The long-term objective of the proposed studies is to define the mechanisms of mammalian heme transport and utilization at the cellular level, and the impact of heme cellular pathways on nutritional iron metabolism. Heme is essential for virtually all organisms. In humans, heme-iron (iron-protopophyrin IX) is a significant dietary source of bio-available iron, and >60% of total body iron is bound to heme in hemoglobin. Within the reticuloendothelial system, macrophages (M&s) play a major role in the recycling of heme-iron via phagocytosis of senescent red blood cells (i.e. erythrophagocytosis), and proteolytic degradation of hemoglobin. How heme is extracted from senescent erythrocytes and transported across the M&phagolysosomal membrane is unknown. Once transported out of the phagolysosome, heme is either degraded by the heme oxygenase family of enzymes for the recycling of iron or incorporated in toto into cellular hemoproteins. Because heme is a hydrophobic and cytotoxic macrocycle, its transport across cellular membranes and delivery to specific intracellular compartments are likely to be tightly regulated. Our laboratory has identified the first eukaryotic heme importer/transporter HRG-1, using the genetic animal model Caenorhabditis elegans (Nature 2008). HRG-1 is conserved in mammals, and studies in our laboratory have demonstrated that both worm and human HRG-1 bind and transport heme. In zebrafish, knockdown of hrg-1 results in yolk tube defects, hydrocephalus, and severe anemia;and these defects are fully rescued by worm HRG-1. Collectively, our studies have identified a new pathway for cellular transport of heme in animals. The studies in this proposal are specifically designed to elucidate the mechanisms of HRG-1-mediated heme transport in M&s during erythrophagocytosis. The degradation of senescent red blood cells occurs in the phagolysosomal compartment of M&s. We seek to test the hypothesis that HRG-1 transports heme out of the phagolysosome following erythrophagocytosis in M&s. We plan to use primary mouse macrophages derived from the bone-marrow to genetically and cell biologically dissect the function of HRG-1. The work described in this proposal is significant because it will provide new mechanistic insights into the processes of heme transport and iron recycling from senescent RBCs - pathways that have remained poorly understood. PUBLIC HEALTH RELEVANCE: Iron-deficiency anemia is the most prevalent nutritional disorder worldwide. According to the World Health Organization, four out of five people in the world may be iron deficient, making nutritional iron deficiency one of the top ten risk factors for losing healthy years of life in both developed and developing countries. In the United States, iron deficiency is most prevalent among minority females and young children. Perinatal iron deficiency negatively impacts intelligence and cognition in children. Heme is the most bioavailable form of iron for human consumption. Understanding the molecular mechanisms of heme recycling from senescent red blood cells will permit the synthesis of novel heme- and porphyrin -based nutritional therapies for patients suffering from anemia and nutritional Fe deficiency, including pregnant mothers and infants.