IRP2 is stabilized under iron-replete conditions after siRNA-mediated suppression of SCF^FBXL5. (A) IRP2 accumulation was assessed using the AlphaScreen assay (top) or by immunoblot analysis of endogenous IRP2 (bottom). Knockdown efficiency of the SKP1, CUL1, and RBX1 siRNAs is shown in fig. S2D. (B) IRP1 stabilization under iron-replete conditions after siRNA-mediated suppression of FBXL5, as measured by immunoblot analysis of HEK 293 cell lines stably transfected with either a myc-tagged wild-type (WT) or 3C>3S IRP1 Tet-inducible expression construct. (C) Measurement of RNA binding activity from lysates after siRNA-mediated suppression of FBXL5 in cells expressing HA-IRP2-FLAG. Because human IRP1-IRE and IRP2-IRE complexes migrate similarly, antibodies were added to supershift individual complexes. (D) Relative TfR1 and FBXL5 mRNA accumulation levels measured by qRT-PCR. Assays were performed in triplicate with data represented as the mean ± SE with P values determined using Student's unpaired t test (***P < 0.001).

IRP2 is a substrate for SCF^FBXL5. (A) FBXL5-V5 coimmunoprecipitates with HA-IRP2-FLAG. (B) Coimmunoprecipitation of endogenous IRP2 and FBXL5-FLAG from HEK 293T cells incubated with either DFO or FAC. (C) Immunoblot analysis of FLAG immunoprecipitates from insect cells coexpressing SKP1, CUL1, and RBX1 with either full-length FLAG-FBXL5 (WT) or FLAG-FBXL5 lacking the F-box domain (ΔF-box). (D) Recombinant SCF^FBXL5 ubiquitinates IRP2 in vitro when supplemented with purified E1 and E2 enzymes, adenosine triphosphate (ATP), and WT ubiquitin (top) or glutathione S-transferase (GST)–tagged ubiquitin lacking lysines (K₀-GST-Ub) and thus unable to form polyubiquitin chains (bottom).

FBXL5 protein accumulation is dependent on iron and oxygen availability. (A) Immunoblot analysis of stably transfected FBXL5-FLAG protein accumulation under iron-deplete (DFO) or iron-replete (FAC) conditions. (B) Time course of IRP2 and FBXL5 responses to changes in iron availability. HEK FBXL5-FLAG cells were incubated 16 hours with either FAC or DFO, then switched to low-or high-iron conditions, respectively. Quantitation is provided in table S1. (C) Endogenous IRP2 and FBXL5 levels were assessed by immunoblot analysis in WT cells (293 TRex) or in a clonal cell line stably expressing a Tet-inducible FBXL5 shRNA. (D) Accumulation of FBXL5 under iron-replete conditions is attenuated (by a factor of 2.5) in cells incubated under low-oxygen conditions (1% O₂) for 16 hours. (E and F) Residues 1 to 161 mediate iron- and oxygen-dependent regulation of FBXL5 accumulation. Assessment of FBXL5 protein accumulation under iron-deplete (DFO) or iron-replete (FAC) conditions by immunoblot analysis of transiently transfected FBXL5 constructs. Quantitation is provided in table S2.

FBXL5 contains an iron- and oxygen-binding hemerythrin domain that regulates its stability. (A) Iron content of recombinant WT or variant (E61A) FBXL5 hemerythrin domains. Assays were performed in triplicate with data represented as the mean ± SE, with P values determined using Student's unpaired t test (***P < 0.001). (B) UV chromatogram of the FBXL5 hemerythrin domain before and after dithionite addition. (C) Circular dichroism spectra of purified FBXL5 hemerythrin domains. (D) Measurementofmean molar residual ellipticity at 222 nm as a function of thermal de-naturation of the WT domain treated with dithionite and iron chelator (o-Phen). (E) Luciferase activity (upper panels) and protein accumulation levels (lower panels; quantitation provided in table S3) in HEK 293T cells transiently transfected with fusion protein expression constructs. E.V., empty vector. Assays were performed in triplicate with data represented as the mean ± SE. (F) Immunoblot analysis of protein accumulation from transiently transfected FBXL5 expression constructs.