Defective Recycling and Exocytosis of Transferrin in Reticulocytes of hbd (Hemoglobin Deficit) Mice.

Mice with hemoglobin deficit (hbd) have a hypochromic, microcytic anemia inherited as an autosomal recessive. The mice also exhibit reticulocytosis, hyperferremia and elevated free protoporphyrin in red cells (Bannerman et al 1986 Proc Soc Exp Biol Med 182:52). The spontaneous mutation has been mapped to chromosome 19 (Bloom et al 1998 Mamm Genome 9:666), making it ripe for identification of the gene, given the power of mouse genetics. Until the actual locus is determined, however, some mice must be identified as +/? as dominant inheritance obscures the second allele. Prior studies established that hbd/hbd reticulocytes take up 125I-labelled transferrin as well as +/? controls, but are less effective at incorporating 59Fe from transferrin (Garrick et al 1987 Exp Hemato 15:671). Scatchard analysis showed the affinity of transferrin receptors for transferrin was similar in hbd/hbd versus +/? reticulocytes; slightly more receptors were present, however, on the surface of hbd/hbd reticulocytes. We now report on aspects of transferrin-dependent iron metabolism in hbd/hbd reticulocytes to resolve the nature of the alterations in the transferrin cycle in these rodent mutants. Improved time resolution for 59Fe uptake from transferrin by hbd/hbd versus +/? reticulocytes reveals that uptake in the first 2.5 min is indistinguishable; but that, by 5 min, incorporation by the mutant cells lags behind. Similarly, endocytosis of 125I-labelled transferrin by mutant versus control reticulocytes occurs at essentially the same rate; while exocytosis is about 4-fold slower for hbd/hbd reticulocytes compared to +/? reticulocytes. We conclude that recycling and exocytosis of transferrin in reticulocytes of hbd/hbd mice is strikingly impaired. The mutation in these mice should be in a gene that causes this impairment.

Identification and Characterization of an Erythrocyte Endosomal Ferrireductase Critical for Transferrin Dependent Iron Uptake.

The transferrin cycle is a critical pathway for iron uptake by developing red blood cells. Defects in the cycle typically result in a hypochromic microcytic anemia due to ineffective hemoglobin synthesis. Recently we described a new microcytic mouse mutant, nm1054, with an autosomal recessive defect in transferrin mediated iron uptake. We now have identified the gene responsible for the defect through positional cloning and complementation experiments. The gene, mammalian ferrireductase 1 (Mfre1) encodes a predicted six pass transmembrane protein with homology to oxidoreductases of archaea and bacteria as well as homology to the yeast family of FRE proteins. Mfre1 is expressed highly in hematopoietic tissues and co-localizes with the transferrin cycle endosome of cells. Over-expression of Mfre1 in cultured cells stimulates reduction of iron while reticulocytes deficient in Mfre1 are defective in conversion of ferric iron to a ferrous form. Here we comprehensively describe the role of Mfre1 in iron metabolism and speculate on the role of several other novel homologues in iron homeostasis.

A Mutation in Sec15l1 Disrupts the Transferrin Cycle and Causes Anemia in Hemoglobin Deficit (hbd) Mice.

Mouse mutants have been used to identify and characterize genes involved in iron homeostasis. The mouse mutant hemoglobin deficit (hbd), has inherited hypochromic, microcytic anemia and elevated zinc protoporphyrin in the setting of replete iron stores, consistent with a selective defect in erythroid iron assimilation. Circulating soluble transferrin receptor, a marker of iron-restricted erythropoiesis, is elevated out of proportion to the anemia. A mapping population of >3300 backcross animals allowed assignment of the hbd locus to a 0.5 Mb region on chromosome 19. Seven candidate genes were identified in mouse genome databases. Only Sec15l1, encoding one of two mouse homologs of yeast Sec15p, carried a mutation unique to hbd. The mutation deleted 4.5 kb of Sec15l1 genomic DNA including a 69 bp exon. Two distinct antibodies elicited against murine Sec15l1 demonstrated that there was little or no Sec15l1 protein detectable in tissues from hbd animals, suggesting that the mutant form was unstable in vivo. To confirm that mutation of Sec15l1 was responsible for the hbd phenotype, hbd bone marrow cells were transduced with a retrovirus expressing wild type Sec15l1 and transplanted into hbd mice. Expression of the wild type protein partially corrected the anemia. For further confirmation, mice carrying a different, targeted mutation in Sec15l1 were bred with hbd mice. Compound mutant mice carrying both the targeted and hbd alleles recapitulated the hbd phenotype. Sec15l1 functions as a component of the mammalian multi-protein exocyst complex. Previous studies in model organisms suggested that the exocyst is critical for a variety of cellular functions; loss of other exocyst proteins caused early lethality. We speculate that Sec15l2, a mammalian homolog of Sec15l1, can substitute in non-erythroid tissues. Expression analysis showed that little or no Sec15l2 was present in hbd spleen, which is massively infiltrated with erythroid precursors. The exocyst is an effector for Rab11, a GTPase involved in recycling of transferrin cycle endosomes. In S. cerevisiae, Sec15p interacts both with Sec10p, another exocyst component, and with a yeast homolog of Rab11. Preliminary studies suggest that the hbd form of mammalian Sec15l1, expressed in yeast, retains its ability to interact with both of its mammalian partner proteins - Rab11 and Sec10. We conclude that depletion of Sec15 in hbd mice alters transferrin receptor recycling and, possibly, enhances transferrin receptor shedding. As a consequence, erythroid iron assimilation is impaired. Sec15L1 should be considered a candidate gene in patients with inherited iron-resistant iron deficiency anemia.

Gene expression of divalent metal transporter 1 and transferrin receptor in duodenum of Belgrade rats

Regulation of iron absorption is thought to be mediated by the amount of iron taken up by duodenal crypt cells via the transferrin receptor (TfR)-transferrin cycle and the activity of the divalent metal transporter (DMT1), although DMT1 cannot be detected morphologically in crypt cells. We investigated the uptake of transferrin-bound iron by duodenal enterocytes in Wistar rats fed different levels of iron and Belgrade (b/b) rats in which iron uptake by the transferrin cycle is defective because of a mutation in DMT1. We showed that DMT1 in our colony of b/b rats contains the G185R mutation, which in enterocytes results in reduced cellular iron content and increased DMT1 gene expression similar to levels in iron deficiency of normal rats. In all groups the uptake of transferrin-bound iron by crypt cells was directly proportional to plasma iron concentration, being highest in iron-loaded Wistar rats and b/b rats. We conclude that the uptake of transferrin-bound iron by developing enterocytes is largely independent of DMT1.