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Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

A Corrigendum to this article was published on 17 April 2013

Abstract

Defects in the availability of haem substrates or the catalytic activity of the terminal enzyme in haem biosynthesis, ferrochelatase (Fech), impair haem synthesis and thus cause human congenital anaemias1,2. The interdependent functions of regulators of mitochondrial homeostasis and enzymes responsible for haem synthesis are largely unknown. To investigate this we used zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anaemia, pinotage (pnt tq209 ). Here we describe a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize haem. The loss of Atpif1 impairs haemoglobin synthesis in zebrafish, mouse and human haematopoietic models as a consequence of diminished Fech activity and elevated mitochondrial pH. To understand the relationship between mitochondrial pH, redox potential, [2Fe–2S] clusters and Fech activity, we used genetic complementation studies of Fech constructs with or without [2Fe–2S] clusters in pnt, as well as pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe–2S] cluster renders vertebrate Fech vulnerable to perturbations in Atpif1-regulated mitochondrial pH and redox potential. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize haem, resulting in anaemia. The identification of mitochondrial Atpif1 as a regulator of haem synthesis advances our understanding of the mechanisms regulating mitochondrial haem homeostasis and red blood cell development. An ATPIF1 deficiency may contribute to important human diseases, such as congenital sideroblastic anaemias and mitochondriopathies.

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Figure 1: Disruption of atpif1 in pinotage (pnttq209) produces hypochromic anaemia.
Figure 2: Functional characterization of the atpif1a gene.
Figure 3: Loss of Atpif1 produces a haemoglobinization defect in mammalian cells.
Figure 4: Atpif1 regulates haem synthesis by modulating Fech activity.

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Acknowledgements

We thank members of our laboratory (M. Cassim, A. Kaplan, G. Hildick-Smith and H. Anderson) and colleagues (S. L. Alper, K. Pepper and N. S. Trede) for critical review of the manuscript, T. C. Law for pnt adult blood characterization, H. Mulhern for help with the electron microscopy, and Christopher Lawrence and his team for the zebrafish husbandry. This research was supported in part by the Cooley’s Anemia Foundation (D.I.S. and C.C.), the March of Dimes Foundation (B.H.P.), the American Heart Association (J.D.C. and A.E.M.), the Dutch National Science Fund (I.J.S.), the Fondation Soldati pour la Recherche en Cancerologie (G.V.), the Burroughs Welcome Fund (N.N.D.), the NIDDK (D.I.S., B.H.P., A.N., J.K., H.A.D. and S.M.H.) and the NHLBI (D.I.S. and B.H.P).

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Authors and Affiliations

Authors

Contributions

D.I.S. and B.H.P. originally conceived the project, designed and performed the experiments, analysed data and wrote the manuscript. N.T.-M., A.S., L.L., D.M.W. and J.K. measured 59Fe uptake in mitochondria and complexed in haem, PPIX levels, Fech activity, xanthine oxidase and aconitase activities and the haem levels in a yeast knockout for Inh1 and participated in scientific discussions. J.D.C., I.J.S., E.L.P. and N.B.L. did zebrafish embryo microinjections. S.K.H., G.V., C.C. and W.C. helped with zebrafish colony maintenance and protein experiments. Y.Z. helped with high resolution meiotic mapping. A.N., S.N.H. and B.L.E. helped with silencing ATPIF1 in human primary CD34+ cells. S.M.H. helped with silencing Atpif1 in MFPL cells. A.E.M., T.A.D. and H.A.D. created Fech constructs for injection, measured Fech activity as a function of pH, analysed Fech structure for [2Fe–2S] clusters and participated in scientific discussions. D.F., J.M.W., M.C. and N.N.D. helped to design and measure mitochondrial physiological parameters. C.B. analysed pnt adult blood parameters. D.R.C. and M.D.F. helped with electron microscopic analysis of mitochondrial structure and disease.

Corresponding authors

Correspondence to Harry A. Dailey, Jerry Kaplan or Barry H. Paw.

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The authors declare no competing financial interests.

Additional information

Sequences are available in GenBank/EMBL/DDBJ as follows: zebrafish atpif1a (NM_001089521.1), zebrafish atpif1b (NM_001044859), mouse Atpif1 (NC_000070.5) and human ATPIF1 (NC_005104.2).

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Shah, D., Takahashi-Makise, N., Cooney, J. et al. Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts. Nature 491, 608–612 (2012). https://doi.org/10.1038/nature11536

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