Iron transport in K562 cells: a kinetic study using native gel electrophoresis and 59Fe autoradiography.
The exact mechanisms of iron transport from endosomes to the target iron containing cellular proteins are currently unknown. To investigate this problem, we used the gradient gel electrophoresis and the sensitive detection of 59Fe by autoradiography to detect separate cellular iron compounds and their iron kinetics. Cells of human leukemic line K562 were labeled with [59Fe]transferrin for 30-600 s and cellular iron compounds in cell lysates were analyzed by native electrophoretic separation followed by 59Fe autoradiography. Starting with the first 30 s of iron uptake, iron was detectable in a large membrane bound protein complex (Band I) and in ferritin. Significant amounts of iron were also found in labile iron compound(s) with the molecular weight larger than 5000 as judged by ultrafiltration. Iron kinetics in these compartments was studied. Band I was the only compound with the kinetic properties of an intermediate. Transferrin, transferrin receptor and additional proteins of the approximate molecular weights of 130000, 66000 and 49000 were found to be present in Band I. The labile iron compounds and ferritin behaved kinetically as end products. No evidence for low molecular weight transport intermediates was found. These results suggest that intracellular iron transport is highly compartmentalized, that iron released from endosomal transferrin passes to its cellular targets in a direct contact with the endosomal membrane complex assigned as Band I. The nature of the labile iron pool and its susceptibility to iron chelation is discussed.
Vyoral
D
D
Institute of Hematology and Blood Transfusion, U nemocnice 1, 128 20, Praha 2, Czech Republic. vyoral@uhkt.cz
Petrák
J
J
D001345:Autoradiography; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D007501:Iron / Q000493:pharmacokinetics*; D007504:Iron Radioisotopes; D008954:Models, Biological; D011990:Receptors, Transferrin / Q000378:metabolism; D014168:Transferrin / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D007504:Iron Radioisotopes; D011990:Receptors, Transferrin; D014168:Transferrin; D007501:Iron