Transferrin Uptake by Cultured Rat Embryo Fibroblasts

The uptake of doubly labelled ( 59 Fe and 3 H] rat serum transferrin by rat embryo fibroblasts in culture has been studied. 59 Fe is taken up by the cells in a process dependent on time and temperature which, at 37° C, is almost saturable with transferrin concentration. The iron is incorporated in part into ferritin. In contrast, the uptake of 3 H label at 37° C is much less than that of 59 Fe and is associated with a saturable uptake of demonstrably intact transferrin and with the time‐dependent and concentration‐dependent release of transferrin degradation products from the cells. Low temperature binding studies indicate that 60–100 × 10 3 transferrin molecules are bound to high‐affinity specific plasma‐membrane receptors ( K a : 1.1 × 10 7 M −1 ), and that a large number of low‐affinity sites also exist. Transferrin bound to low‐affinity sites can be readily released from the cells by washing or by reincubation in fresh medium. Short‐term kinetic experiments at 37° C imply that a mechanism exists whereby iron is rapidly taken up from transferrin and the iron‐depleted transferrin is released intact to the extracellular medium. After 4 min (1‐min incubation at 37° C, 1‐min washing and 2‐min reincubation) half of the [ 3 H]transferrin is released into the medium while almost all of the 59 Fe remains associated with the cells. On the basis of our results we suggest that iron uptake from transferrin involves both receptor‐mediated and flluid‐phase endocytosis. In the former, the fusion of the endocytic vacuole with a lysosome would provide a favourable environment for iron release; the iron‐depleted transferrin would then be returned to the extracellular medium during the recycling of the membrane. In the latter, after the pinocytic vacuole gains access to lysosomes the iron would be released and the protein digested. In both mechanisms the iron would pass into the cytosol and be incorporated into ferritin or be used for mitochondrial haem synthesis.