Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells.
The subcellular location of radiolabeled transferrin (125I-Tf), internalized during cellular iron uptake, and the cellular distribution of transferrin (Tf) receptors were studied in cultured HeLa cells. Cells were incubated at 37 degrees C with 125I-Tf(Fe)2. Forty per cent of the labeled ligand was associated with cell surface receptors. The remaining 60% was internalized as shown by the inability to dissociate 125I-Tf from cells by competition with excess Tf(Fe)2 or treatment of cells with 0.2 M acetic acid containing 0.5 M NaCl. Subcellular fractionation studies using sucrose density gradients indicated that internalized Tf was localized in a membranous vesicle distinct from lysosomes, Golgi apparatus, endoplasmic reticulum, or plasma membranes. The subcellular distribution of Tf receptors was studied using an assay for detergent solubilized receptors. Even without preincubation with ligand, the majority of cellular Tf receptors were localized intracellularly in a vesicle with the same buoyant density as the vesicle containing internalized 125I-Tf. Using an assay for occupied receptors, we demonstrated that the same vesicle contained both internal receptors and internalized ligand. A portion (20%) of the intracellular receptor pool was insensitive to trypsin treatment of whole cells at 37 degrees C suggesting that during the experimental time period (20-30 min) this portion did not recycle to the cell surface. We propose that during cellular iron uptake, Tf receptor-ligand complexes are internalized and directed to a nonlysosomal compartment where iron is released, followed by recycling to the cell surface of an intact Tf receptor-apo-Tf complex.
D001692:Biological Transport; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011990:Receptors, Transferrin; D013347:Subcellular Fractions / Q000378:metabolism; D014168:Transferrin / Q000302:isolation & purification / Q000378:metabolism*
D007457:Iodine Radioisotopes; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin