Fig 2: Evaluating NCOA4 iron binding.(A) NCOA4383−509 and HERC22553−2639 were expressed in E. coli and the purified protein solution is depicted with NCOA4383−509 solution having a brown color suggestive of an iron-containing protein. (B) Coomassie stained SDS-PAGE gel of purified NCOA4383−509 and HERC22553−2639.DOI: http://dx.doi.org/10.7554/eLife.10308.010

Fig 3: Knockdown of ncoa4 impacts erythropoiesis in zebrafish and tissue culture cells.(A, B) Expression of HGBG (A), HGBA (B) determined by quantitative RT-PCR in K562 cells expressing a control (shGFP) or two independent shRNAs targeting NCOA4. Hemin treatment (25 μM, 72 hr) as indicated. (C) Immunoblot analysis of K562 cells as in (A) and (B) with indicated antibodies. Hemin treatment (25 μM, 72 hr) as indicated. (D) Appearance of K562 pellets as in (A, B, and C). Red coloration indicates appropriate hemoglobinization of cells after hemin differentiation. Brown coloration indicates accumulation of iron without hemoglobinization. (E) Expression of ncoa4 in circulating erythroid cells relative to sites of primitive hematopoiesis. Abbreviations: hpf = hours post-fertilization, ICM = intermediate cell mass, YS = yolk sac, H = heart, L = liver. (F) Circulating RBCs are visualized in globin-LCR:eGFP erythrocyte reporter zebrafish at 30 hpf. Morpholino-mediated knockdown of ncoa4 severely disrupts erythropoiesis (n > 30 each condition). Inset shows erythrocytes circulating in the caudal artery and caudal vein plexus. (G) FACS quantification of erythrocytes in globin-LCR:eGFP reporter zebrafish at 30 hpf following control or ncoa4-morpholino-mediated knockdown (ncoa4 MOa and MOb). ***p < 0.001 and **p < 0.002 by two-tailed unpaired t-test. (H) o-dianisidine staining (brown) for hemoglobinized red cells in zebrafish embryos. Embryos were grown from zygotes injected at the one- to two-cell stage with ncoa4-targeting morpholinos (MOa and MOb) or control injected zygotes (control). Ncoa4 MOa: 15/16 embryos with diminished staining in comparison to control. Ncoa4 MOb: 18/22 embryos with diminished staining in comparison to control.DOI: http://dx.doi.org/10.7554/eLife.10308.011

Fig 4: Iron-dependent NCOA4 turnover via the HERC2 E3 Ubiquitin Ligase.(A) Endogenous IP of NCOA4 from 293T cells treated with DFO or FAC for 6 hr. Lysate input for control IgG IP is the untreated sample. HERC2 and NCOA4 binding was evaluated by immunoblot. (B) Schematic of HERC2 functional domains. To interrogate NCOA4 binding to HERC2, six fragments spanning the HERC2 protein (F1-6) were subcloned and expressed in 293T cells. (C) Endogenous NCOA4 binding to NTAP-HERC2 fragments. Extracts from 293T cells stably expressing the indicated proteins were immunoprecipitated with anti-Flag (Flag-IP) and immunoblotted with the indicated antibodies. (D) Identification of the minimal HERC2 domain responsible for NCOA4 binding. HERC2F3 was divided into six sub-fragments as indicated. (E) NCOA4 binds the CUL7 (CPH) homology domain of HERC2. HERC2 FLAG-tagged fragments 3-1 through 3–6 were expressed in 293T cells followed by Flag-IP, and immunoblot with the indicated antibodies. (F) HERC2 CUL7 domain binds endogenous NCOA4 in an iron-dependent manner. FLAG-tagged HERC2F3 was expressed in 293T cells, followed by DFO or FAC treatment for 9 hr and Flag-IP as in (C). Lysate input for the control IP (normal mouse serum) corresponds to the untreated sample. (G) siRNA-mediated knockdown of HERC2 expression promotes NCOA4 protein accumulation. U2OS cells were transfected with control or HERC2-specific siRNAs for 72 hr, harvested, and subjected to SDS-PAGE and immunoblot with the indicated antibodies to evaluate HERC2 knockdown and NCOA4 abundance. (H) NCOA4 half-life is extended upon depletion of HERC2. Immunoblot of NCOA4 protein levels in U2OS cells following control or HERC2 siRNA delivery and cycloheximide treatment (CHX) as indicated. (I) Quantification of NCOA4 protein levels in 2 independent biological CHX experiments. Error bars represent +/− standard deviation, *, p < 0.05 by two-tailed unpaired t-test at each time point. (J) Expression of HERC22540−2700 abrogates iron-mediated NCOA4 downregulation. 293T cells were transfected with FLAG-tagged HERC2F3−6 or FLAG-tagged GFP as a control, followed by FAC treatment as indicated. NCOA4 protein level was evaluated by immunoblot.DOI: http://dx.doi.org/10.7554/eLife.10308.007

Fig 5: FTH1 R23A forms functional ferritin cages and abrogates NCOA4 binding.(A) Size exclusion chromatogram of recombinant wild-type FTH1-only complexes with main peak noted at approximately 14 ml corresponding to an apparent 450 kDa molecular weight. (B) Size exclusion chromatogram of recombinant R23A FTH1-only complexes with main peak noted at approximately 14 ml corresponding to an apparent 450 kDa molecular weight. (C) Control or FTH1-deficient cells (three independent CRISPR clones) were transduced with FTH1WT or FTH1R23A lentivirus and stable cell lines were generated. Endogenous NCOA4 and FTL binding was evaluated by immunoblot following FLAG-IP of WT or mutant FTH.DOI: http://dx.doi.org/10.7554/eLife.10308.006