Iron regulation through the back door: iron-dependent metabolite levels contribute to transcriptional adaptation to iron deprivation in Saccharomyces cerevisiae

Abstract

Budding yeast (Saccharomyces cerevisiae) responds to iron deprivation both by Aft1-Aft2-dependent transcriptional activation of genes involved in cellular iron uptake and by Cth1-Cth2-specific degradation of certain mRNAs coding for iron-dependent biosynthetic components. Here, we provide evidence for a novel principle of iron-responsive gene expression. This regulatory mechanism is based on the modulation of transcription through the iron-dependent variation of levels of regulatory metabolites. As an example, the LEU1 gene of branched-chain amino acid biosynthesis is downregulated under iron-limiting conditions through depletion of the metabolic intermediate alpha-isopropylmalate, which functions as a key transcriptional coactivator of the Leu3 transcription factor. Synthesis of alpha-isopropylmalate involves the iron-sulfur protein Ilv3, which is inactivated under iron deficiency. As another example, decreased mRNA levels of the cytochrome c-encoding CYC1 gene under iron-limiting conditions involve heme-dependent transcriptional regulation via the Hap1 transcription factor. Synthesis of the iron-containing heme is directly correlated with iron availability. Thus, the iron-responsive expression of genes that are downregulated under iron-limiting conditions is conferred by two independent regulatory mechanisms: transcriptional regulation through iron-responsive metabolites and posttranscriptional mRNA degradation. Only the combination of the two processes provides a quantitative description of the response to iron deprivation in yeast.

Fig. 1.

Aft1 alone plays no significant role in the downregulation of LEU1, CYC1, and HEM15. (A) Northern blot analysis of LEU1, CYC1, and HEM15 transcription. Total RNA was isolated from exponentially growing wild-type cells (WT) cultivated under iron-replete conditions in the presence of 50 μM FeCl₃ (+Fe) or under iron-deprived conditions in the presence of 50 μM bathophenanthroline for 16 h (−Fe) and from Atm1- or Yah1-depleted Gal-ATM1 (ATM1) or Gal-YAH1 (YAH1) cells grown under iron-replete conditions in synthetic complete minimal (SD) medium. RNA was separated on agarose gels, blotted onto nylon membranes, and hybridized with ³²P-labeled probes. ACT1 served as a loading control (20). (B) Cell extracts of wild-type cells cultivated for 16 h in the presence of 50 μM FeCl₃ (+Fe) or 50 μM bathophenanthroline (−Fe) and wild-type cells overproducing Aft1p from vector p424MET3-AFT1 (AFT1↑) were analyzed for Leu1 activity. The inset shows an immunostaining for Leu1 and Por1 in wild-type and Aft1-overproducing cells cultivated under iron-replete and iron-deprived conditions. (C) Mitochondria were isolated from iron-replete, iron-starved, or Aft1-overproducing wild-type cells (AFT1↑). Protein levels of cytochrome c (Cyc1) and ferrochelatase (Hem15) were assessed by immunostaining. An immunostaining for Isa2 served as a loading control. (D) Ferrochelatase activities of mitochondria were determined by monitoring the insertion of Zn²⁺ into protoporphyrin IX. (E) The wild-type strain (W303A), the Atm1-depleted Gal-ATM1 strain (ATM1), and the W303 wild-type strain overproducing Aft1 from the methionine-regulated vector p424MET3-AFT1 harboring reporter plasmid pFET3-GFP were cultivated under iron-replete and iron-depleted conditions. Aft1-overproducing cells were cultivated under repressive conditions in the presence of methionine (AFT1↓) or under inducing conditions in the absence of methionine (AFT1↑). FET3 promoter activities were determined by measuring the GFP-specific fluorescence emission of logarithmically grown cells. Error bars indicate the standard errors of the means (n ≥ 4).

Fig. 2.

Both promoter and terminator mediate the iron-responsive expression of LEU1. (A) W303A wild-type cells cultivated for 24 h in the presence of 50 μM FeCl₃ (+Fe) or 50 μM bathophenanthroline (−Fe) and Atm1-, Ssq1-, or Yah1-depleted Gal-ATM1, Gal-SSQ1, and Gal-YAH1 cells harboring reporter plasmid pLEU1-Luc2 (containing the CYC1 terminator; see box on top) were grown to mid-log phase. The luciferase-derived luminescence of clarified cell extracts was quantified. (B) Wild-type (W303A) and Aft1-overproducing cells (AFT1↑) harboring reporter plasmid pLEU1/LEU1-term were cultivated for 24 h under iron-replete and iron-depleted conditions, and the luciferase activity of cell extracts was quantified. (C) luc2 transcript levels from the MET25 promoter under iron-replete and iron-limiting conditions were determined in wild-type (W303) cells. The reporter plasmids harboring the luciferase gene contained either no terminator (pMET25-Luc2, none), or the 3′ UTRs (term) of LEU1 (pMET25/LEU1-term) or CYC1 (p416MET25-luc2), respectively. (D) Wild-type (BY4742) and the respective cth1Δ, cth2Δ, and cth1Δ/cth2Δ (cth1/2Δ) deletion strains were transformed with the reporter plasmid pLEU1/LEU1-term. Cells were cultivated under iron-replete and iron-depleted conditions for 24 h, and luc2 transcript levels were determined. Error bars indicate the SEM (n ≥ 4). ORF, open reading frame.

Fig. 3.

Iron-responsive transcription of LEU1 is mediated by cellular levels of α-isopropylmalate. (A) Wild-type cells (BY4742) were transformed with luciferase-based reporter plasmids harboring truncated versions of the LEU1 promoter as indicated (left), and luciferase activities of cell extracts were quantified (right). (B) LEU1 expression was determined in wild-type cells (W303) cultivated under iron-replete and iron-depleted (−Fe) conditions for 24 h and in the leu3Δ deletion strain (BY4742) harboring reporter plasmid pLEU1/LEU1-term (see box on top). (C) LEU1 expression was determined in strains (W303) carrying deletions of the indicated genes of the biosynthetic pathway for leucine, isoleucine, and valine; a W303 strain harboring a wild-type LEU2 allele (LEU2); and the leu3Δ strain (BY4742) (Fig. 4C). Cells were transformed with plasmid pLEU1/LEU1-term (box on top) and cultivated under iron-replete and iron-depleted conditions for 24 h. (D) Enzyme activities of the yeast dihydroxy-acid dehydratase Ilv3 were determined in mitochondria isolated under anaerobic conditions from wild-type cells cultivated under iron-replete and iron-depleted conditions. (F) Wild-type (BY4741) and ilv3Δ cells harboring a genomic copy of a terminatorless LEU1 gene with a C-terminally fused GFP were cultivated under iron-replete and iron-depleted conditions for 24 h. Promoter activities were determined by measuring the GFP-specific fluorescence emission of logarithmically grown cells. (F) LEU1 expression was determined in leu4/9Δ cells cultivated in SD medium supplemented with increasing amounts of α-IPM under iron-replete or iron-depleted conditions. Cells contained reporter plasmid pLEU1/LEU1-term. Error bars indicate the SEM (n ≥ 4). ORF, open reading frame; WT, wild type.

Fig. 4.

Dual regulation of the iron responsiveness of LEU1. (A) The ratio of LEU1 expression under iron-replete conditions to that under iron-depleted conditions in the indicated strains as derived from Fig. 3C (right panel) was compared with that of a LEU1 core promoter construct (pLEU1^P-175/LEU1-term) in W303 wild-type cells. (B) Expression levels of the LEU1 core promoter were determined under iron-replete (+Fe) and iron-depleted (−Fe) conditions in wild-type cells (BY4742) and the cth1Δ, cth2Δ, and cth1Δ/cth2Δ (cth1/2Δ) deletion strains harboring reporter plasmid pLEU1^P-175/LEU1-term (box on top). (C) Model for the iron-responsive expression of LEU1 in S. cerevisiae. Abbreviations: α,β-DIV, α,β-dihydroxyisovalerate; α-KIV, α-ketoisovalerate; β-IPM, β-isopropylmalate; α-KIC, α-ketoisocaproate. WT, wild type; ORF, open reading frame.

Fig. 5.

The iron responsiveness of CYC1 is mediated by transcription and Cth1/Cth2-dependent mRNA degradation. (A) Model for the CYC1 promoter (17). (B and C) The wild-type strain (BY4742) and the cth1Δ, cth2Δ, and cth1Δ/cth2Δ (cth1/2Δ) isogenic deletion strains were transformed with the reporter plasmid pΔUAS1/2-hRluc (B) or pCYC1-hRluc (C) (boxes on top). Cells were cultivated under iron-replete (+Fe) and iron-depleted (−Fe) conditions in the presence of raffinose for 24 h, and the Renilla luciferase level was determined in cell extracts. Error bars indicate the SEM (n ≥ 4). ORF, open reading frame.

Fig. 6.

Hap1 and Hap4 are involved in the iron-responsive expression of CYC1. (A) Wild-type (BY4742) cells and the hap1Δ and hap2Δ isogenic deletion strains were transformed with reporter plasmid pCYC1-hRluc, pΔUAS1-hRluc, or pΔUAS2-hRluc carrying the indicated CYC1 promoters (Fig. 5A). Cells were cultivated under iron-replete (+Fe) and iron-depleted (−Fe) conditions for 24 h, and the Renilla luciferase-derived luminescence was determined. (B) Expression of the different CYC1 promoter constructs was determined in hap4Δ and wild-type cells overproducing Hap4 (Hap4↑) as described for panel A. (C) The ratio of the iron-responsive expression was calculated from the quotas of the expression of the different CYC1 promoter constructs under iron-replete and iron-deprived conditions for the indicated strains in panels A and B. (D) Wild-type cells expressing the C. elegans heme importer CeHRG4, which harbored the reporter plasmid pCYC1-Luc2, were cultivated under iron-depleting conditions and supplemented with increasing amounts of hemin for 16 h, and the luciferase-derived luminescence was determined. Error bars indicate the SEM (n ≥ 4). ORF, open reading frame.