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.