The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C) Binding Protein 1 (PCBP1) is an iron chaperone for ferritin;both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and 2 exhibited high-affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and 4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron sensitive, ccc1 yeast strain intensified the toxic effects of iron, while expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and 2 form a complex for iron delivery to ferritin and all PCBPs may share iron chaperone activity. PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the eukaryotic translation factor eIF5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo-form of the enzyme in cells, particularly when cells were made mildly iron deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells, but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA binding activity, a pattern suggesting the incomplete disassembly of the 4Fe-4S cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic non-heme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.
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