Abstract
The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.
Highlights
Transferrin by its resistance to release during treat- The siteof iron release fromtransferrin in immature erythment with Pronase at 4 "C
When a steady state habseen reached between Recent biochemical evidence supports the contention that endocytosisand exocytosis of the ligand, about 90% of receptor-mediated endocytosis of transferrin is involved in the totacl ell-bound transferrin is internalE. ndocytosis iron uptake ina number of cell types, possibly by transporting of transferrin was found to be negligible below 10"C. the transferrin to an intracellular environment which is fa
Like cytosis is 33.0 2 2.7 kcal/mol, whereas from 26-39 "C many ligands internalized by receptor-mediated endocytosis the activation energy isconsiderably lower, at 12.3 f [25], transferrin has been shown to pass through lysosome
Summary
The kinetics of uptake and endocytosisof '251-apotransferinternalization of transferrin was performed [28]. "k,,", was calculated for each temperature from theslopes of semilogarithmic plots of the decrease in internalized transferrin (Fig. 3B).The k,, values obtained were 0.08 min" at 22 "C, 0.19 min" a t 30 "C, and 0.48 min" at 37 "C. These values are almost identical with kthinevalues fortransferrin endocytosis, and imply that endocytosis and exocytosis of transferrin proceed at the same rate. In order to determine whether transferrin was broken down during its uptake by reticulocytes, samples of the cells were incubated with lZ5I-. Therate constant, "kin",was calculated foreach temperatureaccording to the equation: where CI and C, are percentages of the Co value (100%) and represent internal transferrin at times tl and tP,respectively.
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