Intraendosomal Transferrin Saturation Governs Interorganellar Association for Iron Delivery In Erythroid Cells

Abstract 4253

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 Fe is released from Tf by a process involving endosomal acidification. Iron, following its reduction to Fe²⁺ by Steap3 (six-transmembrane epithelial antigen of the prostate 3), is then transported across the endosomal membrane by the divalent metal transporter, DMT1. Unfortunately, the post-endosomal path of Fe 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 must enter mitochondria since ferrochelatase, the final enzyme in the heme biosynthetic pathway that inserts Fe²⁺ into protoporphyrin IX, resides in the inner part of the inner mitochondrial membrane. In erythroid cells, strong evidence exists for specific targeting of Fe toward mitochondria: 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). In the present study, we developed two additional experimental strategies to seek further support for the above hypothesis. First, instead of using two-dimensional (2D) confocal microscopy (Sheftel et al. 2007), we generated 3D confocal images using a Quorum WaveFX Spinning Disc Confocal System: a laser-based system using optimized Nipkow Spinning Disc Technology capable of acquiring confocal images at video rates, allowing us to capture images of endosomes interacting with mitochondria in space, excluding the possibility of “pseudo-interactions” caused by coincidental overlapping. This technique clearly revealed contact of endosomes with mitochondria in 3D space. Moreover, we developed a novel method using a flow subcytometry to analyze lysates obtained from reticulocytes with fluorescently labeled endosomes (Alexa Green Transferrin) and mitochondria (MitoTracker Deep Red). Using this strategy, we have identified three distinct flow particulate populations: endosomes, mitochondria, and a population double labeled with both fluorescent markers. The size of the double-labeled population (presumably representing endosomes associated with mitochondria) increases with incubation time and reaches a plateau in ∼20 min. Reticulocyte re-incubation with unlabeled Fe₂-Tf leads to a time-dependent decrease of the dual-labeled population, indicating a reversible nature of mitochondria-endosome interactions. Bafilomycin A1, which blocks endosome acidification and Fe release from Tf, slows down the generation of the double-labeled population. One likely explanation for this observation is that bafilomycin A1 blocks Fe²⁺ export from endosomes already associated with mitochondria, extending their residence time on these organelles, thus causing a “traffic jam” that decelerates the movement of endosomes containing fluorescent Tf towards mitochondria. Similar effects were obtained using heme, which was shown to feedback inhibit the release of iron from Tf within reticulocytes endosomes (Ponka et al. Biochem J 251:105, 1988). The presence of heme during the re-incubation of double-labeled reticulocytes with unlabeled Fe₂-Tf slowed down the disappearance of the double population, suggesting that the efficient dissociation of endosomes requires them to be iron-free. Taken together, our new data in support of this “kiss-and-run” behavior of organelles in erythroid cells suggest that a molecular mechanism is in place which coordinates the iron status of intraorganellar Tf with endosomal trafficking.