all procedures were approved by the animal care and use committee of boston children s hospital . we performed complete blood count and wright - geimsa analyses on peripheral blood recovered from adult pnt . genetic mapping and positional cloning were utilized to identify zgc : 162207 ( atpif1a ) as the candidate gene for the pnt locus on zebrafish chr . we employed qrt - pcr using taqman gene expression assays ( applied biosystems , carlsbad , ca ) to measure levels of atpif1a and atpif1b mrna . morpholinos ( gene tools , philomath , or ) against splice - site of atpif1a and atpif1b were designed and injected in wt embryos to verify loss - of - function phenotype . the crna for atpif1a , atpif1a - e26a , and atpif1b were injected in pnt embryos for complementation . the cdna prepared from wt and pnt embryos was sequenced , and the polymorphism in the 3utr of the atpif1a sequence was verified using sscp gels . the atpif1-silenced , differentiated hcd34 + and mel cells were stained with o - dianisidine to measure hemoglobinized cells , while mpfl cells were treated with drabkin s reagent to measure relative hemoglobin content . the loss of atpif1 protein and the state of mitochondrial structural proteins in mel cells were verified using western and electron microscopic analyses . we analyzed fluorescent intensities of tmre as a function of mitochondrial membrane potential , mg green as a function of atp levels , and ratio of carboxy snarf-1 to mitotracker green as a function of the mitochondrial matrix ph . we prepared fe - saturated transferrin , and measured fe incorporated in mitochondria and complexed in heme using a gamma counter . we examined ppix levels and the catalytic efficiency of fech in mel cells using spectrophotometric analysis . the mel cells were treated with fccp and 2,4-dnp , followed by analysis for hemoglobinized cells . human and yeast fech were treated with dtn , and subsequently their catalytic efficiency were measured . the crna for zebrafish fech or yeast fech was injected in pnt embryos , and their efficiency to rescue the anemia in pnt was measured using o - dianisidine staining and verified by using sscp analysis .
summarydefects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis , ferrochelatase ( fech ) , impair heme synthesis , and thus cause human congenital anemias1,2 . 
 the inter - dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown . to uncover this unmet need 
 , we utilized zebrafish genetic screens and cloned mitochondrial atpase inhibitory factor 1 ( atpif1 ) from a zebrafish mutant with profound anemia , pinotage ( pnt tq209 ) . 
 we now report a direct mechanism establishing that atpif1 regulates the catalytic efficiency of vertebrate fech to synthesize heme . 
 the loss of atpif1 impairs hemoglobin synthesis in zebrafish , mouse , and human hematopoietic models as a consequence of diminished fech activity , and elevated mitochondrial ph . to understand the relationship among mitochondrial ph , redox potential , [ 2fe-2s ] clusters , and fech activity , we used ( 1 ) genetic complementation studies of fech constructs with or without [ 2fe-2s ] clusters in pnt , and ( 2 ) pharmacological agents modulating mitochondrial ph and redox potential . 
 the presence of [ 2fe-2s ] cluster renders vertebrate fech vulnerable to atpif1-regulated mitochondrial ph and redox potential perturbations . 
 therefore , atpif1 deficiency reduces the efficiency of vertebrate fech to synthesize heme , resulting in anemia . 
 the novel mechanism of atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development . 
 a deficiency of atpif1 may contribute to important human diseases , such as congenital sideroblastic anemias and mitochondriopathies .