Effect of wtFPN and FPN mutants on iron accumulation and iron release. (A-B) Iron accumulation. 293T cells were transfected as for Figure 1, grown for 8 hours, and then fed 40 μg/mL ⁵⁹Fe-Tf for 20 hours. Cells were then washed 3 times and counted, and cellular ⁵⁹Fe in the pellets was measured. Each point represents the uptake into 1 aliquot of 10⁶ cells. Panels A and B show that cells transfected with wtFPN accumulate 35% to 40% as much radioactive iron as untransfected control or CD8 transfectants. Mutants A77D, V162Δ, L170F, and G490D are able to accumulate ⁵⁹Fe to the same levels as CD8 control cells, while in the case of C326Y, N144D, Y64N, and N144H levels are lower and comparable to wild type. (C-D) Iron release. Cells were grown in 40 μg/mL ⁵⁹Fe for 24 hours, then washed and transfected as in panels A and B. Fifteen hours later cells were harvested, washed, and resuspended at 10⁶ cells/mL in serum-free medium, and ⁵⁹Fe export was measured at the intervals shown up to 36 hours after harvesting. Data are presented as a percentage of starting radioactivity when cells were harvested. Panels C and D show that cells transfected with wtFPN release up to 3 times as much ⁵⁹Fe as untransfected control or CD8 transfectants. Mutants A77D, V162Δ, L170F, and G490D are only able to release ⁵⁹Fe to levels similar to CD8 control cells, while in the case of C326Y, N144D, Y64N, and N144H iron release is higher and comparable to wtFPN. These results are representative of 3 experiments.