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We have used succinylacetone (4,6-dioxoheptanoic acid), a specific inhibitor of delta-aminolevulinic acid dehydrase, to gain insight into the defect in iron metabolism in the Belgrade anemia. The Belgrade rat has an inherited microcytic, hypochromic anemia associated with poor iron uptake into developing erythroid cells. Succinylacetone inhibits heme synthesis, leading to nonheme iron accumulation in mitochondria and cytosol of normal reticulocytes. When succinylacetone is used to inhibit Belgrade heme synthesis, iron from diferric transferrin does not accumulate in the stromal fraction that contains mitochondria, nor does 59Fe accumulate in the nonheme cytosolic fraction. Hence, the defect in the Belgrade rat reticulocyte occurs in the endocytic vesicle or in a step subsequent to iron transit from the vesicle but before the nonheme cytosolic or mitochondrial iron fractions. Therefore, the mutation affects either the release of iron from transferrin or iron transport from the vesicle to the mitochondrion.

Proteins are required for the efflux of heme from mitochondria and liposomes. The efflux from liposomes is independent of the heme-binding affinity of the protein (Biochem. 23:3715, 1984). We tested whether heme-binding proteins increase efflux of newly synthesized heme from structurally and functionally intact rat liver mitochondria. Mitochondria whose heme was labeled with 14C-delta-aminolevulinic acid, were incubated in the presence of glutathione transferases (GSTs), serum albumin (RSA) or heme-binding protein (HBP), all from the rat. HBP caused a 6-8 fold increase in efflux of newly synthesized heme as compared to that effected by RSA or GSTs. This result indicates that heme efflux from intact mitochondria, unlike that from liposomes, depends on the type of protein present and that HBP may specifically facilitate heme efflux from mitochondria.

Electron-microscopic examination of rat reticulocytes and normoblasts incubated with transferrin conjugated to ferritin or ferritin-labeled antitransferrin revealed binding of ferritin conjugates to the surface membrane, and uptake of ferritin conjugates in micropinocytotic vesicles. No binding or endocytosis of ferritin was visualized when rat reticulocytes or normoblasts were incubated with ferritin alone or ferritin conjugated to nonspecific rabbit IgG. These observations support the concept that transferrin binds to a surface membrane receptor and is subsequently internalized by the developing red cell. Time course and temperature dependence studies suggest the endocytosis of transferrin may be an important mechanism in delivery of iron to the developing red cell.