Further Evidence for the “Kiss and Run” Hypothesis of Iron Delivery to Mitochondria in Erythroid Cells,

Abstract 3178

Delivery of iron (Fe) to most cells occurs following the binding of diferric transferrin (Tf) to its cognate receptors on the cell membrane. The Tf-receptor complexes are then internalized via endocytosis, and iron is released from Tf by a process involving endosomal acidification and reduction by Steap3. Iron is then transported across the endosomal membrane by the divalent metal transporter, DMT1. Unfortunately, the post-endosomal path of iron within cells remains elusive or is, at best, controversial. It has been commonly accepted that a low molecular weight intermediate chaperones iron in transit from endosomes to mitochondria and other sites of utilization; however, this much sought iron binding intermediate has never been identified. In erythroid cells, more than 90% of iron has to enter mitochondria where ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe²⁺ into protoporphyrin IX, resides. Indeed, strong evidence exists for specific targeting of Fe toward mitochondria in developing red blood cells in which iron acquired from Tf continues to flow into mitochondria even when the synthesis of protoporphyrin IX is suppressed. Based on this, we have formulated the hypothesis that, in erythroid cells, a transient mitochondrion-endosome interaction is involved in Fe translocation to its final destination and have collected experimental support for this proposition (Zhang et al. Blood 105:368, 2005; Sheftel et al. Blood 110: 125, 2007).

We have previously shown, using 3D live confocal imaging, that the iron delivery pathway in reticulocytes involves a transient interaction of endosomes with mitochondria. Moreover, we have demonstrated the interaction of these organelles by a novel method exploiting flow cytometry to analyze reticulocyte lysates labeled with Alexa Green Transferrin (AGTf) and MitoTracker Deep Red (MTDR). By using this new technique of flow subcytometry, we identified a double-labeled population representing endosomes interacting with mitochondria. The dynamic nature of this interaction was shown by chase experiments in which a time-dependent decrease of the double-labeled population was observed when reticulocytes were washed and re-incubated with unlabeled Fe₂-Tf. Furthermore, we have shown that the iron status of endosomes governs the efficacy of endosome-mediated iron delivery to mitochondria. Experiments with heme, which feedback inhibited the release of iron from Tf in endosomes, slows the generation of the double-labeled population. Additionally, treatment of cells with heme in chase experiments retarded the dissociation of endosomes from mitochondria.

In this study, we provide further evidence for an interaction between mitochondria with endosomes. Fluorescently-labeled mitochondria isolated from mouse reticulocytes with MTDR and AGTf were analyzed using 2D confocal microscopy. Results from these experiments confirmed that mitochondria indeed come in physical contact with endosomes. In addition, we have used different constructs of fluorescently labeled, recombinant human Tf, which either remain permanently bound to iron (recombinant diferric-transferrin; rTf) or cannot bind to iron (recombinant apotransferrin; rapoTf), in flow subcytometry studies. As expected, these studies showed that reticulocytes incubated with MTDR and rapoTf failed to produce a double-labeled population in uptake experiments. Interestingly, when reticulocyte lysates were incubated with MTDR and rTf, compared to controls using wild type human Tf, the size of the double-labeled population was decreased. This suggests that failure of iron release from rTf may interfere with the process of endocytosis or endosomal trafficking.