PROJECT SUMMARY The overall objective of this proposal is to identify and characterize novel proteins involved in erythroid iron/heme metabolism. Erythropoiesis is a massive exercise in cellular proliferation and synthesis of a single protein, hemoglobin. As a consequence, there is a tremendous demand for iron and heme to be efficiently trafficked within the developing erythron. Despite advances in our understanding of extra-cellular iron trafficking and proto-porphyrin biosynthesis, significant gaps remain, especially with respect to components involving the egress of iron from the endosomes to the mitochondria, the trafficking of iron/heme within the mitochondria, the transporters required for proto-porphyrin genesis, the cofactors that facilitate the intracellular trafficking of iron/heme, and the eventual export of heme from the mitochondria for its incorporation in hemoglobin. Using complementary approaches of genetics and bioinformatics from transcriptional profiling, we previously identified several proteins, such as Mitoferrin1 (Mfrn1), Sorting Nexin3 (Snx3), Tmem14c, Lat3, and Clpx1, as new components in the intracellular trafficking of iron, heme and nutrients crucial to red cell development. Although transcriptional profiling as provided insights, we showed that post-translational mechanisms play equally critical roles in the expression and function of proteins involved in iron and heme metabolism. Using quantitative mass spectrometry, we examined changes in the mitochondrial proteome as erythroid cells undergo maturation. We identified several solute carriers and transmembrane proteins, whose function in erythropoiesis have not been previously ascribed, that were induced with hemoglobinization. We propose to study the expression and loss-of-function phenotype of these 7 candidate genes (Aim 1). In particular, we plan to focus previously identified gene, Fam210b (c20orf108), and its interacting partners in red cell development (Aim 2). Functional elucidation of these structural genes will expand our knowledge into the unknown additional steps in intracellular solute, iron and heme trafficking crucial for erythropoiesis. The results of our proposal will provide us with new genetic tools to explore human disorders of anemias.