Iron Metabolism Anemia hbd Mice Have a Mutation in the Sec15l1 Gene for Vesicle Docking.

Defects in iron absorption and utilization lead to iron deficiency and anemia. Although iron transport in transferrin receptor-mediated endocytosis is well understood, it is not clear how iron is transported from the endosome to mitochondria where it is used to synthesize heme. We undertook a positional cloning project to identify the causative mutation for the hemoglobin deficit (hbd) mouse which suffers from a microcytic, hypochromic anemia associated with a defect in reticulocyte iron uptake. The hbd locus was previously mapped to Chromosome 19 in mouse. We established a mating of B6MOLDF1-+/hbd with C57BL/6J-hbd/hbd mice to generate a high resolution map from 2,454 backcross mice. The hbd critical region was defined by the flanking marker with 6 crossover backcross mice, Kif11, at 0.24 cM on the proximal side, and by the very informative marker with only 1 crossover backcross mouse, Fer1l3 at 0.04 cM on the distal side. The calculated size of the hbd critical region is 836 kb as determined by the sequence available in GenBank contig NT 039689. Our approach for mutation analysis was to amplify exons for each gene in the hbd critical region by genomic PCR using primers derived from flanking sequence. The results were examined for gene deletions, insertions, or large rearrangements as determined by the size of the PCR product generated from normal and mutant DNA. We now report the identification of a strong candidate gene for hbd: Sec15l1, a homologue to yeast SEC15 which has been shown to be a key protein in vesicle docking. The Sec15l1 gene was the only gene in this region with a defect and was shown to have a deletion of exon 8 in hbd genomic DNA. The Sec15l1 partial deletion was verified by Southern blotting. Sec15l1 RNA expression in hbd mice was examined by RT-PCR and subsequent sequencing of the amplicon product. The exon 8 deletion leaves the coding sequence in-frame and a truncated SEC15L1 protein (3 kDa smaller) is predicted to be produced in hbd mice. The deletion in Sec15l1 removes 23 amino acids which includes two amino acids important for tertiary structure: a cysteine residue at position 283 and a proline residue at position 287. Studies of mutant hbd reticulocytes have shown that the binding of transferrin to its receptor and the formation of the endosome is normal but accumulation of iron is deficient in these cells. Iron and transferrin enter the hbd cells but iron does not build up in the cell. The efficient transfer of iron from endosome to mitochondria in normal reticulocytes has been proposed to result from the endosome traversing the cell to dock onto the mitochondria in order to directly transfer iron to it ("kiss and run" hypothesis). In hbd reticulocytes iron may not be transferred efficiently from the endosome to mitochondria because vesicle docking is impaired. This is the first known mutation of a SEC gene homologue in mammals and our findings suggest that SEC15L1 plays a crucial role in the transport of iron in the endocytosis cycle and may involve iron targeting to mitochondria.