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How ferritin-Fe becomes available for cell functions is unknown. Our previous studies with rat hepatoma cells indicated ferritin had to be degraded to release its Fe. In these studies, we investigated whether this occurs in other cell types and whether lysosomes are required. Release of ferritin-Fe was induced with desferoxamine (DFO) in (59)Fe-preloaded hepatoma, Caco2, and erythroid K562 cells and measured by rocket immunoelectrophoresis and autoradiography. The half-lives for ferritin-(59)Fe and protein were parallel (23, 16, and 11 h for the hepatic, Caco2, and K562 cells, respectively). Co-treatment with 180 microM Fe, leupeptin, chymostatin, or chloroquine markedly decreased rates of ferritin-Fe release and ferritin degradation. Lactacystin had no effect except for a small one in erythroid cells. Fractionation of hepatoma cell lysates on iodixanol gradients showed rapid depletion of cytosolic ferritin by DFO treatment but no accumulation in lysosomes. We conclude that regardless of cell type, release of Fe from ferritin occurs mainly through lysosomal proteolysis.

Iron can be released from the interior of its storage protein, ferritin, in vitro, through reduction and chelation by low molecular weight agents that penetrate structural pores. However, we showed in previous work (J Biol Chem 276: 48775-80, 2001; Am J Physiol 291: C445-55, 2006) that cells with different modes of Fe metabolism primarily access ferritin Fe by digesting the protein in lysosomes. Here we show in cultured rat hepatoma cells and livers of mature male Sprague Dawley rats that when Fe is needed, high-Fe ferritin is preferentially transferred from cytoplasm to lysosomes, and some DMT1 also shifts to the lysosomal compartment. Using separation of cytoplasm and lysosomes by differential centrifugation or in gradients, and determining Fe:protein ratios of ferritin purified from the cytoplasm and lysosomes, we found that Fe deprivation caused a marked drop in Fe:protein ratio for cytoplasmic ferritin and movement of ferritin into lysosomes but no accumulation. However, leupeptin (inhibitor of lysosomal proteases) did cause lysosomal ferritin accumulation. Confocal microscopy showed that some ferritin was always in lysosomes, and some DMT1 in lysosomal membranes. However, Fe deprivation increased DMT1 association and bafilomycinA1 prevented it, implying involvement of protons in regulation of this process. [ABSTRACT FROM AUTHOR]