IRON ACQUIRED FROM TRANSFERRIN BY K562 CELLS IS DELIVERED INTO A CYTOPLASMIC POOL OF CHELATABLE IRON(II)

The release of iron from transferrin (Tf) in the acidic milieu of endosomes and its translocation into the cytosol are integral steps in the process of iron acquisition via receptor-mediated endocytosis (RME), The translocated metal is thought to enter a low molecular weight cytoplasmic pool, presumed to contain the form of iron which is apparently sensed by iron responsive proteins and is the direct target of iron chelators, The process of iron delivery into the cytoplasmic chelatable pool of K562 cells was studied in situ by continuous monitoring of the fluorescence of cells loaded with the metal-sensitive probe calcein, Upon exposure to Tf at 37 degrees C, intracellular fluorescence decayed, corresponding to an initial iron uptake of 40 nM/min, The Tf-mediated iron uptake was profoundly inhibited by weak, bases, the protonophore monensin, energy depletion, or low temperatures (<25 degrees C), all properties characteristic of RME, Cell iron levels were affected by the slowly permeating chelator desferrioxamine only after prolonged incubations, Conversely, rapidly penetrating, lipophilic iron(II) chelators such as 2,2'-bipyridyl, evoked swift increases in cell calcein fluorescence, equivalent to sequestration of 0.2-0.5 mu M cytosolic iron, depending on the degree of pre-exposure to Tf, Addition of iron(III) chelators to permeabilized 2,2'-bipyridyl-treated cells, failed to reveal significant levels of chelatable iron(III), The finding that the bulk of the in situ cell chelatable pool is comprised of iron(II) was corroborated by pulsing K562 cells with Tf-Fe-55, followed by addition of iron(II) and/or iron(III) chelators and extraction of chelator-Fe-55 complexes into organic solvent. Virtually all of the accumulated Fe-55 in the chelatable pool could be complexed by iron(II) chelators, The cytoplasmic concentration of iron(II) fluctuated between 0.3 and 0.5 mu M, and its mean transit time through the chelatable pool was 1-2 h, me conclude that after iron is translocated from the endosomes, it is maintained in the cytosol as a transit pool of chelatable iron(II), The ostensible absence of chelatable iron(III) implicates the intracellular operation of vigorous reductive mechanisms.