated in the box. C. Quantitation of the three hybrid results. L40-coat cells co-transformed with (1) pACT2-CTH1 and (2) pIIIA/MS-1 alone or fused to the 3’-UTR of SDH4, SDH4-mt1, SDH4-mt2 were grown on SC-Ura-Leu and assayed for β-galactosidase activity. Cells were also co-transformed with (1) pACT2-CTH1-C225R and (2) the 3’-UTR SDH4 and β-galactosidase activity was assayed. D. Cth1 accelerates the rate of decay of the SDH4 mRNA. cth1Δcth2Δsdh4Δ cells were co-transformed with either p415GAL1:SDH4 and pRS416 (vector), pRS416-CTH2:CTH1 and pRS416-CTH2:CTH1-C225R, or p415GAL1:SDH4-mt2 and pRS416-CTH2:CTH1. Cells were grown in medium containing galactose and 100 µM BPS. Glucose was added to stop SDH4 transcription and RNA analyzed by RNA blotting. SDH4 levels were normalized to ACT1 and mRNA half-lives determined from three independent experiments.

Expression of Cth2 decreases the half-life of SDH4 mRNA from ~14 to ~7 min in cells grown in low Fe (Puig et al., 2005). To ascertain if Cth1 also stimulates the turnover of SDH4 mRNA, SDH4 was conditionally expressed using the galactose-inducible and glucose-repressible GAL1 promoter in cth1Δcth2Δsdh4Δ yeast cells and co-expressed with either the CTH2:CTH1 plasmid or vector alone (Figure 3D). Cells were grown in galactose and 100 µM of the Fe-specific chelator, BPS, to induce the expression of both the GAL1-SDH4-3’-UTR and the CTH2:CTH1 transcription units, respectively. Transcription of the SDH4 gene was shut-off by addition of glucose and steady state mRNA levels were detected and quantitated over time by RNA blotting. As shown in Figure 3D, SDH4 mRNA half-life decreased from 15.5 ± 1.6 minutes to 9.1 ± 1.7 minutes when Cth1 was expressed. In contrast, there was no decrease in SDH4 mRNA half-life when the CTH1-C225R mutant allele was expressed, nor was the SDH4 mRNA half-life decreased in the presence of wild type Cth1 when all SDH4 ARE sequences were mutated (SDH4-mt2) (Figure 3D). Taken together, these data demonstrate that Cth1, like Cth2, can promote the accelerated turnover of mRNAs in a manner that is dependent upon a functional Cth1 TZF RNA binding domain and ARE sequences within the 3’-UTR of the target mRNA.

Cth1 target mRNAs mostly encode mitochondrial proteins

In response to Fe-limiting conditions Cth2 promotes the degradation of many mRNAs that encode proteins involved in Fe-homeostasis and Fe-dependent metabolism, thereby mediating a global remodeling of metabolism that optimizes growth in the presence of limited available Fe (Puig et al., 2005). Results presented here (Figure 1) suggest that Cth1 is able to partially compensate for loss of Cth2, suggesting that Cth1 and Cth2 might play at least partially overlapping roles under Fe-deficiency. The inability of Cth1 to completely rescue cth2Δ phenotypes could be due to differences in expression levels of both proteins or to functional differences.

To begin to dissect the individual contribution of Cth1 and Cth2 in the response to Fe-deficiency, we compared the genome-wide expression patterns of cth1Δcth2Δ cells expressing physiological levels of CTH1, CTH2, or vector alone. Figure 4 shows a schematic representation of the results of these experiments from triplicate Affymetrix DNA microarray studies. A total of 60 messenger RNAs were down-regulated by at least 1.5 fold in the presence of CTH1. Twenty of the 60 mRNAs down-regulated in cells expressing Cth1 harbor putative 3’-UTR ARE-sequences (Supplemental Table S1 and Figure 4). Among the 20 ARE-containing transcripts that are down-regulated by Cth1, we identified 13 that encode mitochondrial proteins, including 7 proteins involved in respiration, 3 in amino acid biosynthesis, and 3 proteins of uncharacterized function that have been localized to mitochondria. The remaining 7 ARE-containing transcripts include 2 encoding proteins involved in rRNA biosynthesis and 5 of diverse function. Down-regulation of the remaining 40 non-ARE containing mRNAs in the presence of Cth1 may be an indirect effect of Cth1 function or Cth1 binding to a non-canonical ARE. It is interesting to note that roughly 25% of these mRNAs (11 out of 40) encode proteins that have been identified as localized to the mitochondria. These results suggest that in response to Fe-deprivation Cth1 promotes the preferential down-regulation of mRNAs involved in critical Fe-dependent mitochondrial processes including respiration and amino acid biosynthesis.

Figure 4. Cth1 and Cth2 have non-identical mRNA targets.

Figure 4

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(Left) Representation of the total number of mRNAs down-regulated in cells expressing either Cth1 or Cth2, as well as the functional categories of their encoded proteins. mRNAs corresponding to 223 genes were down-regulated by at least 1.5 fold in Cth2-expressing cells and a total of 60 mRNAs in Cth1-expressing cells. The intersect shows that mRNAs from 16 genes exhibited lower steady-state levels in both Cth1- and Cth2-expressing cells. The overlapping set of genes primarily encodes mitochondrial proteins involved in respiration and amino acid biosynthesis. (Right) Representation of the subset of ARE-bearing mRNAs down-regulated in Cth1- and Cth2-expressing cells. Ninety-five mRNAs that showed decreased expression levels in the presence of Cth2 harbor ARE-sequences as well as 20 of the mRNAs down-regulated in Cth1-containing cells. Thirteen of the 16 overlapping genes contain 3’-UTR ARE-sequences.

The microarray results from Cth2-expressing cells confirm our previous observations (Puig et al., 2005) and increases the number of mRNAs likely to be direct Cth2 targets (Supplemental Table S2). A total of 223 mRNAs were down-regulated in Cth2-containing cells under Fe-limiting conditions when compared to cells harboring an empty vector (Supplemental Table S2 and Figure 4). Ninety-four of these transcripts are potentially direct targets of Cth2, as they harbor either 5’-UAUUUAUU-3’ or 5’UUAUUUAU-3’ ARE consensus sequences within their 3’-UTR. Twenty-two of the 94 ARE-containing mRNAs encode proteins involved in respiration, 9 in amino acid metabolism, 6 in heme/Fe-S cluster biosynthesis, 7 in fatty acid metabolism, 6 mitochondrial proteins and 3 encoding proteins involved in Fe homeostasis. The remaining ARE-containing mRNAs encode proteins involved in distinct cellular processes including stress responses, transport, nucleic acid metabolism and other processes (Supplemental Table S2 and Figure 4). Down-regulation of the remaining 128 non-ARE containing mRNAs could be due to secondary effects of Cth2 activity, or Cth2 binding to non-consensus AREs. Taken together, these mRNA expression data indicate that 13 of the 20 target mRNAs of Cth1 are contained within the set of mRNAs targeted by Cth2 (Figure 4 and Supplemental Table S3). Interestingly, this overlapping subset of 13 ARE-containing mRNAs, down-regulated by both Cth1 and Cth2, primarily encodes proteins involved in mitochondrial functions, including oxidative phosphorylation and amino acid biosynthesis.

Cth1/Cth2-dependent changes in carbohydrate metabolism

As a facultative anaerobe, S. cerevisiae generates ATP through both glycolysis and mitochondrial oxidative phosphorylation. Consistent with important roles for Fe in cellular metabolism, a common functional category for 11 of 13 mRNAs targeted for degradation by both Cth1 and Cth2, and which harbor consensus AREs, are established or proposed to encode proteins that play key roles in mitochondrial respiration or in other pivotal mitochondrial functions. Based on these results, Fe deprivation could lead to rapid loss of mitochondrial function via Cth1 and Cth2-stimulated mRNA degradation. Moreover, we observed that the steady state level of 17 mRNAs in CTH1-expressing cells, and approximately 40 distinct mRNAs in CTH2 wild type cells is elevated, but not in cth1Δcth2Δ cells, in response to Fe deficiency (Figure 5A and Supplementary Table S4 and S5). Among these mRNAs are those that encode proteins involved in glucose uptake (HXT5, HXT6 and HXT7, medium and high affinity glucose transporters), glycolysis (HXK1, hexokinase), reserve carbohydrate metabolism (GPH1 glycogen phosphorylase, GSY1 glycogen synthase, PGM2 phosphoglucomutase, SOL4 6-phosphogluconolactonase, YMR090W dTDP-glucose 4, 6 dehydratase), anaerobic metabolism (COX5b subunit 5b of cytochrome oxidase expressed anaerobically, ALD3 aldehyde dehydrogenase) and other functions proposed to be required for adaptation to low glucose conditions (Saccharomyces Genome Database). Furthermore, their of the genes identified as up-regulated in these microarrays, such as HSP12, are known to be induced by glucose deprivation. These experiments also identified genes involved in DNA repair and meiosis (APN1, MMS1, MSC1, REC104, SAE3), as well as genes involved in RNA metabolism (SNR46, SEN1, PPE1). These regulatory changes, and their dependency on Cth1 and Cth2, have been confirmed by RNA blotting (Figure 5B). Furthermore, this up-regulation is dependent on both Cth1 and Cth2, as it is not observed in a cth1Δcth2Δ strain (Figure 5B and data not shown). A similar pattern of up-regulation of mRNAs that function in glucose uptake, glycolysis and reserve carbohydrate metabolism is also observed in wild type cells in response to Fe deprivation or in cells defective in Fe-S cluster biogenesis (Puig et al., 2005; Hausmann et al., 2008). Taken together, these results suggest that Cth1/Cth2- mediated down-regulation of Fe-dependent metabolic pathways might lead to a secondary energy limitation due to the degradation of mRNAs encoding key mitochondrial proteins. This may result in the enhanced expression of mRNAs encoding high-affinity glucose transporters, glycolytic enzymes and factors involved in the metabolism of reserve carbohydrates.

Figure 5. Summary of mRNAs up-regulated in cells expressing either Cth1 or Cth2.

Figure 5

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A. Forty mRNAs, whose encoded proteins correspond to the indicated functional categories, showed increased levels by at least 1.5 fold in Cth2-expressing cells, and a total of 17 mRNAs in cells transformed with Cth1 during Fe-deficiency when compared to cells harboring an empty vector. The intersect represents the number of common mRNAs up-regulated between Cth1- and Cth2-expressing cells. B. Microarray validation. Wild-type and cth1Δcth2Δ cells transformed with pRS426, pRS416-CTH1 or pRS416-CTH2 were grown in medium containing either 300 µM FAS (Fe+) or 100 µM BPS (Fe−) and analyzed by RNA blotting. FET3 was used as a control for Fe- deprivation, and ACT1 as loading control. C. Representation of a partial list of genes up-regulated in CTH2 wild type cells in response to Fe-deficiency grouped by transcription factors experimentally demonstrated to regulate their expression that include Msn2/4, Rox1 and Sok2.

Fe-deficiency elicits changes in carbohydrate metabolism

Our results suggest that S. cerevisiae cells grown under Fe-limiting conditions have elevated levels of mRNAs encoding proteins involved high-affinity glucose uptake, glycolysis, reserve carbohydrate metabolism, and anaerobic metabolism, as well as mRNAs known to be highly-expressed in response to low glucose conditions (Figure 5A). We found that concomitant with the increased levels of these mRNAs, the HXT1 mRNA, encoding a low affinity glucose transporter known to be down-regulated during glucose limitation (Ozcan and Johnston, 1999), was down-regulated during Fe-starvation conditions (Supplemental Table S2). Similarly, RMD5 and VID24 mRNAs, encoding two proteins required for the ubiquitination and proteasome-dependent degradation of Fructose 1,6-BisPhophatase (FBPase) during high glucose conditions, were also found to be down-regulated under Fe-starvation conditions (Supplemental Table S2). Based on these observations, we hypothesized that Fe-deprivation might lead to a secondary glucose starvation. Additionally, a recent report suggests that cells with impaired mitochondrial Fe-S cluster assembly and export systems have increased levels of mRNAs encoding proteins involved in glucose transport (Hausmann et al. 2008).

The serine/threonine protein kinase, Snf1, is a member of the AMP-activated protein kinase family required for the adaptation to glucose starvation in yeast. During glucose limitation Snf1 is phosphorylated, thereby becoming activated and promoting the up-regulation of high affinity glucose transport systems, increased glycolysis and glycogen biosynthesis, among others activities (Celenza and Carlson, 1986; Wilson et al., 1996; Hardie et al, 1998). We ascertained the phosphorylation status of Snf1 kinase in response to Fe-deprivation by immunobloting using antibodies that specifically recognize the phosphorylated form of Snf1. This experiment revealed that Snf1 from cells grown under Fe-limited, but glucose replete, conditions is more highly phosphorylated than that from cells grown under Fe-supplemented conditions (Figure 6A). This suggests that Fe-limitation could lead to a secondary glucose deficiency, which in turn triggers the phosphorylation-mediated activation of Snf1 kinase. To examine if Fe-deficiency stimulated Snf1 phosphorylation correlates with changes in cellular carbohydrate metabolism, and whether this response is dependent on Cth1 and Cth2, we analyzed glycogen content in wild type and cth1ΔΔ cells under Fe-sufficient and Fe-deplete conditions. As shown in Figure 6B, cth1Δcth2Δ cells, or the same mutant harboring wild type CTH1 and CTH2 genes, grown under Fe-supplemented conditions, accumulate similar levels of glycogen. In contrast, under Fe-deficient conditions cells containing the wild type CTH1 and CTH2 genes accumulate approximately 7 fold higher levels of glycogen when compared to isogenic cth1Δcth2Δ cells. These results suggest that during Fe-deficiency, wild type cells both activate Snf1 kinase and hyper-accumulate glycogen, and this hyper-accumulation is dependent on the presence of Cth1 and Cth2 activity.

Figure 6. Fe-deficiency elicits changes on carbohydrate metabolism.

Figure 6

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A. Fe deficiency-dependent phosphorylation of Snf1. Snf1-HA was immunoprecipitated from cells grown in 300 µM FAS (Fe +) or 100 µM BPS (Fe −). Immunoprecipitations were analyzed by immublotting using anti-Snf1 and phospho-specific anti-Snf1 antibodies. As negative and positive controls for Snf1-HA phosphorylation, cells were also grown on 4% or 0.05% glucose medium, respectively. B. Fe deficiency-induced glycogen hyper-accumulation. Cells were grown in the presence of 300 µM FAS (Fe +) or 100 µM BPS (Fe −) and total glycogen content was analyzed as described by Parrou and Francoise (1997).

Discussion

Fe functions as a co-factor for a broad range of enzymes that are essential for normal growth, maintenance and development, and organisms must continually adapt to changes in Fe availability. Previous studies have identified regulatory mechanisms in response to Fe availability at the level of gene transcription, mRNA stability, translation, protein degradation and protein trafficking (Escolar et al., 1999; Moseley et al., 2002; Hentze et al., 2004; Lee et al., 2005; Kaplan et al., 2006; Rouault, 2006; Philpott and Protchenko, 2008). Under conditions of Fe scarcity, S. cerevisiae cells induce the expression of the Cth2 mRNA binding protein, which stimulates the turnover of many mRNAs encoding proteins that function in Fe homeostasis, storage and Fe-dependent metabolism. While cth2Δ cells exhibit a growth defect under conditions of Fe starvation and defective target mRNA turnover, the additional deletion of the CTH1 gene, encoding a homologous RNA binding protein, exacerbates these defects.

Observations reported here support a specific role for CTH1 during Fe deficiency and include the Aft1/2-dependent activation of the CTH1 promoter in response to Fe deficiency. In addition, a Flag-Cth1 fusion protein is expressed rapidly, but transiently, after cells are deprived of Fe. Preliminary observations suggest that Cth2 can bind to and promote the destabilization of the CTH1 transcript (Puig, Vergara, and Thiele, unpublished data). While this mechanism has not been explored, it could at least in part account for the decreased steady-state levels of the Cth1 protein after 2hr of Fechelation (Figure 3E; unpublished data) and suggests tight regulatory mechanisms for Cth1 expression during Fe-deficiency. Furthermore, this rapid but transient expression of Cth1 would explain why previous experiments, using prolonged Fe deficiency growth conditions, did not identify CTH1 as an Fe-responsive gene (Rutherford et al., 2003; Shakoury-Elizeh et al., 2004; Puig et al., 2005).

While at present we cannot eliminate the possibility that Cth1 and Cth2 play roles outside of Fe homeostasis, we propose that a primary function for these two proteins, may be to allow rheostatic adaptation to changes in Fe availability. Small fluctuations in Cth1 levels could be sufficient for adaptation to modest or transient Fe deficiency by first targeting mitochondrial functions. If the Fe deficiency is more severe or sustained, Cth2 is strongly induced and provides a means for more widespread metabolic changes that, together, would lead to a shift away from mitochondrial oxidative phosphorylation and toward the down-regulation of Fe storage and other Fe-dependent metabolic activities. Further experiments are required to rigorously test this hypothesis.

While both Cth1 and Cth2 are transcriptionally induced by Aft1/2, bind ARE mRNA sequences to stimulate mRNA turnover and contribute to growth under conditions of Fe deficiency, our results strongly suggest that these proteins have only partial functional overlap in the response to Fe deficiency. Analysis of the mRNA expression pattern of cth1Δcth2Δ cells expressing physiological levels of either CTH1 or CTH2 indicates that Cth1 and Cth2 share a subset of mRNA targets. Only 13 common mRNAs with recognizable AREs are down-regulated in cells expressing either Cth1 or Cth2. These targets common to both Cth1 and Cth2 include mRNAs encoding proteins involved in mitochondrial respiration and mitochondrial-localized amino acid metabolism (Table S3). It is currently unclear what determinants give rise to distinct mRNA target specificity for these two similar RNA binding proteins.

Given the critical role that Fe plays in mitochondrial oxidative phosphorylation, the results presented here suggest an additional important role for Cth1 and Cth2 in facilitating a transition between oxidative phosphorylation and glycolytic metabolism in response to Fe deficiency. Wild type cells, but not cth1Δcth2Δ cells, accumulate transcripts encoding proteins involved in glucose uptake, glycolysis and reserve carbohydrate metabolism in concert with the down regulation of those encoding proteins essential for mitochondrial oxidative phosphorylation and other critical mitochondrial processes. To begin to understand the nature of this coordinate up-regulation of these mRNAs, their cognate genes were grouped according to their validated regulation by gene-specific transcription factors. As shown in Figure 5C, of the genes annotated in the Yeastract database (36 of 40), 50% are activated by the Msn2/4 stress-responsive transcription factors, 40% by Sok2 and 36% by Rox1. Notably, Msn2/4 are known induce gene expression in response to glucose deprivation, Sok2 responds to cAMP levels and Rox1 is a heme-dependent repressor of hypoxic genes. Currently, there is no evidence supporting the notion that proteins belonging to the Cth-family can stabilize mRNAs. As such, we hypothesize that Fe deficiency that leads to a Cth1/Cth2-dependent mitochondrial inactivation, through their mRNA-destabilizing activity, would activate each of the signaling pathways that regulate the Msn2/4, Sok2, and Rox1 transcription factors, thus promoting an elevation in the steady state levels of the mRNAs encoding proteins involved in glucose metabolism (Liang-chuan et al., 2005; Ward et al., 1995; Lowry and Zitomer, 1984; Ter Linde and Steensma, 2002).

Experimental Procedures

Yeast strains and growth conditions

The AFT1-TAP strain was obtained from the TAP-tag collection (Ghaemmaghami et al., 2003) and verified by PCR and immunoblotting. The snf1Δ strain used was DTY434 (Matα ade2-101 trp1Δ1 lys2-801 ura3-52 snf1Δ). All other yeast strains used in this study have been previously described (Puig et al., 2005). For spot assays, cells were grown in synthetic complete medium (SC) minus specified nutrients to mid-exponential phase and spotted in 10-fold serial dilutions starting at A600 = 0.1 on SC or SC plus 750 µM of the membrane permeable Fe-chelator, ferrozine, to impose Fe-starvation. For liquid cultures, 100µM of the Fe-specific chelator BathoPhenanthroline diSulfonate (BPS) was used to impose Fe-deficiency, and 300µM Ferrous Ammonium Sulfate (FAS) was used to create Fe-replete conditions (cultures grown under these conditions for 6 to 8 hrs reach an OD600 = ~0.4 to 0.6). Thus, all conditions described as Fe− and Fe+ refer to the supplementation of either BPS or FAS in liquid cultures. Yeast three hybrid experiments were carried out as previously described (Puig et al., 2005).

Plasmids

The overlap extension method was used to create the CTH2 : CTH1 fusion and to create gene, promoter and 3’-UTR mutations. The coding sequences from wild type CTH2 and CTH1 were cloned into the p416TEF vector using standard cloning methods. Wild-type CTH1 and the CTH1-C225R alleles were fused to the Gal4 activation domain in the pACT2 vector and used for the yeast three-hybrid assay. Other plasmids used in this work have been previously described (Puig et al., 2005). Both the 2xFLAG-CTH1 and the CTH2 genes are under the regulation of their endogenous promoter and 3’UTRs. All plasmid inserts were verified by sequencing. Plasmid pSNF1-316 was a gift from Dr. Martin Schmidt at the University of Pittsburg.

Chromatin Immunoprecipitation

Overnight cultures of AFT1-TAP cells (Ghaemmaghami et al., 2003) were re-inoculated to A600 = 0.1 in 50 mL SC medium supplemented with either 300 µM FAS (Fe+) or 100 µM BPS (Fe−), and grown for 8 hours at 30°C. Formaldehyde crosslinking and ChIP using 500 µg of total protein was carried out as described (Keller et al., 2005) in three independent experiments. Promoter occupancy by Aft1 was ascertained by PCR using primers annealing at the promoter region of CTH1. Primers for FET3 and CTH2 promoters were used as positive controls, and CMD1 as negative controls. PCR products were visualized on 2% agarose gels using ethidium bromide.

Messenger RNA half-life determination

Cells were grown overnight in SC-Ura-Leu-Raffinose (2% raffinose; no glucose) and re-inoculated in SC-Ura-Leu-Galactose (2% galactose; no glucose) supplemented with 100 µM BPS until exponential growth phase (OD600 = ~0.4 – 0.6). Glucose was added to a final concentration of 4% to extinguish transcription of the GAL1-SDH4 fusions and aliquots were taken after glucose addition, total RNA extracted and analyzed by RNA blotting using SDH4 and ACT1 probes. SDH4 levels were quantified using a STORM 840 phosphoImager (Amersham) and normalized to ACT1 levels. The mRNA half-life was determined from at least three independent experiments.

Protein analyses

cth1Δcth2Δ cells were co-transformed with pRS415-CTH2 and pRS416-2xFLAG-CTH1. Overnight cultures were re-inoculated to A600 = 0.05 in SC-Ura-Leu and allowed to grow to A600 = 0.10, at which point 100µM BPS was added, and began time course at the indicated times. Total protein was extracted using the Triton-X 100/glass bead method, and 50 µg of total protein were resolved in a 10% SDS-PAGE and transferred onto a nitrocellulose membrane. Flag-Cth1 fusion protein was detected using an HRP-conjugated α-Flag anti-body from Sigma. An α-Pgk1 antibody was used as loading control. Two chemiluminescence substrates were used for the detection of the HRP-conjugated anti-body, Pico and Fempto (Pierce). To detect Snf1-phosphorylation, DTY434 cells (snf1Δ) were transformed with plasmid pSNF1-316 (SNF1-3HA) or vector alone, and grown in SC-ura overnight. Overnight cultures were re-inoculated to A600 = 0.2 in SC-Ura medium supplemented with either 300 µM FAS or 100 µM BPS and grown for 6 hrs at 30°C. Protein extractions were done using ice-cold RIPA buffer and glass beads supplemented with protease inhibitors (Roche mini-tablet) and phosphatase inhibitors (30mM Na4P207, 50mM NaF, 100uM Na3VO4, 25mM β-glycerolphosphate). 500ug of total protein were used to immunoprecipitate Snf1-HA using 20µL of monoclonal anti-HA agarose conjugate beads (Sigma), using a standard IP protocol. Immunoprecipitated samples were fractionated in 10% SDS-PAGE gels, transferred onto nitrocellulose membranes, and phosphospecific anti-bodies against Snf1 were used (anti-PT210 (M. Schmidt) or anti-P-AMPKalpha (Cell Signaling)) as well as anti-Snf1 (yK-16 Santa Cruz) as loading control. For positive and negative controls of Snf1 phosphorylation, additional cultures were grown in SC-ura medium containing either 4% or 0.05% glucose, respectively.

Microarray analyses

cth1Δcth2Δ cells independently transformed with pRS416, pRS416-CTH1 or pRS416-CTH2 were grown in triplicate in SC-Ura containing 100 µM BPS until exponential growth phase, RNA was extracted, labeled and hybridized to Yeast Genome S98 Afffymetrix arrays. For further information about sample preparation, synthesis of labeled cDNA, hybridization, scanning and data acquisition, and quality control steps, visit the Duke Microarray Core Facility at http://www.genome.duke.edu/cores/microarray/. All data were analyzed using GeneSpring 6.1 (Silicon Genetics), using volcano plot with minimum of 1.5 fold change and a p-value < 0.05. Microarray data have been deposited in NCBI’s Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE11236.

Glycogen measurements

cth1Δch2Δ mutants were transformed with vectors (pRS416 + pRS415) or with both CTH (pRS416-CTH1 + pRS415-CTH2), inoculated at OD=0.1 in SC-ura-leu and grown for 8 hours in medium containing either 300µM FAS or 100µM BPS. Extraction and glycogen determination was performed as described in Parrou and Francoise (1997).

Supplementary Material

01

Acknowledgements

We are grateful to Eric Askeland for outstanding technical assistance. We thank Drs. P. Blaiseau, and M. Wickens for generously providing yeast strains and plasmids used in this study, Dr. Holly Dressman from the Duke Microarray Core Facility, Drs. Jack Keene and Emilia Matallana for helpful discussions and comments on the manuscript. Dr. Martin Schmidt from the University of Pittsburg for generously providing the SNF1-HA plasmid and the PT210 antibody. S.P. is the recipient of a Ramón y Cajal contract with the Universitat de València, Spain. This work was supported by a grant from the Spanish Ministerio de Educación y Ciencia (BIO2005-07120) and FEDER funds from the European Community to S.P., NIH grant GM41840 from the National Institutes of Health to D.J.T., and NIH pre-doctoral fellowship FDK081304A to S.V.V., a trainee of the Duke University Graduate Program in Genetics and Genomics.

Footnotes

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