Divalent metal transporter 1 (DMT1) is responsible for dietary-iron absorption from apical plasma membrane in the duodenum and iron acquisition from the transferrin cycle endosomes in peripheral tissues. Two isoforms of the DMT1 transcript generated by alternative splicing of the 3' exons have been identified in mouse, rat, and human. These isoforms can be distinguished by the different C-terminal amino acid sequences and by the presence (DMT1A) or absence (DMT1B) of an iron response element located in the 3' untranslated region of the mRNA. However, it has been still unknown whether the structural differences between the two DMT1 isoforms is functionally important. Here, we report that each DMT1 isoform exhibits a differential cell type-specific expression patterns and distinct subcellular localizations. DMT1A is predominantly expressed by epithelial cell lines, whereas DMT1B is expressed by the blood cell lines. In HEp-2 cells, GFP-tagged DMT1A is localized in late endosomes and lysosomes, whereas GFP-tagged DMT1B is localized in early endosomes. Using site-directed mutagenesis, a Y(555)XLXX sequence in the cytoplasmic tail of DMT1B has been identified as an important signal sequence for the early endosomal-targeting of DMT1B. In polarized MDCK cells, GFP-tagged DMT1A and DMT1B are localized in the apical plasma membrane and their respective specific endosomes. Disruption of the N-glycosylation sites in each of the DMT1 isoforms affects their polarized distribution into the apical plasma membrane but not their correct endosomal localization. Our data indicate that the cell type-specific expression patterns and the distinct subcellular localizations of two DMT1 isoforms may be involved in the different iron acquisition steps from the subcellular membranes in various cell types.

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.