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. 2008 May 30;320(5880):1207-10.
doi: 10.1126/science.1157643.

A cytosolic iron chaperone that delivers iron to ferritin

Haifeng Shi  1 , Krisztina Z Bencze, Timothy L Stemmler, Caroline C Philpott
Affiliations

A cytosolic iron chaperone that delivers iron to ferritin

Haifeng Shi et al. Science. .

Abstract

Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)-binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.

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Figures

Figure 1
Figure 1
PCBP1 delivers iron to ferritin in Saccharomyces cerevisiae. (A) Activation of an iron-regulated, Aft1-responsive promoter (FeRE) fused to the HIS3 coding sequence when cytosolic iron is transferred into ferritin. (B) PCBP1-dependent activation of the FeRE/HIS3 reporter in yeast expressing ferritin, but not in yeast without ferritin. Transformed strains were plated in serial dilutions on media with and without histidine. (C and D) PCBP1 increases ferritin mineralization. Strains transformed as in B were grown with 55FeCl3, ferritin was isolated by native gel electrophoresis and iron within ferritin was detected by autoradiography. In D, replicates were normalized to the vector transformed strain (n=5). (E) PCBP1 did not affect ferritin protein levels (n=10). (F) PCBP1-dependent increase in surface ferric reductase activity in yeast expressing ferritin, but not in yeast without ferritin (n=5). *P<0.002. This and subsequent p values were determined using 2-tailed t test.
Figure 2
Figure 2
Depletion of PCBP1 impairs ferritin iron loading in Huh7 cells. Huh7 cells transfected with PCBP1 or control siRNAs were labeled with 55FeCl3 for the indicated times. Ferritin was detected by autoradiography (A), or immunoblotting (B). (C) Relative quantitation of ferritin protein, iron, and iron/protein ratio (n=5). *P<0.0002. (D) Degradation of IRP2 in cells lacking PCBP1. HEK293 cells expressing IRP2 were transfected with PCBP1 and control siRNAs. IRP2, PCBP1, and actin were detected by immunoblotting. (E) Increased labile iron pool in cells lacking PCBP1. PCBP1 was depleted in Huh7 cells, and the relative amounts of the chelatable intracellular iron pool measured (n=6). *P=0.003.
Figure 3
Figure 3
PCBP1 binds to ferritin in vivo. Yeast cells expressing ferritin H- and L-chains, PCBP1, or both were lysed and subjected to immunoprecipitation with anti-PCBP1 or anti-ferritin antibody in buffers containing ferrous iron (A and B), no iron (B), or ferrous iron and a ferrous iron chelator (BPS, B). Immune complexes were detected with antibodies against PCBP1 and ferritin. Note that PCBP1 migrates more rapidly in whole lysates than after IP (A).
Figure 4
Figure 4
PCBP1 binds ferrous iron and increases ferritin iron loading in vitro. (A and B) PCBP1 binds ferrous iron with micromolar affinity by isothermal titration calorimetry (ITC). A, raw ITC spectrum; B, heat evolved per addition of titrant. Metal binding was enthalpically favorable (ΔH E − 9.9 kcal/mol) with an overall favorable ΔG. Binding was entropically unfavorable (ΔS E -10.7 cal/K•mol). (C and D) Increased ferritin mineralization in the presence of PCBP1. Equine apoferritin was incubated with 55Fe(II) and PCBP1 or albumin (BSA). Ferritin iron was detected by autoradiography. (D) Relative 55Fe incorporated into ferritin. Samples were normalized to the sample without added PCBP1 (n=6). *P<0.007.
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