Pcbp1 and Ncoa4 Regulate the Flux of Iron through Ferritin in Developing Erythroid Cells

In general human cell models, Poly(rC)-binding proteins (Pcbp) mediate the delivery and integration of iron into the iron storage protein. Conversely, nuclear receptor coactivator 4 (Ncoa4) initiates the liberation of ferritin iron by localizing the protein to autophagosomes. The erythron is the major consumer of body iron. Whether these regulators of ferritin iron are functional in developing red cells remains unclear, and thus was investigated. Both Pcbp1 and Ncoa4 were readily detectable in immunoprecipitates of erythroid ferritin. The peak of Pcbp1-binding to ferritin preceded the stage of maximal Ncoa4-ferritin interaction, which coincided with the induction of β-globin, during erythroid maturation. Both Pcbp1 and Ncoa4 deficiency resulted in lower iron incorporation into heme and hemoglobin by differentiation, without compromised Alas2 gene expression. In Pcbp1-deficent cells, less iron loading into ferritin accompanied the erythropoietic defects. Ncoa4-lacking cells exhibited a massive accumulation of iron in ferritin. No apparent interaction between Pcbp2 and ferritin was detectable in erythroid cells. However, Pcbp2 depletion affected ferritin mineralization in a manner opposite to Pcbp1 deficiency. This was associated with an increase in Pcbp1-ferritin complex formation detected by coimmunoprecipitation. Supplemental iron led to decreases in the amount of both Pcbp1 and Ncoa4 interacting with ferritin. The iron-responsiveness of Ncoa4's ferritin-binding activity was primarily due to post-transcriptional changes in protein abundance. The down-regulation of Ncoa4 protein by high iron required intact lysosome activity, and the expression of an E3 ubiquitin protein ligase, Herc2. Collectively, the present studies provide evidence of the metallochaperone activity of Pcbp1 for ferritin mineralization, and the involvement of Ncoa4 in the mobilization process of stored iron in erythroid cells. Furthermore, the impaired hemoglobinization by insufficiency of these genes identifies ferritin as an essential source of iron for heme production during terminal erythroid differentiation.