Potential Roles of DMT1 and Zip8 in Release of Iron from Lysosomes During Mobilization of Iron Stores in Mammalian Cells

In mammalian cells, excess iron is stored in ferritin. Iron stores are mobilized when a cell or organism becomes deficient. Iron deprivation is accompanied by movement of ferritin from the cytosol to the lysosomes, and inhibition of the activity of lysosomal proteases reduced or prevented release of iron from ferritin. This indicated that the iron inside ferritin only becomes available once the protein has been degraded. 

The question is then how the iron returns to the cytosol for utilization by the cell and/or release into the blood. We are testing the hypotheses that divalent metal transporter 1 (DMT1) and Zip8 could be mediating export from lysosomes, since both of these transporters occur in the membrane of that organelle.

These ideas were tested using a rat hepatoma cell line (H4-II-E-C3), knocking down expression of both transporters with siRNA, checking knockdown with quantitative PCR and immunoblotting, and testing for retention of iron by lysosomes in cells using separation of organelles on density gradients prepared with iodixanol.

siRNA knockdown over 72 hr indicate no change in DMT1 protein expression. However, double treatment with siRNA, twice over 6 days, seemed to reduce expression by about half in the case of both DMT1 and Zip8. DMT1 responded to iron deprivation by increasing its association with lysosomal membranes – shown by confocal microscopy.

The results so far suggest that DMT1 has a long half-life and is the main transporter involved in returning iron from lysosomes back to the cytoplasm to meet cellular and organismal needs.