Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37°C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5/~m. Inside these large MVEs are round bodies of ~50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nrn bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the ~50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation.It is well known that the transferrin receptor is lost during the maturation of the reticulocyte into the erythrocyte (1-4). Recent studies have shown that the maturation process can be followed in vitro (4, 5-7). The loss of the transferrin receptor can be used as a marker of maturation (4-6), the transferrin receptor being released, undegraded, to the medium in vesicular form (6, 7). The transferrin receptor is known to recycle many times during the course of Fe 3÷ delivery without degradation of either the receptor or the natural ligand, transferrin (8-l 1). It has been shown in several systems (8,(12)(13)(14)(15)(16)(17)(18)(19)(20), using either labeled transferrin or antibody against the transferrin receptor, that the ligand and the receptor are internalized into vesicles (endosomes) during incubation at temperatures above 10*C and that this internalization may constitute part of the iron delivery mechanism.Since the transferrin receptor is largely lost from sheep reticulocytes during 24 h of incubation in vitro (5-7), intermediate stages associated with vesicle externalization during receptor elimination might become apparent during the longterm incubation of reticulocytes. No studies to date have addressed the question of the processing that may occur to prepare the receptor for externalization during reticulocyte maturation. We have previously shown (6) that a polyclonal 942 antibody against the transferrin receptor is internalized (as judged by resistance to acid treatment), and that during the course of long-term incubation (several hours at 37"C), this internalized antibody and the surface-bound antibody are lost from the cells along with the antibody-binding capacity of the cells. Since no degraded antibody was found, both internal and surface antibody appear to be eliminated from the cells without degradation.The rate of loss of internalized 125I-antibody as we...

Sheep reticulocytes from phlebotomized animals have a total transferrin binding potential that may exceed by an order of magnitude the surface binding capacity. Steady state uptake of transferrin at 37 degrees C is generally less than 50% of the total transferrin binding capacity. During long-term incubation of the reticulocytes, all transferrin binding ability is lost, the ability to internalize being lost most rapidly. The loss in ability to bind transferrin during long-term incubation is independent of the number of surface transferrin binding sites, since removal of surface receptors with pronase does not affect the rate of loss of the internal pool of receptors during long-term incubation. Moreover, after removing surface receptors with pronase, only a fraction of the original number of receptors is restored to the surface, despite the presence of a large pool of internal receptors. These data suggest that only a fraction of the internal pool of receptors is capable of recycling to the cell surface in sheep reticulocytes.