Intracellular kinetics of iron in reticulocytes: evidence for endosome involvement in iron targeting to mitochondria

In erythroid cells the vast majority of iron (Fe) released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase that inserts Fe into protoporphyrin IX. In the present study, we developed a method whereby a cohort of Fe-59-transferrin (Tf)-laden endosomal vesicles were generated, from which we could evaluate the transfer of Fe-59 into mitochondria. Iron chelators, dipyridyl or salicylaldehyde isonicotinoyl hydrazone (SIH), were able to bind the Fe-59 when they were present during a 37degreesC incubation; however, addition of these agents only during lysis at 4degreesC chelated virtually no Fe-59. Bafilomycin All (which prevents endosome acidification) and succinylacetone (an inhibitor of 5-aminolevulinate dehydratase) prevented endosomal Fe-59 incorporation into heme. Importantly, both the myosin light chain kinase inhibitor wortmannin and the calmodulin antagonist, N-(6-amino-hexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), caused significant inhibition of Fe-59 incorporation from Fe-59-Tf-labeled endosomes into heme, suggesting that myosin is required for Tf-vesicle movement. Our results reaffirm the astonishing efficiency of Tf-derived Fe utilization in hemoglobin (Hb)-producing cells and demonstrate that very little of this Fe is present in a chelatable pool. Collectively, these results are congruent with our hypothesis that a transient endosome-mitochondrion interaction mediates iron transfer between these organelles. (C) 2005 by The American Society of Hematology.