Mitochondrial Atpif1 Regulates Heme Synthesis in Developing Erythroblasts
Authors: Shah Dhvanit I. , Takahashi-Makise Naoko , Cooney Jeffrey D. , Li Liangtao , Schultz Iman , Pierce Eric L. , Narla Anupama , Hattangadi Shilpa , Langer Nathanial , Chen Wen , Dailey Tamara A. , Hurst Slater N. , Faccenda Danilo , Wiwczar Jessica , Law Terrence C. , Vogin Guillaume , Brugnara Carlo , Zhou Yi , Ward Diane M. , Ebert Benjamin L. , Campanella Michelangelo , Danial Nika , Lodish Harvey F. , Dailey Harry A. , Kaplan Jerry , Paw Barry H. 
Published: October 2019

Abstract
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.