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Dysfunctional mitochondria characterise Parkinson’s Disease (PD). Uncovering etiological molecules, which harm the homeostasis of mitochondria in response to pathological cues, is therefore pivotal to inform early diagnosis and therapy in the condition, especially in its idiopathic forms. This study proposes the 18 kDa Translocator Protein (TSPO) to be one of those. Both in vitro and in vivo data show that neurotoxins, which phenotypically mimic PD, increase TSPO to enhance cellular redox-stress, susceptibility to dopamine-induced cell death, and repression of ubiquitin-dependent mitophagy. TSPO amplifies the extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signalling, forming positive feedback, which represses the transcription factor EB (TFEB) and the controlled production of lysosomes. Finally, genetic variances in the transcriptome confirm that TSPO is required to alter the autophagy–lysosomal pathway during neurotoxicity.

•IF1 regulates MCU-dependent mitochondrial Ca2+ uptake.\ud \ud •IF1 loss induces MCU upregulation through activation of the AMPK/CREB pathway.\ud \ud •OMA1 depletion restores physiological MCU levels and mitochondrial Ca2+ entry.

Mitochondria play a vital role in cellular metabolism and are central mediator of intracellular signalling, cell differentiation, morphogenesis and demise. An increasingly higher number of pathologies is linked with mitochondrial dysfunction, which can arise from either genetic defects affecting core mitochondrial components or malfunctioning pathways impairing mitochondrial homeostasis. As such, mitochondria are considered an important target in several pathologies spanning from neoplastic to neurodegenerative diseases as well as metabolic syndromes. In this review we provide an overview of the state-of-the-art in mitochondrial pharmacology, focusing on the novel compounds that have been generated in the bid to correct mitochondrial aberrations. Our work aims to serve the scientific community working on translational medical science by highlighting the most promising pharmacological approaches to target mitochondrial dysfunction in disease.

British journal of pharmacology 178(2), 298-311 (2021). doi:10.1111/bph.15279, Published by Wiley, Malden, MA

Mitochondrial dysfunction occurs in most neurodegenerative diseases, contributing to both their onset and progression. A recent breakthrough unveiled that propagation of the inflammatory response and subsequent neuronal injury are also mediated extracellularly by damaged mitochondria, which are released from active microglial cells into the brain milieu. These extracellular fragmented mitochondria can therefore generate sufficient toxicity to trigger neuronal death and widespread brain damage through activation of naïve astrocytes. Besides suggesting potential new pharmacological strategies of therapeutic intervention in neurodegeneration, this original work indicates that mitochondria might act as bioactive ligands exerting paracrine functions. This is an interesting, novel and impactful concept that deserves consideration by the scientific community, as the attention should now be focused on the identification of the specific receptors through which mitochondria mediate such an important extracellular signalling mechanism in neurological conditions.

A glutamic acid to lysine (E40K) residue substitution in superoxide dismutase 1 (SOD1) is associated with canine degenerative myelopathy: the only naturally occurring large animal model of amyotrophic lateral sclerosis (ALS). The E40 residue is highly conserved across mammals, except the horse, which naturally carries the (dog mutant) K40 residue. Here we hypothesized that in vitro expression of mutant dog SOD1 would recapitulate features of human ALS (ie, SOD1 protein aggregation, reduced cell viability, perturbations in mitochondrial morphology and membrane potential, reduced ATP production, and increased superoxide ion levels); further, we hypothesized that an equivalent equine SOD1 variant would share similar perturbations in vitro, thereby explain horses' susceptibility to certain neurodegenerative diseases. As in human ALS, expression of mutant dog SOD1 was associated with statistically significant increased aggregate formation, raised superoxide levels (ROS), and altered mitochondrial morphology (increased branching (form factor)), when compared to wild-type dog SOD1-expressing cells. Similar deficits were not detected in cells expressing the equivalent horse SOD1 variant. Our data helps explain the ALS-associated cellular phenotype of dogs expressing the mutant SOD1 protein and reveals that species-specific sequence conservation does not necessarily predict pathogenicity. The work improves understanding of the etiopathogenesis of canine degenerative myelopathy.

The mitochondrial FThe chemical structure of the FNHS selectively blocks the consumption of ATP by mitochondria leading a subtle cytotoxicity associated via the concomitant engagement of autophagy which impairs cell viability. NHS achieves such a function independently of the FThe novel sulfoximine analogue of BTB-06584, NHS, acts as a selective pharmacological inhibitor of the mitochondrial F

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus to promote cytoprotective mechanisms. This process of inter-organelles interplay is known as the Mitochondrial Retrograde Response (MRR) and is enacted by alterations in the mitochondrial function which result into the nuclear stabilization of transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here, we demonstrate that this adaptive response is relying on the formation of contact sites between mitochondria and nucleus. These hot-spots of communication allow for the redistribution of cholesterol to the nucleus which favours nuclear residency and activity of the NF-kB. The expression level of the cholesterol Translocator Protein (TSPO), resident on the Outer Mitochondrial Membrane (OMM), is key to the formation of these contact sites and the implementation of the cytoprotective response. This work provides therefore a new paradigm in the intracellular communication during stress by reporting the first evidence for a functional and biochemical tethering between mitochondria and nucleus which governs cellular adaptation.

Mitochondria drive cellular adaptation to stress by retro-communicating with the nucleus. This process is known as Mitochondrial Retrograde Response (MRR) and is induced by mitochondrial dysfunctions which perturb cell signalling. MRR results in the nuclear stabilization and activation of pro-survival transcription factors such as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). Here we demonstrate that MRR is facilitated by the formation of contact sites between mitochondria and the nucleus which establish microdomains of communication between the two organelles. The 18kD Translocator Protein (TSPO), which de-ubiquitylates and stabilizes the mitochondrial network preventing its mitophagy-mediated segregation, is required for this interaction. The tethering TSPO enacts is mediated by the complex formed with the Protein Kinase A via the A-kinase anchoring protein Acyl-CoA Binding Domain Containing 3 (ACBD3) and allows the redistribution of cholesterol which sustains the pro-survival response by blocking NF-kB de-acetylation. This work proposes a new paradigm in the mitochondrial retro-communication by revealing the existence of contact sites between mitochondrial and the nucleus and a signalling role for cholesterol.

Summary: Transglutaminase type 2 (TG2) is a multifunctional enzyme that plays a key role in mitochondria homeostasis under stressful cellular conditions. TG2 interactome analysis reveals an enzyme interaction with GRP75 (glucose-regulated protein 75). GRP75 localizes in mitochondria-associated membranes (MAMs) and acts as a bridging molecule between the two organelles by assembling the IP3R-GRP75-VDAC complex, which is involved in the transport of Ca2+ from the endoplasmic reticulum (ER) to mitochondria. We demonstrate that the TG2 and GRP75 interaction occurs in MAMs. The absence of the TG2-GRP75 interaction leads to an increase of the interaction between IP3R-3 and GRP75; a decrease of the number of ER-mitochondria contact sites; an impairment of the ER-mitochondrial Ca2+ flux; and an altered profile of the MAM proteome. These findings indicate TG2 is a key regulatory element of the MAMs. : TG2 is an enzyme that plays a key role in mitochondria homeostasis. D’eletto et al. found that TG2 interacts with GRP75, a protein localized in mitochondria-associated membranes (MAMs). TG2 regulates the number of ER-mitochondria contact sites and Ca2+ flux, suggesting a key regulatory role in MAMs. Keywords: mitochondrial Ca2+, MAMs, GRP75, TG2, ER-mitochondria contact sites

In vertebrates, mitochondria are tightly preserved energy producing organelles, which sustain nervous system development and function. The understanding of proteins that regulate their homoeostasis in complex animals is therefore critical and doing so via means of systemic analysis pivotal to inform pathophysiological conditions associated with mitochondrial deficiency. With the goal to decipher the role of the ATPase inhibitory factor 1 (IF1) in brain development, we employed the zebrafish as elected model reporting that the Atpif1a−/− zebrafish mutant, pinotage (pnt tq209 ), which lacks one of the two IF1 paralogous, exhibits visual impairment alongside increased apoptotic bodies and neuroinflammation in both brain and retina. This associates with increased processing of the dynamin-like GTPase optic atrophy 1 (OPA1), whose ablation is a direct cause of inherited optic atrophy. Defects in vision associated with the processing of OPA1 are specular in Atpif1−/− mice thus confirming a regulatory axis, which interlinks IF1 and OPA1 in the definition of mitochondrial fitness and specialised brain functions. This study unveils a functional relay between IF1 and OPA1 in central nervous system besides representing an example of how the zebrafish model could be harnessed to infer the activity of mitochondrial proteins during development.

Restoring pluripotency using chemical compounds alone would be a major step forward in developing clinical-grade pluripotent stem cells, but this has not yet been reported in human cells. We previously demonstrated that VPA_ AFS cells, human amniocytes cultivated with valproic acid (VPA) acquired functional pluripotency while remaining distinct from human embryonic stem cells (hESCs), questioning the relationship between the modulation of cell fate and molecular regulation of the pluripotency network. Here, we used single-cell analysis and functional assays to reveal that VPA treatment resulted in a homogeneous population of self-renewing non-transformed cells that fulfill the hallmarks of pluripotency, i.e., a short G1 phase, a dependence on glycolytic metabolism, expression of epigenetic modifications on histones 3 and 4, and reactivation of endogenous OCT4 and downstream targets at a lower level than that observed in hESCs. Mechanistic insights into the process of VPA-induced reprogramming revealed that it was dependent on OCT4 promoter activation, which was achieved independently of the PI3K (phosphatidylinositol 3-kinase)/ AKT/ mTOR (mammalian target of rapamycin) pathway or GSK3 beta inhibition but was concomitant with the presence of acetylated histones H3K9 and H3K56, which promote pluripotency. Our data identify, for the first time, the pluripotent transcriptional and molecular signature and metabolic status of human chemically induced pluripotent stem cells.

The ubiquitously expressed ATPase inhibitory factor 1 (IF1) is a mitochondrial protein that blocks the reversal of the F1Fo-ATPsynthase, preventing dissipation of cellular ATP and ischemic damage. IF1 suppresses programmed cell death, enhancing tumor invasion and chemoresistance, and is expressed in various types of human cancers. In this study, we examined its effect on mitochondrial redox balance and apoptotic cristae remodeling, finding that, by maintaining ATP levels, IF1 reduces glutathione (GSH) consumption and inactivation of peroxiredoxin 3 (Prx3) during apoptosis. This correlates with inhibition of metallopeptidase OMA1-mediated processing of the pro-fusion dynamin-related protein optic atrophy 1 (OPA1). Stabilization of OPA1 impedes cristae remodeling and completion of apoptosis. Taken together, these data suggest that IF1 acts on both mitochondrial bioenergetics and structure, is involved in mitochondrial signaling in tumor cells, and may underlie their proliferative capacity.

Background and Purpose Ischaemia compromises mitochondrial respiration. Consequently, the mitochondrial F1Fo-ATPsynthase reverses and acts as a proton-pumping ATPase, so maintaining the mitochondrial membrane potential (ΔΨm), while accelerating ATP depletion and cell death. Here we have looked for a molecule that can selectively inhibit this activity without affecting ATP synthesis, preserve ATP and delay ischaemic cell death. Experimental Approach We developed a chemoinformatic screen based on the structure of BMS199264, which is reported to selectively inhibit F1Fo-ATPase activity and which is cardioprotective. Results suggested the molecule BTB06584 (hereafter referred to as BTB). Fluorescence microscopy was used to study its effects on ΔΨm and on the rate of ATP consumption following inhibition of respiration in several cell types. The effect of BTB on oxygen (O2) consumption was explored and protective potential determined using ischaemia/reperfusion assays. We also investigated a potential mechanism of action through its interaction with inhibitor protein of F1 subunit (IF1), the endogenous inhibitor of the F1Fo-ATPase. Key Results BTB inhibited F1Fo-ATPase activity with no effect on ΔΨm or O2 consumption. ATP consumption was decreased following inhibition of respiration, and ischaemic cell death was reduced. BTB efficiency was increased by IF1 overexpression and reduced by silencing the protein. In addition, BTB rescued defective haemoglobin synthesis in zebrafish pinotage (pnt) mutants in which expression of the Atpif1a gene is lost. Conclusions and Implications BTB may represent a valuable tool to selectively inhibit mitochondrial F1Fo-ATPase activity without compromising ATP synthesis and to limit ischaemia-induced injury caused by reversal of the mitochondrial F1Fo-ATPsynthase.

Mitochondrial structure has a central role both in energy conversion and in the regulation of cell death. We have previously shown that IF1 protects cells from necrotic cell death and supports cristae structure by promoting the oligomerisation of the F1Fo-ATPsynthase. As IF1 is upregulated in a large proportion of human cancers, we have here explored its contribution to the progression of apoptosis and report that an increased expression of IF1, relative to the F1Fo-ATPsynthase, protects cells from apoptotic death. We show that IF1 expression serves as a checkpoint for the release of Cytochrome c (Cyt c) and hence the completion of the apoptotic program. We show that the progression of apoptosis engages an amplification pathway mediated by: (i) Cyt c-dependent release of ER Ca(2+), (ii) Ca(2+)-dependent recruitment of the GTPase Dynamin-related protein 1 (Drp1), (iii) Bax insertion into the outer mitochondrial membrane and (iv) further release of Cyt c. This pathway is accelerated by suppression of IF1 and delayed by its overexpression. IF1 overexpression is associated with the preservation of mitochondrial morphology and ultrastructure, consistent with a central role for IF1 as a determinant of the inner membrane architecture and with the role of mitochondrial ultrastructure in the regulation of Cyt c release. These data suggest that IF1 is an antiapoptotic and potentially tumorigenic factor and may be a valuable predictor of responsiveness to chemotherapy.

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.

The aggressiveness of glioblastoma multiforme (GBM) is defined by local invasion and resistance to therapy. Within established GBM, a subpopulation of tumor-initiating cells with stem-like properties (GBM stem cells, GSCs) is believed to underlie resistance to therapy. The metabolic pathway autophagy has been implicated in the regulation of survival in GBM. However, the status of autophagy in GBM and its role in the cancer stem cell fraction is currently unclear. We found that a number of autophagy regulators are highly expressed in GBM tumors carrying a mesenchymal signature, which defines aggressiveness and invasion, and are associated with components of the MAPK pathway. This autophagy signature included the autophagy-associated genes DRAM1 and SQSTM1, which encode a key regulator of selective autophagy, p62. High levels of DRAM1 were associated with shorter overall survival in GBM patients. In GSCs, DRAM1 and SQSTM1 expression correlated with activation of MAPK and expression of the mesenchymal marker c-MET. DRAM1 knockdown decreased p62 localization to autophagosomes and its autophagy-mediated degradation, thus suggesting a role for DRAM1 in p62-mediated autophagy. In contrast, autophagy induced by starvation or inhibition of mTOR/PI-3K was not affected by either DRAM1 or p62 downregulation. Functionally, DRAM1 and p62 regulate cell motility and invasion in GSCs. This was associated with alterations of energy metabolism, in particular reduced ATP and lactate levels. Taken together, these findings shed new light on the role of autophagy in GBM and reveal a novel function of the autophagy regulators DRAM1 and p62 in control of migration/invasion in cancer stem cells.

The functional integrity of mitochondria is strictly dependent on the molecular dynamics governing their shape, size, and structure. A sequence of fusion and fission events serve to tailor the mitochondrial reticulum and to preserve its morphological and functional homeostasis.1 The mitochondrial ultrastructure, defined by the alignment and density of cristae, plays a central role in mitochondrial function, dictating both the efficiency of oxidative phosphorylation, through production of adenosine 5-triphosphate (ATP), and the execution of programmed cell death (or apoptosis) by tuning the release of pro-apoptotic molecules stored within the intermembrane space, such as cytochrome c (Cyt c). Concomitantly with the dismantling of the inner mitochondrial structure, during activation of the intrinsic pathway of apoptosis, a signaling platform assembles on mitochondria, driving the permeabilization of the outer mitochondrial membrane (OMM) and sustaining the release of pro-apoptotic proteins. Alterations in the structural dynamics of mitochondria therefore influence the rate and efficiency of apoptosis. We have recently found that the protein IF1 (the inhibitor of the F1 complex of the ATP synthase) contributes to the regulation of Cyt c release and the signaling cascade that culminates in apoptosis.2 The biochemistry of this small mitochondrial protein (~10 kDa) has been studied in exquisite detail as an inhibitor of the hydrolysis of adenosine triphosphate (ATP) by the F1Fo-ATPsynthase (see ref. 3 and references therein). This event is likely to be important during conditions of oxygen deprivation, making IF1 protective in ischemia by slowing the rate of ATP depletion. Its effect on the activity of the F1Fo-ATPsynthase during physiological oxidative phosphorylation remains controversial.4 Quite distinct from its role as an inhibitor of the F1Fo-ATPase, we found that overexpression of IF1 increased cristae density and number, while knockdown of the protein could leave mitochondria with only minimal cristae structures, although they could maintain a considerable potential.5 It seems likely that this results from a promotion of dimerization of the F1Fo-ATPsynthase, which, in turn, increases membrane curvature and hence cristae density. Strikingly, we also found that increased IF1 expression levels limit the progression of apoptotic cell death by greatly delaying the release of Cyt c. This is consistent with the rise in cristae density, which limits cristae remodeling, an event that is required for complete cytochrome c release. This closely resembles the mechanism by which the pro-fusion protein optic atrophy type 1 (Opa1) reduces the mobilization of Cyt c and delays apoptosis by retaining cristae shape during the activation of apoptosis,6 while the pro-fission dynamin-related protein 1 (Drp1) mediates an opposite effect on apoptosis, supporting oligomerization of the pro-apoptotic protein Bax and hence augmenting release of the Cyt c. We found that the overexpression of IF1 preserved mitochondrial shape during pharmacological activation of apoptosis, delaying the mobilization of Cyt c and the downstream signaling apoptotic cascade. The anti-apoptotic role of IF1 involves other elements in the regulation of apoptosis, namely the multidomain pro-apoptotic proteins Bax and Drp1. During apoptosis, Bax behaves similarly to Drp1, being recruited to the outer mitochondrial membrane (OMM), where it inserts and oligomerizes to form foci that are functionally linked to the permeabilization of the OMM.7 Under apoptotic conditions, we found that the overexpression of IF1 limited the formation of these foci, reducing the accumulation of Bax as well as that of Drp1 on mitochondria; whereby, in cells in which IF1 expression was knocked down, increased mitochondrial translocation of Drp1 was accompanied by increased oligomerization of Bax.2 Suppression of the apoptotic foci and limitation of the formation of constriction sites, is therefore consistent with the idea that IF1 plays an anti-apoptotic role by limiting mitochondrial fragmentation. In the scheme illustrated in Figure 1, we propose that the ratio of expression of IF1 relative to that of the F1Fo-ATPsynthase controls the release of Cyt c from mitochondria and hence regulates the completion of the apoptotic program. The progression of apoptosis leading to cell death engages an amplification pathway mediated by (1) Cyt c-dependent release of ER Ca2+,8, (2) Ca2+-dependent recruitment of the GTPase Dynamin-related protein 1 (Drp1), (3) Bax insertion into the outer mitochondrial membrane, and (4) further release of Cyt c. This cascade, which amplifies and accelerates Cyt c release, was significantly slowed down by the overexpression of IF1, and apoptotic cell death was suppressed. Figure 1. (A) In resting conditions, the pro-apoptotic protein Bax resides inactive in the cytosol, while the anti-apoptotic proteins Bcl-2/Bcl-xL are associated with the mitochondrial outer membrane, where they prevent the translocation and the ... The preservation of mitochondrial morphology by IF1 during apoptosis is consistent with its influence on the inner membrane architecture, regulating Cyt c release. Such a role appears to be independent from the protein’s canonical role as an inhibitor of the F1Fo-ATPase. Thus, we found that the dissipation of the ΔΨm during apoptosis occurred concomitantly with the release of Cyt c and the permeabilization of mitochondrial membranes independently of the IF1 level of expression. Interestingly, IF1 is strongly overexpressed in many human tumors, suggesting that the suppression of apoptotic cell death by this small protein may prove highly significant in the metabolic and structural adaptations associated with tumor development.

The mitochondrial F1Fo-ATPase Inhibitory Factor 1 (IF1) blocks the reversal of the F1Fo-ATPsynthase to prevent detrimental consumption of cellular ATP and associated demise. Herein, we infer further its molecular physiology by assessing its protective function in neurons during conditions of challenged homeostatic respiration.By adopting in vitro and in vivo protocols of hypoxia/ischemia and re-oxygenation, we show that a shift in the IF1:F1Fo-ATPsynthase expression ratio occurs in neurons. This increased IF1 level is essential to induce recruitment of the mitophagic ubiquitin ligase PARK-2 and promote autophagic “control” of the mitochondrial population. In IF1 overexpressing neurons ATP depletion is reduced during hypoxia/ischemia and the mitochondrial membrane potential resilient to re-oxygenation as well as resistant to electrogenic, Ca2+ dependent depolarization.These data suggest that in mammalian neurons mitochondria adapt to respiratory stress by upregulating IF1, which exerts a protective role by coordinating pro-survival cell mitophagy and bioenergetics resilience.

The ATPase inhibitory factor 1 (IF1) is an ubiquitously expressed mitochondrial protein that blocks the reversal of the F1Fo-ATPsynthase, preventing dissipation of cellular ATP and ischaemic damage. Many human cancers express high levels of IF1, which suppress cell death and enhance tumour cell invasion and chemoresistance. In this study, we assessed the effect of IF1 over-expression on mitochondrial redox balance and apoptotic cristae remodelling. We found that IF1 maintains ATP levels under apoptosis and reduces glutathione (GSH) loss and inactivation of peroxiredoxin 3 (Prx3). This correlates with inhibition of the metallopeptidase OMA1-mediated processing of the pro-fusion dynamin related protein optic atrophy 1 (OPA1), impeding cristae remodelling and apoptosis completion. IF1 therefore has a pivotal antioxidant activity that hinders the OMA1/OPA1-dependent deconstruction of mitochondrial morphology and cellular demise. The data presented here highlight a dual regulatory activity of IF1 on both mitochondrial bioenergetics and structure, resulting in increased proliferative capacity of tumour cells and significantly contributing to the molecular signalling of mitochondria in cancer.

Nature 491, 608–612 (2012); doi:10.1038/nature11536 On page 610 of this Letter, the overview comments concerning the action of ATP synthase should read: “Under physiological conditions, the F1F0-ATP synthase generates ATP by consuming the proton motive force existing across the inner mitochondrial membrane, transporting H+ ions from the intermembrane space into the mitochondrial matrix8,15.

Abstract 343 Iron and protoporphyrin IX (PPIX) are key substrates used by ferrochelatase (Fech) to produce heme, which subsequently binds to globin to generate hemoglobin in red blood cells. Defects in the transport of iron, synthesis of PPIX, or catalytic activity of Fech impair heme synthesis, and thus cause human congenital anemias. However, the precise mechanisms regulating transporters and enzymes facilitating heme synthesis and their inter-dependent actions remain largely unknown. The zebrafish mutant pinotage (pnttq209) exhibits profound hypochromic, microcytic anemia. Erythrocytes from viable adult pnt fish have reduced hemoglobin content and cell volume. Positional cloning, morpholino-induced loss-of-function, cRNA over-expression, quantitative RT-PCR, and mutational analysis show that mitochondrial ATPase inhibitory factor1 (Atpif1) is the gene disrupted in pnt. Previous studies have demonstrated the role of Atpif1 in the regulation of mitochondrial proton motive force, pH, and ATP synthesis. Here, we report direct evidence that Atpif1 regulates mitochondrial heme synthesis. Knock down of the human and murine orthologs of Atpif1 using shRNAs in mammalian erythroid tissues, human CD34+, mouse Friend erythroleukemia (MEL) and primary fetal liver cells, impairs hemoglobinization. Atpif1 protein levels are reduced in stable MEL cells silenced for Atpif1; however, the protein levels of other mitochondrial structural proteins, β-subunit of ATP synthase (AtpB), voltage-dependent anionic-selective channel protein 1 (Vdac1), complex IV (CoxIV), and heat shock protein 60 (Hsp60), are normal, indicating an intact mitochondrial structure in Atpif1 silenced cells. Moreover, Atpif1 silenced cells have an increased mitochondrial membrane potential, an elevation in mitochondrial pH, and depleted ATP levels. Differentiating MEL cells silenced for Atpif1 display reduced incorporation of 59Fe into heme, although their mitochondria have sufficient amounts of Fech substrates, iron and PPIX. This is due to diminished in vivo Fech activity, despite having normal levels of Fech protein in Atpif1 silenced cells. We further establish that the Fech activity is reduced at pH 8.5, corresponding to the more alkaline mitochondrial pH in Atpif1-silenced cells. The over-expression of either yeast Fech or zebrafish Fech in pnt embryos shows that only yeast Fech rescues the anemia in pnt. This demonstrates that the catalytic activity of [Fe-S] bound zebrafish Fech, and not yeast Fech, which lacks the [Fe-S] cluster, is vulnerable to the elevation in mitochondrial pH due to the loss of Atpif1. Therefore, the loss of Atpif1 reduces the catalytic ability of vertebrate Fech to incorporate iron into PPIX to make heme, resulting in hypochromic anemia. The newly uncovered role of Atpif1 to regulate Fech provides a new insight on the mitochondrial regulation of heme synthesis and a potential cause of sideroblastic anemias. Mechanistic model of Atpif1 function in heme synthesis. The mitochondrial Atpif1 normally preserves mitochondrial pH. Loss of Atpif1 alkalinizes mitochondrial pH, the [Fe-S] cluster binding makes Fech sensitive to mitochondrial pH changes, and consequently reduces its catalytic efficiency for the production of heme. Disclosures: No relevant conflicts of interest to declare.