Control of iron homeostasis by an iron-regulated ubiquitin ligase

Abstract

Eukaryotic cells require iron for survival and have developed regulatory mechanisms for maintaining appropriate intracellular iron concentrations. The degradation of iron regulatory protein 2 (IRP2) in iron-replete cells is a key event in this pathway, but the E3 ubiquitin ligase responsible for its proteolysis has remained elusive. We found that a SKP1-CUL1-FBXL5 ubiquitin ligase protein complex associates with and promotes the iron-dependent ubiquitination and degradation of IRP2. The F-box substrate adaptor protein FBXL5 was degraded upon iron and oxygen depletion in a process that required an iron-binding hemerythrin-like domain in its N terminus. Thus, iron homeostasis is regulated by a proteolytic pathway that couples IRP2 degradation to intracellular iron levels through the stability and activity of FBXL5.

Fig. 1

FBXL5 forms a SCF complex that associates with IRP1 and IRP2. (A) Flp-In TREx-293 cells (Invitrogen, Carlsbad, CA) stably expressing HA-FLAG-FBXL5 were transfected with Myc-CUL1, Myc-SKP1, or empty vector. Myc-CUL1 and Myc-SKP1 were immunoprecipitated with antibody to c-Myc. Whole-cell extracts (WCEs) and immunoprecipitates (IPs) were immunoblotted with antibodies to FLAG and c-Myc. (B) HA-FLAG-FBXL5 was immunoprecipitated from stable Flp-In TREx-293 cells using antibody to HA. WCEs and IPs were immunoblotted with antibodies to FLAG, IRP1, IRP2, and β-tubulin. (C) HEK293 cells were cotransfected with FLAG-IRP2 and Myc-FBXL5, Myc-FBXL5-ΔF-box, or empty vector and treated for 8 hours with FAC or DFO. FLAG-IRP2 was immunoprecipitated using antibodies to FLAG. WCEs and IPs were immunoblotted with antibodies to FLAG, c-Myc, and β-tubulin.

Fig. 2

FBXL5 regulates IRP2 ubiquitination and iron-dependent degradation. (A) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5 or control cells were treated for 8 hours with FAC or DFO. WCEs were probed with antibodies to FLAG, IRP2, ferritin, and β-tubulin. (B) HEK293 cells were transfected with nonspecific or FBXL5 siRNAs and then treated with or without FAC for 8 hours. WCEs were immunoblotted with the specified antibodies. (C) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5 or HA-FLAG-FBXL5-ΔF-box or control cells were pulsed with ³⁵S-met/cys for 1 hour and then chased in medium supplemented with or without additional FAC. ³⁵S-labeled endogenous IRP2 was immunoprecipitated with antibody to IRP2 and half-lives (t 1/2) are shown as average ± SD (n = 2 independent experiments). (D) Flp-In TREx-293 cells stably expressing FLAG-IRP2 were treated with nonspecific or FBXL5 siRNAs, pulsed with doxycycline overnight so as to induce FLAG-IRP2 expression, and then chased in medium supplemented with FAC. WCEs were probed with antibodies to FLAG and actin. FLAG-IRP2 half-lives (t 1/2) are shown as average ± SEM (n = 3 independent experiments). (E) HEK293 cells were cotransfected with HA-Ub, FLAG-IRP2 and Myc-FBXL5, Myc-FBXL5-ΔF-box, or empty vector and then treated with FAC and MG132 for 4 hours and ubiquitin conjugates immunoprecipitated by using antibodies to HA. HA-immunoprecipitates (IP: HA) and WCEs were immunoblotted with antibodies to FLAG, c-Myc, and β-tubulin.

Fig. 3

Hypoxia and iron depletion promote the proteasomal degradation of FBXL5. (A) Flp-In TREx-293 control cells were treated with FAC, DFO, or 1% O₂ (Hyp) for 8 hours. WCEs were immunoblotted with antibodies to FBXL5, IRP2, and β-tubulin. (B and C) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5 were treated overnight with doxycycline so as to induce HA-FLAG-FBXL5 expression and then chased in control medium (−) in 21% O₂ or 1% O₂ (hypoxia) or in medium supplemented with FAC or DFO. (B) Cells were supplemented with or without MG132 for 6 hours. WCEs were immunoblotted with antibodies to FLAG and actin. (C) WCEs were immunoblotted with antibodies to FLAG, IRP2, and actin. HA-FLAG-FBXL5 half-lives (t 1/2) are shown as average ± SEM (n = 3 independent experiments).

Fig. 4

FBXL5 stability is regulated by an iron-binding hemerythrin-like domain. (A) An N-terminal fragment of FBXL5 encompassing amino acids 1 to 199 (FBXL5-N199) or Smt3p (negative control) was expressed in Escherichia coli, and the amount of copurifying iron was measured by means of ICP-MS. (B) HEK293 cells were transfected with HA-FLAG-FBXL5, HA-FLAG-FBXL5-N199, or HA-FLAG-FBXL5-C492 (amino acids 200 to 691) and treated with FAC or DFO for 8 hours. WCEs were immunoblotted with antibodies to FLAG or β-tubulin. (C) HEK293 cells were cotransfected with HA-FLAG-FBXL5-N199, HA-FLAG-FBXL5-N199-H15A, or HA-FLAG-FBXL5-N199-H57A, and a plasmid expressing glutathione S-transferase (GST) as a marker for transfection efficiency, and treated with FAC (F) or DFO (D) for 8 hours. WCEs were immunoblotted with antibodies to FLAG and GST. (D) Flp-In TREx-293 cells stably expressing HA-FLAG-FBXL5-C492 or control cells were treated for 8 hours with FAC or DFO. HA-FLAG-FBXL5-C492 was immunoprecipitated by use of antibodies to HA. WCEs and IPs were probed with antibodies to FLAG, IRP2, ferritin, and β-tubulin.