1. Volume 19, Issue 2, Pages 171-181 (July 2005)
Involvement of Heme Regulatory Motif in Heme-Mediated Ubiquitination and Degradation of IRP2 
Haruto Ishikawa, Michiko Kato, Hiroshi Hori, Koichiro Ishimori, Takayoshi Kirisako, Fuminori Tokunaga, Kazuhiro Iwai 
Molecular Cell 
Volume 19, Issue 2, Pages (July 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 Heme Synthesis Induces IRP2 Downregulation
(A) IRP2 levels correlate inversely with heme content. RD4 cells were treated with 100 μg/mL FAC (lane 2) or 100 μM Df + 5 mM SA (lane 3) for 16 hr. After removing the media, the Df + SA-treated cells were then treated for 6 hr with 100 μM Df (D) or 5 mM SA (S), or with 100 μg/mL FAC (F) or 20 μM hemin (H) in the presence or absence of 5 mM SA, as indicated in lanes 4–9. The cells were lysed, as were untreated cells (lane 1), and 50 μg of the lysates were subjected to 6% SDS-PAGE followed by immunoblotting with anti-IRP2 antibody.
(B) Heme content of RD4 cells. RD4 cells were treated as indicated in Figure 1A (cells treated with H or H + SA were excluded) or treated with 100, 200, and 500 μg/mL FAC (F) for 16 hr without any pretreatment. The data represent the mean ± SD for three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 The IDD Domain Is Required for IRP2 Degradation
(A) Schematic representation of IRP1 and IRP2. The amino acid sequence of the IDD domain and the HRM (underlined) are shown.
(B and C) Effects of Df or hemin on the stability of IRP2-myc or its mutants. (B) RD4 cells were induced by DEX to express IRP2 or its mutants. Two clones with different expression levels were chosen to study IRP2-73 (IRP2-73 Cl.1 and Cl.2) and IRP2-79 (IRP2-79 Cl.1 and Cl.2). Cells were treated 100 μM Df (D) or 20 μM hemin (H) for 12 hr. (C) 293 cells were induced by DEX to express IRP2 or IRP2-73. Cells were treated with 100 μM Df (D) or 50 μM hemin (H) for 6 hr. The cells were harvested and subjected to immunoblot analysis with anti-myc or anti-tubulin antibodies.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Effect of the Cys and/or His in the IDD Domain on the Heme-Mediated Degradation of IRP2
(A and B) Various heme-protein complexes were generated in vitro from purified IRP proteins, and their absorption spectra were recorded between 250 and 800 nm using a 3 μM protein solution. Shown are the absorption spectra of IRP2/hemin (solid line) and IRP2-73/hemin (dotted line) (A), and IRP1/hemin (solid line) and IRP1+73/hemin (dotted line) (B).
(C and D) Mutations of both Cys201 and His204 to Ala stabilize IRP1+73 and IRP2. RD4 cells were induced by DEX to express mutant IRP1+73 (C) or IRP2 (D). After a 12 hr treatment with 100 μM Df (D) or 20 μM hemin (H), the cells were lysed, and 50 μg of the lysates were subjected to 6% SDS-PAGE and immunoblot analysis with anti-IRP2 (C) or anti-myc antibody (D). Band intensity was quantified by a LAS3000 Bioimaging analyzer and expressed relative to the band intensity of cells treated with Df, which was set at 1.0.
(E) IRP2 C201AH204A was stable in hemin-treated cells. Pulse chase analysis of IRP2 and IRP2 C201AH204A in RD4 cells treated with 100 μM Df (D) or 20 μM hemin (H) was performed as described in the Experimental Procedures. The closed and open squares represent the hemin- or Df-treated cells, respectively. The residual radioactivities in the graphs were calculated from three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Spectroscopic Features of Hemin-Treated MBP-IDD
(A and B) Absorption spectra of wt (solid line) and C201A (dotted line) MBP-IDD (A) and of H204A (solid line) and C201AH204A (dotted line) MBP-IDD (B). The absorption spectra were measured between 250 and 800 nm using a 10 μM protein solution.
(C) Absorption spectra of 5 μM hemin after MBP-IDD is added at varying amounts up to a 2 mol equivalent of the hemin amount. The dotted line represents the free hemin.
(D) Titration curves of hemin after increasing amounts of MBP-IDD were added, as represented by the absorbance peak at 370 nm.
(E and F) EPR spectra of heme bound IRP2 (trace A) and IRP1+73 (trace B) at 15K (E) and 5K (F). EPR spectrum measurements were carried out at the X-band (9.22 GHz) microwave frequency.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Absorption Spectra of Heme Bound IRP2 and MBP-IDD under Reducing Conditions
(A–D) Absorption spectra of ferrous (solid line) and ferrous-CO (dotted line) heme bound IRP2 (A), wt MBP-IDD (B), and H204A MBP-IDD (C), and absorption spectra of 10 μM free heme (D). Absorption spectra were measured between 250 and 800 nm using 10 μM protein solutions. An asterisk indicates an absorption peak due to the presence of dithionite.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 Effect of Mutations in the HRM on Heme-Mediated Degradation
(A) IRP1+73 HAP was not decreased by hemin. IRP1+73 and its mutants in RD4 cells treated with 100 μM Df (D) or 20 μM hemin (H) for 12 hr, or untreated (−), were probed with anti-IRP2 antibody.
(B) HOIL-1 recognizes wt IDD, but not IDD HAP or IDD C201AH204A, in yeast two-hybrid analysis. AH109 cells carrying pGBKT7 (vector), pGBKT7-IDD (IDD wt), or IDD mutants (IDD C201AH204A or IDD HAP) together with pGADT7-HOIL-1 were plated onto SD plates lacking LW (left), or onto SD plates lacking AHLW but containing 70 mM 3AT, 200 μM FAS, and 0.5 μg/ml dpIX.
(C and D) Absorption spectra for ferric heme bound MBP-IDD HAP (C) and of MBP-IDD HAP containing ferrous (solid line) and ferrous-CO (dotted line) heme (D). The absorption spectra of the MBP-IDD HAP mutants were measured between 250 and 800 nm using 10 μM protein solutions. An asterisk indicates an absorption peak due to the presence of dithionite.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions