Mitochondrial Atpif1 regulates haem synthesis in developing erythroblasts

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 anaemias. 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.

Fig. 1. Disruption of atpif1 in pinotage (pnt^tq209) produces hypochromic anemia

a, pnt embryos are severely anemic. Wild-type (WT) embryo at 72 hpf exhibits o-dianisidine stained (brown) hemoglobinized cells. b, The positional cloning of the pnt locus on zebrafish chromosome (Chr.) 19. A positional cloning effort with 1,912 diploid pnt embryos identified the closest linked genetic marker, z42828b. We initiated a chromosomal walk, at a distance of ~0.01 centimorgan (cM) from the pnt locus. The BAC clone, encompassing the pnt locus, is shown below, along with the annotated genes within the critical physical contig. c, Phylogenetic dendrogram showing the amino acid homology between the various atpif1 genes. D. rerio (Dr) atpif1a aligns with its related paralog, atpif1b, on zebrafish Chr. 17. Both atpif1a and atpif1b are shown clustering with their functional mammalian orthologs from mouse (Mm) and human (Hs). d, qRT-PCR analysis of atpif1a and atpif1b mRNA in pnt and WT embryos, showing reduced atpif1a and normal atpif1b mRNA level in pnt. *p<0.05 (t-test, n=3)

Fig. 2. Functional characterization of the atpif1a gene

a, Splice blocking morpholino (MO) knock down of atpif1a phenocopies the anemia observed in pnt embryos. b, qRT-PCR analysis shows that the anemic phenotype is due to the accurate knockdown of atpif1a. c, Ectopic expression of atpif1a or atpif1b cRNA functionally complements the anemia in pnt embryos at 72 hpf. WT control, pnt, and rescued pnt embryos complemented with atpif1a or atpif1b cRNA are stained with o-dianisidine. The non-functional atpif1a, harboring the E26A mutation, specifically failed to complement the pnt anemia. d, pnt embryos have an AC polymorphism in the 3′ UTR of the atpif1a gene. e, The 3′UTR AC polymorphism co-segregates with the pnt phenotype by SSCP analysis. The SSCP segregation pattern for lanes 1–2 (+/+), lane 3 (+/pnt), and lanes 4–6 (pnt/pnt). f, The mutant AC polymorphism in the 3′UTR of the atpif1a cDNA functionally destabilizes its mRNA. MT construct stably expressed in MEL cells showed reduced atpif1a mRNA levels. *p<0.05 (t-test, n=3)

Fig. 3. Loss of Atpif1 produces a hemoglobinization defect in mammalian cells

a, Silencing of Atpif1 in human CD34+ (left) and MEL (right) cells with shRNAs (Human: H1^sh & H2^sh; Mouse: M1^sh) results in a hemoglobinization defect. b, Western analysis in Atpif1-shRNA (Mouse: M1^sh) silenced MEL cells. Atpif1 protein level is reduced in Atpif1-shRNA (M1^sh)-silenced cells. However, the mitochondrial structural proteins, AtpB, CoxIV, Vdac1, and Hsp60, are not affected. c, Silencing of Atpif1 elevates the mitochondrial membrane potential (ΔΨm), analyzed using a TMRE fluorescence probe in the presence of verapamil and FCCP. d, Silencing of Atpif1 elevates the import of ⁵⁹Fe in the mitochondria, consistent with the increased mitochondrial ΔΨm. e, Silencing of Atpif1 does not influence the formation of protoporphyrin IX (PPIX), indicating PPIX and iron are not limited for heme synthesis. f, The level of ⁵⁹Fe incorporated in heme is greatly reduced in Atpif1-silenced MEL cells, recapitulating the heme synthesis defect in pnt. *p<0.05 (t-test, n=3)

Fig. 4. Atpif1 regulates heme synthesis by modulating Fech activity

a, Ferrochelatase (Fech) protein levels are normal (top right); however, the Fech activity is reduced in Atpif1-silenced MEL cells. b, Silencing of Atpif1 increases the mitochondrial matrix pH to 8.6. The histogram summarizes values at resting conditions and after challenge with oligomycin that normalizes the initial difference. c, Analysis of Fech activity as a function of pH. The elevation of mitochondria pH to 8.5 & 9.0 markedly reduced the Fech activity. d, Drugs lowering mitochondrial membrane potential reverses the anemic phenotype due to loss of Atpif1. Treatment of FCCP and 2, 4-DNP complements the anemic phenotype of Atpif1-silenced MEL cells. e, Aconitase activity, a marker for mitochondrial [2Fe-2S] cluster synthesis, is normal in Atpif1-silenced MEL cells. f, The presence of [2Fe-2S] cluster makes Fech susceptible to mitochondrial pH alteration in the absence of Atpif1. Yeast Fech, lacking [2Fe-2S], complements pnt anemia, indicating a resistance to pH changes in the absence of atpif1. Zebrafish Fech, containing [2Fe-2S], does not complement pnt anemia, indicating its susceptibility to mitochondrial alkalinization. g, The [2Fe-2S] of Fech is sensitive to the reduction in redox potential. The treatment of DTN reduces human Fech activity, and does not affect yeast Fech activity. h, Reactive oxygen species (ROS) are not responsible for pnt anemia. Treatment of pnt embryos with N-acetyl cysteine (NAC) does not reverse pnt anemia. i, A proposed mechanistic model of Atpif1 function in the maintenance of mitochondrial pH and regulation of Fech activity for heme synthesis. Mitochondrial heme synthesis requires Fech to incorporate iron into PPIX at physiological pH. Atpif1 normally preserves the mitochondrial pH. Loss of Atpif1 alkalinizes mitochondrial pH, the presence of [2Fe-2S] cluster makes Fech susceptible to mitochondrial pH and redox potential perturbations, and consequently reduces its catalytic efficiency for the production of heme. *p<0.05 (t-test, n=3); †p<0.05 vs. control cells at pH 8.5 (t-test, n=3)