Transferrin Receptor 2 Protein Is Expressed in Mitochondrial-Associated Lysosomes During Human Erythropoiesis.

Abstract 628

Regulated expression of transferrin receptor 1 (TFR1) provides for the major mechanism of iron binding and absorption by endocytosis in erythroblasts. In contrast, the expression of transferrin receptor 2 (TFR2) in erythroid cells is not well understood. Fourteen-day cultures of human CD34+ cells were utilized to explore TFR2 gene and protein expression during erythropoiesis. Transcriptome analyses revealed an expression of the TFR2 gene on culture days 7 and 14. Protein analyses were performed with a TFR2 antibody that demonstrated a doublet band at 105 Kd in K562 and HepG2 cells, but no band in Jurkat cells. Western analyses of the primary erythroblasts demonstrated maximum TFR2 levels on culture days 6-8 coinciding with the onset of high-level glycophorin A expression and just prior to hemoglobin accumulation in the cells. Cellular localization of TFR2 was assessed by dual-staining confocal microscopy. More than 15 cells were analyzed for the calculation of correlation coefficients from 4-6 separate pictures per each sample. Unlike TFR1, expression of TFR2 on the plasma membrane was low or absent at all stages of erythroblast maturation. Expression of both proteins was detected in the endosomes throughout the cytoplasm on culture days 2-6. After culture day 6, TFR1 and TFR2 localization became divergent, with TFR2 localized almost exclusively to a single perinuclear compartment near the Golgi stack in the region of the centrosome. Co-localization experiments indicated that TFR2 was most highly co-localized with LAMP1, a lysosome marker. DMT1, the divalent metal transporter was also colocalized with the TFR2-lysosomes. The TFR2-lysosomes were additionally surrounded by and closely associated with mitochondria. Live cell imaging revealed dynamic interactions between the lysosomes and surrounding mitochondria. Pulse-chase experiments using Alexafluor 594-labeled transferrin (Tf) showed that endocytosed Tf was initially co-localized with TFR1. However, Tf was transported to the TFR2 localized region after a chase period of 10 minutes. In summary, TFR2 protein expression is highly regulated during erythropoiesis and localized to centrosomal lysosomes. These studies further suggest that iron regulation in erythroblasts includes trafficking to TFR2 and DMT1 containing lysosomes, which are dynamically associated with mitochondria.