Nuclear translocation of ferritin in corneal epithelial cells

Our previous studies have shown that ferritin within developing avian corne al epithelial cells is predominantly a nuclear protein and that one functio n of the molecule in this location is to protect DNA from UV damage, To elu cidate the mechanism for this tissue-specific nuclear translocation, cultur ed corneal epithelial cells and corneal fibroblasts were transfected with a series of deletion constructs for the heavy chain of ferritin, ferritin-H, tagged with a human c-myc epitope, The subcellular localization of the fer ritin was determined by immunofluorescence for the myc-tag, For the corneal epithelial cells, the first 10 or the last 30 amino acids of ferritin-a co uld be deleted without affecting the nuclear localization. However, larger deletions of these areas, or deletions along the length of the body of the molecule, resulted largely in retention of the truncated proteins within th e cytoplasm. Thus, it seems that no specific region functions as an NLS, Im munoblotting analysis of SDS-PAGE-separated extracts suggests that assembly of the supramolecular form of ferritin is not necessary for successful nuc lear translocation, because one deletion construct that failed to undergo s upramolecular assembly showed nuclear localization. In transfected fibrobla sts, the endogenous ferritin remained predominantly in the cytoplasm, as di d that synthesized from transfected full-length ferritin constructs and fro m two deletion constructs encoding truncated chains that could still assemb le into the supramolecular form of ferritin, However, those truncated chain s that were unable to participate in supramolecular assembly generally show ed both nuclear and cytoplasmic localization, indicating that, in this cell type, supramolecular assembly is involved in restricting ferritin to the c ytoplasm, These data suggest that for corneal epithelial cells, the nuclear localization of ferritin most likely involves a tissue-specific mechanism that facilitates transport into the nucleus, whereas, in fibroblasts, the c ytoplasmic retention involves supramolecular assembly that prevents passive diffusion into the nucleus.