Abcb10 Physically Interacts with Mitoferrin1 to Enhance Its Stability for Heme Synthesis in the Erythroid Mitochondria

We previously described a zebrafish mutant, frascati (frs), with hypochromic anemia and erythroid maturation arrest. Using positional cloning, we identified mitoferrin1 (mfrn1, slc25a37) as the gene disrupted in frs mutants (Shaw GC, et al. 2006 Nature 440:96–100). Mfrn1, the principle iron-importer in the mitochondria, is essential for heme and iron-sulphur (Fe/S) cluster syntheses in erythroblasts, and is required for primitive and definitive erythropoiesis. The biochemistry for Mfrn1-mediated iron acquisition into the mitochondria of developing erythroblasts, however, is poorly understood. In order to identify Mfrn1-associated proteins involved in mitochondrial iron homeostasis, we employed the strategy of in vivo epitope-tagged affinity purification and mass spectrometry (MS). A “bait protein,” Biotag-mouse Mfrn1 (BT-Mfrn1), was engineered to affinity-purify the associated proteins. A series of control experiments were first conducted to demonstrate that the BT-Mfrn1 protein properly targeted to the mitochondria and functionally complemented the anemia in frs embryos. A two-dimensional Blue Native gel followed by SDS-PAGE and western blot showed Mfrn1 forms higher-order protein complexes with interacting proteins in the mitochondrial fraction. A stable mouse erythroleukemia (MEL) clone expressing BT-Mfrn1 protein was derived as a source for protein purification. Affinity-purified BT-Mfrn1 protein complexes were analyzed by MS in triplicate runs in comparison to control MEL cells. Abcb10, a GATA-1 induced ATP-binding cassette transporter highly expressed in hematopoietic tissues and involved in heme biosynthesis (