The long-term objective of the proposal is to understand the mechanisms of iron transport into erythroid cells. All mammalian cells have a requirement for iron and acquire this iron from the plasma iron transport protein transferrin. Transferrin binds to specific receptors on the surface of cells and is then internalized by a process of receptor mediated endocytosis, journeying first from coated pits to clathrin coated vesicles and then, depending on the cell type, to the transreticular portion of the Golgi or to multilaminated vesicles prior to exocytosis. Throughout this passage the transferrin-receptor complex is always within a vesicle so that iron released from transferrin has to cross vesicle membrane to gain access to the cytosol. In a series of studies with rabbit reticulocytes a complex route for iron has been defined in which: 1) the most rapid step after binding of transferrin to its receptor is the release of iron from transferrin with kinetics consistent with substrate utilization; 2) the released iron passes as an intermediate through plasma membrane, cytosolic and mitochondrial compartments; 3) iron is incorporated into home with the kinetics of a final product; and 4) the rate limiting step is the release of apo-transferrin from the receptor. This process will be further examined by isolating and characterizing both the vesicles in which transferrin releases its iron and the membrane iron transport protein. Vesicles will be isolated by isotonic lysis of reticulocytes followed by Percoll gradient fractionation and characterized for proton pump and oxidoreductase activities, both of which are necessary for iron release from transferrin. These processes will be examined in earlier erythroid cells, represented by the hematopoietic cell lines K562 and MEL, defining the kinetics of iron transport and localizing the compartment in which iron is released. In both cell types mathematical modeling will be attempted in an effort to put forth testable hypotheses explaining kinetic observations. In addition, mature erythrocytes which, having been infected with P. falciparum, have regained the capacity to transport iron from transferrin will be examined to determine if the malaria parasite unmasks a hidden iron uptake mechanism or synthesizes the iron transport mechanism de novo. In particular, evidence for the transferrin receptor in P. falciparum DNA will be sought by hybridization techniques.
