A synergistic role of IRP1 and FBXL5 proteins in coordinating iron metabolism during cell proliferation

Iron-regulatory protein 1 (IRP1) belongs to a family of RNAbinding proteins that modulate metazoan iron metabolism. Multiple mechanisms are employed to control the action of IRP1 in dictating changes in the uptake and metabolic fate of iron. Inactivation of IRP1 RNA binding by iron primarily involves insertion of a [4Fe-4S] cluster by the cytosolic ironsulfur cluster assembly (CIA) system, converting it into cytosolic aconitase (c-acon), but can also involve iron-mediated degradation of IRP1 by the E3 ligase FBXL5 that also targets IRP2. How CIA and FBXL5 collaborate to maintain cellular iron homeostasis through IRP1 and other pathways is poorly understood. Because impaired Fe-S cluster biogenesis associates with human disease, we determined the importance of FBXL5 for regulating IRP1 when CIA is impaired. Suppression of FBXL5 expression coupled with induction of an IRP1 mutant (IRPI3C>3S) that cannot insert the Fe-S cluster, or along with knockdown of the CIA factors NUBP2 or FAM96A, reduced cell viability. Iron supplementation reversed this growth defect and was associated with FBXL5-dependent polyubiquitination of IRP1. Phosphorylation of IRP1 at Ser-138 increased when CIA was inhibited and was required for iron rescue. Impaired CIA activity, as noted by reduced c-acon activity, was associated with enhanced FBXL5 expression and a concomitant reduction in IRP1 and IRP2 protein level and RNA-binding activity. Conversely, expression of either IRP induced FBXL5 protein level, demonstrating a negative feedback loop limiting excessive accumulation of ironresponse element RNA-binding activity, whose disruption reduces cell growth. We conclude that a regulatory circuit involving FBXL5 and CIA acts through both IRPs to control iron metabolism and promote optimal cell growth.