Regulation of Ribonucleotide Reductase in Response to Iron Deficiency Nerea Sanvisens, M. Carmen Bañó, Mingxia Huang, Sergi Puig  Molecular Cell  Volume 44, Issue 5, Pages 759-769 (December 2011) DOI: 10.1016/j.molcel.2011.09.021 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Yeast dATP and dCTP Levels Increase upon Nutritional or Genetic Iron Deficiency Wild-type BY4741 and fet3Δfet4Δ cells were grown for 7 hr in SC (Fe) or SC with 100 μM BPS (–Fe), and dATP and dCTP levels were determined by the DNA polymerase-based enzymatic assay. The values of dATP and dCTP are represented as relative to those of the wild-type strain grown under Fe-sufficient conditions. The average and standard deviation of at least three independent experiments is represented. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Yeast Rnr2 and Rnr4 Redistribute to the Cytoplasm in Response to Nutritional or Genetic Fe Deficiencies (A and B) Wild-type BY4741 cells were grown for 6 hr in SC (Fe) or SC with 100 μM BPS (–Fe). Then DNA was stained with 1 μg/ml DAPI for 3 min, and Rnr2 and Rnr4 localization determined by IMF with anti-Rnr2 (A) and anti-Rnr4 (B) antibodies, respectively. An overlap of DAPI and IMF signals is also shown (Merge). (C and D) Quantitative analysis of Rnr2 (C) and Rnr4 (D) subcellular localization patterns in wild-type cells grown as indicated in (A) and (B). Percentages of cells with distinct localization patterns were represented as follows: black bars, cells with a predominantly nuclear IMF signal; gray bars, cells with both nuclear and cytoplasmic IMF signal; and white bars, cells with a predominantly cytoplasmic IMF signal. (E and F) Quantitation of Rnr2 (E) and Rnr4 (F) subcellular localization patterns in fet3Δfet4Δ cells grown for 6 hr in SC (Fe) or SC with 100 μM FAS (+Fe). Cells were processed and the results represented as indicated in (C) and (D). In all cases, at least 200 cells were counted for each independent experiment performed by triplicate. The average and the standard deviation are represented. See also Figure S1. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 The Mec1 and Rad53 Kinases Do Not Participate in the Redistribution of Rnr2 and Rnr4 to the Cytoplasm in Response to Fe Deficiency The phosphorylation stage of Rad53 (A) and Dun1 (B) proteins was determined. Wild-type BY4741 (A) and Dun1-MYC13 (B) cells were grown on SC without (Fe) or with 100 μM BPS (–Fe) for 6 hr, SC with 0.04% MMS for 2 hr, or SC with 0.2 M HU for 2 hr. Proteins were extracted and analyzed by western blotting with anti-Rad53 (A) and anti-c-Myc (B) antibodies. Pgk1 was used as a loading control. Wild-type Y300 (C and D), mec1Δsml1Δ (E and F), and rad53Δsml1Δ cells (G and H) were grown as described in panel A, and cells were processed and the data analyzed as described in Figure 2. See also Figure S2. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Cells Defective in CTH1 and CTH2 Exhibit a Defect in the Redistribution of Rnr2 and Rnr4 to the Cytoplasm in Response to Fe Deficiency cth1Δcth2Δ cells cotransformed with pRS416-CTH1 and pRS415-CTH2 (CTH1 + CTH2; A), pRS416 and pRS415 (cth1Δ + cth2Δ; B), or pRS416-CTH1-C225R and pRS415-CTH2-C190R (CTH1-C225R + CTH2-C190R; C), were grown for 6 hr in SC (Fe) or SC with 100 μM BPS (–Fe), and then processed and the data analyzed as described in Figure 2. See also Figure S3. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Cth1 and Cth2 Promote the Downregulation of Wtm1 Levels in Response to Fe Deficiency cth1Δcth2Δ cells cotransformed with pRS416 and pRS415 (vector + vector), pRS416-CTH1 and pRS415-CTH2 (CTH1 + CTH2), or pRS416-CTH1-C225R and pRS415-CTH2-C190R (C225R + C190R), were grown for 8 hr in SC-ura-leu (+) or SC-ura-leu with 100 μM BPS (–). (A) Total RNA was extracted and analyzed by northern blotting with a WTM1 specific probe. SCR1 was used as a loading control. (B) Proteins were extracted and analyzed by western blotting with anti-c-Myc. Ponceau staining was used as a loading control. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Cth1 and Cth2 Proteins Specifically Interact with WTM1 mRNA to Promote Rnr2 and Rnr4 Redistribution to the Cytoplasm and Sustain RNR Function in Response to Fe Deficiency (A) The Y3H assay was used to monitor in vivo interactions between Cth1 or Cth2 proteins and the ARE-containing fragment of the WTM1 3′ UTR mRNA. L40-coat cells were cotransformed with (1) pIIIA/MS2-1 containing the 3′ UTR of WTM1, WTM1-mt1, WTM1-mt2, WTM1-mt3, SDH4 (as a positive control), or vector alone (as a negative control) and (2) pACT2 vector alone or fused to CTH1, CTH1-C225R, CTH2, or CTH2-C190R. Cells were grown on SC-ura-leu (+His), and SC-ura-leu-his (–His) containing 250 or 750 μM 3-aminotriazol (3-AT) plates for 2–6 days at 30°C. (B) Yeast cells mutagenized in both WTM1 ARE patches exhibit a defect in the redistribution of Rnr2 and Rnr4 to the cytoplasm in response to Fe deficiency similar to CTH1- and CTH2-defective cells. WTM1-mt3 cells were grown for 6 hr in SC (Fe) or SC with 100 μM BPS (–Fe), and then processed and the data analyzed as described in Figure 2. (C) Determination of dATP and dCTP levels in cth1Δcth2Δ and WTM1-mt3 cells. Yeast cells were grown for 7 hr in SC (Fe) or SC with 100 μM BPS (–Fe), and analyzed as described in Figure 1. The values of dATP and dCTP from the wild-type strain, previously shown in Figure 1, have been included for clarity. See also Figure S4. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 Cth1 and Cth2 Control the Levels of Rnr2 and Rnr4 Subunits in Response to Iron Deficiency The levels of Rnr2 and Rnr4 mRNAs (A) and proteins (B) were determined in cth1Δcth2Δ cells cotransformed, grown, and processed as detailed in Figure 5. The levels of RNA and protein were quantified and normalized to SCR1 (RNA loading control) and Pgk1 (protein loading control), respectively. The Y3H assay was used to monitor in vivo interactions between Cth1 and Cth2 (C) proteins and the ARE-containing fragment of the RNR1 and RNR2 3′-UTR mRNAs. L40-coat cells were cotransformed with (1) pIIIA/MS2-1 containing the 3′ UTR of RNR2, RNR4, SDH4, and vector alone and (2) pACT2 vector alone or fused to CTH1, CTH1-C225R, CTH2, or CTH2-C190R. Cells were grown as described in Figure 6. See also Figure S5. Molecular Cell 2011 44, 759-769DOI: (10.1016/j.molcel.2011.09.021) Copyright © 2011 Elsevier Inc. Terms and Conditions