TRANSFERRIN AND IRON DISTRIBUTION IN SUBCELLULAR-FRACTIONS OF K562 CELLS IN THE EARLY STAGES OF TRANSFERRIN ENDOCYTOSIS

Iron distribution in subcellular fractions was investigated at different times after a single cohort of Fe-59-I-125-labeled transferrin (Tf) endocytosis in K562 cells. Cell homogenates prepared by hypotonic lysis and deoxyribonuclease (DNAase) treatment were fractionated on Percoll density gradients. Iron-containing components in the postmitochondrial supernatant were further fractionated according to their molecular weight using gel chromatography and membrane filtration. In the initial phases of endocytosis, both iron and Tf were found in the light vesicular fraction. After 3 min the labels diverged, with iron appearing in the postmitochondrial supernatant and Tf in the heavy fraction containing mitochondria, lysosomes and nuclei. Iron released from Tf-containing vesicles appeared both in low- and high-molecular-weight fractions in the postmitochondrial supernatant. After 5 min of endocytosis Fe-59 activity in the low-molecular-weight fraction remained constant and Fe-59 accumulated in a high-molecular-weight fraction susceptible to desferrioxamine chelation. After 10 min, Fe-59 radioactivity in this fraction decreased and a majority of cytosolic Fe-59 was found in ferritin. These results do not support the concept of the cytosolic low-molecular-weight iron pool as a kinetic intermediate between transferrin and ferritin iron in K562 cells.