A PCBP1–BolA2 chaperone complex delivers iron for cytosolic [2Fe–2S] cluster assembly

Abstract

Hundreds of cellular proteins require iron cofactors for activity, and cells express systems for their assembly and distribution. Molecular details of the cytosolic iron pool used for iron cofactors are lacking, but iron chaperones of the poly(rC)-binding protein (PCBP) family play a key role in ferrous ion distribution. Here we show that, in cells and in vitro, PCBP1 coordinates iron via conserved cysteine and glutamate residues and a molecule of noncovalently bound glutathione (GSH). Proteomics analysis of PCBP1-interacting proteins identified BolA2, which functions, in complex with Glrx3, as a cytosolic [2Fe–2S] cluster chaperone. The Fe–GSH-bound form of PCBP1 complexes with cytosolic BolA2 via a bridging Fe ligand. Biochemical analysis of PCBP1 and BolA2, in cells and in vitro, indicates that PCBP1–Fe–GSH–BolA2 serves as an intermediate complex required for the assembly of [2Fe–2S] clusters on BolA2–Glrx3, thereby linking the ferrous iron and Fe–S distribution systems in cells.

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Fig. 1: BolA2 forms a complex with PCBP1 and binds in the absence of Glrx3.
Fig. 2: BolA2 requires PCBP1, but not Glrx3, to form an iron-binding complex.
Fig. 3: Stabilization of PCBP1–BolA2 complex by iron and glutathione without inorganic sulfur.
Fig. 4: Requirement of PCBP1 in delivery of iron to Grx3–[2Fe–2S]–BolA2 complex.
Fig. 5: KH3 domain of PCBP1 binds BolA2 and restores BolA2–Glrx3 complex formation.
Fig. 6: Reconstitution of KH3–BolA2 complex with iron and GSH in vitro.

Data availability

The proteomics dataset (Supplementary Table 1) is available at MassIVE Repository (https://massive.ucsd.edu/) with the accession number MSV000083887.

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institutes of Diabetes and Digestive and Kidney Diseases and the Office of Dietary Supplements, Office of the Director, National Institutes of Health. J.A.W. is supported by NIH grant nos. GM089778 and GM112763. We thank C. Outten (University of South Carolina) for plasmids and T. Stemmler (Wayne State University) for plasmids and helpful discussions.

Author information

S.J.P. and A.G.F. conceived and coordinated the study, designed, performed, and analyzed most experiments, prepared figures and wrote the paper. D.J.P. created BolA2-inducible cell lines and S.A. generated KH3 mutant plasmids. K.Z.B., A.V. and J.A.W. performed mass spectrometry experiments. C.C.P. conceived and coordinated the study, prepared the figures and wrote the paper. All authors analyzed results and approved the final version of the manuscript.

Correspondence to Caroline C. Philpott.

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