FIGURE 3.
The N-terminal cytoplasmic region of DMT1 and the C-terminal cytoplasmic region of FPN1 are essential for their binding to PCBP2 in mammalian cells. A, the schematics show the full-length or deletion mutants of DMT1 or FPN1. The N-terminal cytoplasmic region of full-length DMT1A-I consists of 98 amino acids. DMT1ΔN is a deletion mutant of DMT1 that lacks amino acids 2–98. Amino acids 534–572 correspond to the C-terminal cytoplasmic region of FPN1. FPN1ΔC is a deletion mutant of FPN1 that lacks amino acids 535–572. B, the cellular localization of full-length or the deletion mutants of DMT1 or FPN1. The GFP-tagged deletion mutant forms of DMT1 and FPN1 were transiently transfected with mCherry-tagged full-length forms of DMT1, FPN1, and CPR. Cells were stained with anti-PDI mAb or anti-LAMP2 mAb. The arrowhead shows co-localization of full-length and deletion-mutant forms of DMT1 or FPN1 at the plasma membrane. Similar results were obtained from at least three experiments. C, GFP-tagged full-length and deletion-mutant forms of DMT1 and FPN1 were transiently transfected into HEp-2 cells. The cells were lysed in TNE buffer and analyzed by immunoblotting using α-PCBP2 Ab. Co-immunoprecipitation (IP) was also performed with anti-GFP pAb-conjugated protein A beads. These samples were analyzed by immunoblotting (WB) using an anti-PCBP2 mAb. Similar results were obtained from at least three independent experiments. D, iron transport activity of full-length and deletion-mutant forms of DMT1 or FPN1. Full-length or deletion-mutant forms of DMT1 were transfected into HEp-2 cells. Cells were loaded with 100 μm FAC for 6 h at 37 °C. The cells were lysed in 50 mm NaOH for the ferrozine-based iron assay. HEp-2 cells stably expressing DMT1A-I were cultured in serum-free DMEM and preloaded with 100 μm FAC for 6 h at 37 °C. The cells were washed with PBS, transfected with the full-length or deletion mutant of FPN1-GFP expression plasmid, and then incubated in serum-free RPMI 1640 medium for 18 h at 37 °C. The cells were then lysed in 50 mm NaOH for the ferrozine-based iron assay. The results shown on the histogram are presented as the means ± S.E. of three independent experiments. Significance was determined by Student's t test: ‡, p < 0.01; §, p < 0.001.