The uptake of transferrin-bound iron by receptor-mediated endocytosis has been the subject of extensive experimental investigation. However, the path followed by iron (Fe) after release from transferrin (Tf) remains obscure. Once Fe is released from Tf within the endosome, it must be transported across the endosomal membrane into the cell. The present investigation describes the presence of a cytoplasmic Tf-free Fe pool which is detectable only when cells are detached from their culture dishes at low temperature, after initial incorporation of diferric transferrin at 37-degrees-C. This cellular iron pool was greatly reduced if incubation temperatures were maintained at 37-degrees-C or if cells were treated with pronase. Human melanoma cells (SK-MEL-28) in culture were prelabeled by incubation with human I-125-Fe-59-transferrin for 2 h, washed, and reincubated at 4-degrees-C or 37-degrees-C in balanced salt solution in the presence or absence of pronase. The cells were then mechanically detached from the plates and separated into "internalized" and supernatant fractions by centrifugation. Approximately 90% of cellular Fe-59 and 20% of I-125-Tf remained internalized when this reincubation procedure was carried out in balanced salt solution at 37-degrees-C. However, at 4-degrees-C, cellular internalized iron was reduced to approximately 50% of the initial value. The release of this component of cellular Fe-59 (approximately 40% of total cell Fe-59) at 4-degrees-C was completely inhibited in the presence of pronase and other general proteinases at 4-degrees-C and at 37-degrees-C, without affecting internalized transferrin levels. Similar results were obtained in fibroblasts and hepatoma cells, indicating that this phenomenon is not unique to melanoma cells. The characterization of this Tf-free cellular Fe pool which is detectable at low temperature may yield valuable insights into the metabolic fate of iron following its transport across the membrane of the endocytotic vesicle.
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1992|