Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes.

Abstract

At 4°C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37°C receptor-mediated endocytosis of transferrin occurred. Uptake at 37°C exceeded binding at 4°C by 2.5-fold and saturated after 20-30 min. During uptake at 37°C, bound transferrin was internalized into a trypsin-resistant space. Trypsinization at 4°C destroyed surface receptors, but with subsequent incubation at 37°C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37°C colloidal goldtransferrin (Auto clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37°C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatasenegative compartments. AuTf, like transferrin, was released with subsequent incubation at 37°C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.Receptor-mediated binding and endocytosis of transferrin occur in many cell types (4,17,23,24,34,38) and appear to be requisite steps in iron delivery under some conditions (7, 9). Transferrin uptake has been best studied in erythropoietic cells, where the synthesis of hemoglobin requires a large amount of iron. Expression of transferrin receptors in these cells peaks early in development and declines progressively during the maturation of erythroblasts and reticulocytes (23,34,36). Despite their lower level of receptor expression, reticulocytes are a convenient model system, since they may be easily isolated from the blood of anemic animals.Transferrin binding is mediated by protease-sensitive receptors (8) but is not inhibited by glycosidase treatment of either transferrin or its receptor (8,18,22). Transferrin receptors have been identified and characterized by many research groups. The uptake of transferrin is both temperature and energy dependent, and transferrin endocytosis has been demonstrated by the use of EM-autoradiography (9, 21), ferritinor horseradish peroxidase-conjugated transferrin (9, 31), and ferritin-conjugated antitransferrin antibodies (31 ). These techniques reveal that transferrin binds to the plasma membrane at 4"C and appears inside intracellular vesicles during incubation at 37"C. Receptor-mediated transferrin uptake may occur by mechanisms ...

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