A chemical potentiator of copper-accumulation used to investigate the iron-regulons of Saccharomyces cerevisiae

Abstract

The extreme resistance of Saccharomyces cerevisiae to copper is overcome by 2-(6-benzyl-2-pyridyl)quinazoline (BPQ), providing a chemical-biology tool which has been exploited in two lines of discovery. First, BPQ is shown to form a red (BPQ)2 Cu(I) complex and promote Ctr1-independent copper-accumulation in whole cells and in mitochondria isolated from treated cells. Multiple phenotypes, including loss of aconitase activity, are consistent with copper-BPQ mediated damage to mitochondrial iron-sulphur clusters. Thus, a biochemical basis of copper-toxicity in S. cerevisiae is analogous to other organisms. Second, iron regulons controlled by Aft1/2, Cth2 and Yap5 that respond to mitochondrial iron-sulphur cluster status are modulated by copper-BPQ causing iron hyper-accumulation via upregulated iron-import. Comparison of copper-BPQ treated, untreated and copper-only treated wild-type and fra2Δ by RNA-seq has uncovered a new candidate Aft1 target-gene (LSO1) and paralogous non-target (LSO2), plus nine putative Cth2 target-transcripts. Two lines of evidence confirm that Fra2 dominates basal repression of the Aft1/2 regulons in iron-replete cultures. Fra2-independent control of these regulons is also observed but CTH2 itself appears to be atypically Fra2-dependent. However, control of Cth2-target transcripts which is independent of CTH2 transcript abundance or of Fra2, is also quantified. Use of copper-BPQ supports a substantial contribution of metabolite repression to iron-regulation.

Fig. 1

BPQ potentiates copper toxicity toward S. cerevisiae. Growth yield following culture (9 h) of wild type S. cerevisiae in liquid YPAD supplemented with increasing concentrations of CuSO₄ in the presence (closed symbols) and absence (open symbols) of 4 μM BPQ. Mean values from three cultures (± SD). Inset, liquid YPAD supplemented with 0 (−) or 5 (+) mM CuSO₄.

Fig. 2

BPQ forms a red 2:1 complex with Cu(I).A. Absorbance at 505 nm (apo-subtracted) of an aerobic solution of BPQ (80 μM) upon addition of CuSO₄ (160 μM).B. Apo-subtracted UV-vis spectra obtained upon titration of BPQ (80 μM) with Cu(I). Inset, solution of BPQ (80 μM) and Cu(I) (160 μM).C. Binding isotherm of the feature at 505 nm shown in B (triangles) and the binding isotherm produced in an analogous experiment performed in liquid YPAD rather than buffer (circles). Cu(I) in B and C was produced by the hydroxylamine method.

Fig. 3

Crystal structure of the (BPQ)₂Cu(I) complex.A. Chemical formula of BPQ.B. Ball-and-stick representation of the crystal structure of the (BPQ)₂Cu(I) complex solved at 1 Å resolution. Carbon atoms are shown in grey, nitrogen atoms in blue and copper in red. H-atoms are omitted for clarity.

Fig. 4

Copper-BPQ mediates a hyper-accumulation of copper.A. Copper content of wild type (5 h) in liquid YPAD supplemented with 100 μM CuSO₄, 1.7 μM BPQ, both or neither as indicated. Inset, expanded y-axis for selected conditions.B. Mitochondrial copper content of wild type (5 h) in liquid YPAD supplemented with or without 100 μM CuSO₄ and 1.7 μM BPQ as indicated. Mean values from three cultures (plus SD).

Fig. 5

Copper-BPQ mediates a hyper-accumulation of copper and iron in ctr1Δ and BPQ can be detected in the mitochondria of copper-BPQ treated cells.A. Growth of ctr1Δ cells cultured in liquid YPAD supplemented with (closed symbols) or without (open symbols) BPQ (1.7 μM) and CuSO₄ (100 μM). Mean values obtained from three cultures (± SD).B. Copper content of ctr1Δ (5 h) in liquid YPAD supplemented with 100 μM CuSO₄, 1.7 μM BPQ, both or neither as indicated.C. Iron content of ctr1Δ cultured as in B. Mean values from three cultures (plus SD).D. BPQ (1 mM, 2 μl in methanol) LC-MS.E. LC-MS of extract from mitochondria isolated from cells cultured in 100 μM CuSO₄ and 1.7 μM BPQ (5 h).F. LC-MS of mitochondrial extract from untreated cells.

Fig. 6

BPQ mediates hyper-accumulation of iron but zinc is unaltered.A. Iron content of wild type (5 h) in liquid YPAD supplemented with 100 μM CuSO₄, 1.7 μM BPQ, both or neither as indicated. Inset, expanded y-axis for selected conditions.B. Mitochondrial iron content of wild type (5 h) in liquid YPAD supplemented with or without 100 μM CuSO₄ and 1.7 μM BPQ as indicated.C. Zinc content of wild type cultured as in A. Mean values from three cultures (plus SD).

Fig. 7

Iron hyper-accumulation depends on iron uptake pathways and is symptomatic of copper-BPQ toxicity.A. Growth of wild-type, fet3Δ and ccc2Δ cells cultured in liquid YPAD supplemented with (closed symbols) or without (open symbols) BPQ (1.7 μM) and CuSO₄ (100 μM). Mean values obtained from three cultures (± SD).B. Copper content of fet3Δ (5 h) in liquid YPAD supplemented with 100 μM CuSO₄, 1.7 μM BPQ, both or neither as indicated.C. Iron content of fet3Δ cultured as in B.D. Copper content of ccc2Δ cultured as in B.E. Iron content of ccc2Δ cultured as described in B. Insets show selected data from the main panels with an expanded y-axis. Mean values from three cultures (plus SD).

Fig. 8

Phenotypes of cells treated with copper-BPQ imply damage to iron–sulphur clusters.A. Transcript abundance of Aft1/2 targets (ARN1, FET3, CCC2, MRS4) and Yap5 targets (GRX4, CCC1, TYW1) in a common population of RNA collected from wild type cells (5 h) in liquid YPAD supplemented with 1 μM BPQ, 100 μM CuSO₄, neither or both as indicated. Analysis was performed by RT-PCR with ACT1 loading control.B. Growth yield of wild type after 12 h in liquid minimal medium supplemented with 40 μg ml⁻¹ l-lysine, 40 μg ml⁻¹ d-lysine or neither as indicated. Cultures were additionally treated with (closed bars) or without (open bars) 1.7 μM BPQ and 100 μM CuSO₄. Mean values from three cultures (plus SD).C. Specific aconitase activity in wild type cells (5 h) in liquid YPAD supplemented with 100 μM CuSO₄, 1.7 μM BPQ, both or neither as indicated. Mean values from three cultures (± SD).D. Tenfold serial dilution of wild type cultures on solid YPAD or YPAG supplemented with or without 2.7 μM BPQ and 100 μM CuSO₄ as indicated.E. Tenfold serial dilution of wild type on solid YPAD supplemented with 4 μM BPQ and 100 μM CuSO₄ as indicated before growth under aerobic or anaerobic conditions.

Fig. 9

The transcriptional fingerprint following copper-BPQ is consistent with high copper and functional iron deficiency. Fold change in transcript abundance in wild type (5 h) in liquid YPAD supplemented with 1.7 μM BPQ and 100 μM CuSO₄ (x-axis) or 100 μM CuSO₄ alone (y-axis), relative to untreated cells. Each data point represents the mean change in transcript abundance, as determined by RNA-seq, and genes are included based on > 2-fold response to copper-BPQ (Tables S1 and S2). Colours represent membership of Ace1 and Mac1 regulons (copper responsive, blue) or Aft1/2 and Cth1/2 regulons (iron responsive, red), upregulation in response to high metal (triangle), downregulation (inverted triangle). Newly identified candidate Aft1/2 and Cth1/2 targets (open red symbols), genes for which metalloregulation is unknown (grey circles). TSA2 is additionally shown. The red and blue lines are best fit through iron and copper regulated genes respectively.

Fig. 10

Yap5 targets respond to copper-BPQ and iron is elevated in fra2Δ.A. Abundance of Yap5 target transcripts (GRX4, CCC1, TYW1) determined by RNA-seq in RNA populations isolated from wild type in liquid YPAD supplemented with (closed bars) or without (open bars) 1.7 μM BPQ and 100 μM CuSO₄ for 5 h. Mean values obtained from three cultures (plus SD).B. Iron content of selected strains in YPAD (means from three cultures plus SD).C. Iron content (expressed as atoms per total cell volume) of fra2Δ (grey) in liquid YPAD supplemented with 1.7 μM BPQ and 100 μM CuSO₄ compared with wild type (open).D. As ‘C’ with msn5Δ (grey). Means from three cultures (± SD).

Fig. 11

FRA2-dependent and -independent Aft target gene regulation.A. Fold-change in transcript abundance (determined by RNA-seq) of representatives of the Aft1/2 regulons (> 2-fold upregulated on treatment of wild type cells with copper-BPQ) in fra2Δ in liquid YPAD in the presence (closed bars) or absence (open bars) of 1.7 μM BPQ and 100 μM CuSO₄ for 5 h, relative to untreated wild type. Numbers in parenthesis represent the fractional change in transcript abundance in fra2Δ untreated cells relative to fra2Δ copper-BPQ treated cells. Inset, fold-change in transcript abundance of representatives of the Cth2 regulon (> 2-fold downregulated on treatment of wild type cells with copper-BPQ) in the same RNA populations. Mean values from three cultures (plus SD).B. Mechanisms of iron regulation. Fra1/2, Grx3/4 an Fe-S cluster plus ‘compound X’ withhold Aft1/2 from DNA (1). Yap5 responds to the production of the iron-sufficiency signal ‘X’ by activating the transcription of GRX4 (2). Grx3/4, with or without Fra2, associate with Aft1 in the nucleus to withhold Aft1/2 from DNA (3). Cth1/2 encourage degradation of transcripts containing 3′-UTR ARE (4). Reduction in flux through Fe-S dependent pathways downregulates transcripts as an iron-sparing mechanism (5).