Alex Sheftel [1], An-Sheng Zhang[1,2], Orian Shirihai [3], Prem Ponka* [1]

Dept. of Physiology and Medicine, McGill University, Montreal, QC, Canada [1]; Dept. of Cellular and Developmental Biology, Oregon Health Sciences University, Portland, OR, USA [2]; Biocurrents Research Center, Marine Biological Institute, Woods Hole, MA, USA [3]

During differentiation, immature erythroid cells acquire vast amounts of iron at a breakneck rate.  Proper coordination of iron delivery and utilization in heme synthesis is essential and disruption of this process likely underlies iron loading disorders such as sideroblastic anemia and myelodysplastic syndrome with ringed sideroblasts.  Iron is taken up by the cells via receptor mediated endocytosis, a process whereby diferric transferrin (Tf) binds to its cognate receptor (TfR) on the erythroid cell plasma membrane, followed by internalization of the Tf-TfR complex.  Subsequent to endocytosis, the endosome is acidified by a H+-ATPase, allowing the release of iron from Tf.  Through an unknown mechanism, iron is targeted to the inner membrane of the mitochondria, where the enzyme that inserts Fe into protoporphyrin IX, ferrochelatase, resides.  Although it has been demonstrated that the divalent metal transporter, DMT1, is responsible for the egress of reduced Fe from the vesicle, the immediate fate of the iron atoms after their transport across the vesicular membrane remains unknown.  Because reduced iron is a strong pro-oxidant, contributing to free radical formation through Fenton chemistry, it has been predicted that an iron binding molecule shuttles Fe from the endosome to mitochondria.  However, this much sought iron binding intermediate, that would constitute the labile iron pool (LIP), has yet to be identified.  Thus, we hypothesize that, in hemoglobin-producing cells, there is a direct relaying of Fe from the endosomal machinery to that of the mitochondria.  We have taken two strategies in examining this supposition: 1) a biochemical approach by which the cytoplasm of cells was loaded with an impermeant iron chelator, thus intercepting the delivery of Fe by the putative LIP intermediate, and 2) a morphological approach employing time-lapse confocal microscopy which permits the tracking of iron-loaded endosomes and mitochondria with high spatial and temporal resolutions.  To examine whether iron delivered by Tf for heme synthesis can bypass the cytosol, we have loaded reticulocytes with a high-molecular weight version of desferrioxamine, hDFO, prior to incubation with 59Fe-Tf.  The incorporation of transferrin iron into heme was unaffected by hDFO when compared to controls.  Importantly, iron delivered to these cells in a form that freely diffuses across the membrane, iron-salicylaldehyde isonicotinoyl hydrazone (59FeSIH2), was significantly prevented from being used for heme synthesis in hDFO-laden reticulocytes.  Using confocal microscopy, as well as polarized light microscopy, we found that endosomes are very mobile organelles.  Immediately following budding from the plasma membrane, these organelles continuously traverse the cytosol and touch a number of mitochondria multiple times.  Experiments using various pharmacological agents indicate that these movements are mediated by components of the cytoskeleton which are essential for proper iron delivery for use in heme synthesis.  Together, these data suggest that iron is directly delivered to mitochondria by endosomes in a “kiss and run” paradigm.  Our current studies will examine the required components and regulation of this interaction using the same experimental strategies as well as a cell free system consisting of isolated organelles.