REDUCTION SITE OF TRANSFERRIN-DEPENDENT AND TRANSFERRIN-INDEPENDENT IRON IN CULTURED HUMAN FIBROBLASTS

Mammalian cells internalize iron as diferric transferrin (Fe(2)Tf) iro n via receptor-mediated endocytosis (RME) and a redox mechanism under physiological condition and as an iron salt through the Tf-independent iron uptake (Tf-IU) system under morbid conditions. We have previousl y shown that Tf iron is reduced at the Tf molecule through a redox sys tem on the plasma membrane prior to uptake [Oshiro, S., Nakajima, H., Markello, T., Krasnewich, D., Bernadini, I., and Gahl, W. (1993) J. Bi ol. Chem. 268, 21586-21591]. In the present study, the reduction site for Tf iron uptake via RME and for Tf-independent iron uptake via the Tf-IU system were examined using specific iron sources and well-charac terized ferric or ferrous iron chelators in cultured human fibroblasts . At 4 degrees C for 1 h, although [Fe-55](2)Tf was not internalized i nto the cells, Fe-55 from [55Fe](2)Tf-Sepharose was taken up. However, under the same conditions, EDTA or dipyridyl removed the radioactive iron as a ferric or ferrous iron-chelator complex from [55Fe](2)Tf bou nd to the Tf receptor on the cell surface. Moreover, after Fe-55-citra te was loaded into the cells at 4 degrees C for 1 h, Fe-55 was removed from the cell surface by dipyridyl just as observed for [Fe-55](2)Tf- Sepharose. In inhibition experiments, ferric citrate showed a dose-dep endent inhibition of the iron uptake of both Tf iron and Tf-independen t iron. Conversely, Fe(2)Tf dose-dependently inhibited the uptake of F e-55-citrate. These results suggest that Tf-independent iron as well a s Fe(2)Tf is reduced at the plasma membrane prior to uptake, as the fe rric reduction of Fe(2)Tf taken up by the redox mechanism occurs at th e same site.