Structural and functional analysis of SFT, a stimulator of Fe transport

Previous studies demonstrated that SFT (Stimulator of Fe Transport) facilitates both transferrin and nontransferrin-bound iron uptake in HeLa cells (Yu, J,, and Wessling-Resnick, M, (1998) J, Biol. Chem, 273, 6909-6915), To further characterize the structure and function of SFT, we studied this human factor in rodent BHR cells. Kyte-Doolittle analysis suggests that SFT has six transmembrane-spanning segments. This transport protein also displays an REXXE motif resembling domains involved in iron binding by ferritin and in iron uptake mediated by the yeast transporter Ftr1, Using N- and C-terminal epitope tags, we have identified that modification of either protein terminus does not interfere with SFT function in nontransferrin-bound iron uptake. The N- and C-terminal domains are intracellularly disposed since antibodies against these epitopes fail to recognize expressed proteins unless BHK cells are solubilized with detergents. To define the topology of two large extramembranous loop domains, anti-peptide antibodies were employed; anti-loop 4 antibodies show no immunoreactivity unless cells are permeabilized but anti-loop 5 antibodies recognize and bind surface SFT. Thus, loop 4 must be intracellular while loop 5 is extracellular, These topological studies situate the putative iron-binding REXXE domain on the cytosolic face of the plasma membrane, However, Fe-55-binding studies reveal that the ability of SFT to bind and mediate transport of extracellular iron is defective in mutants with Glu --> Ala conversions in this motif, Curiously, we also find that depletion of intracellular iron by desferrioxamine impairs SFT transport and iron-binding functions. These observations lead to the speculation that the REXXE motif may play an important role in regulating SFT activity through interaction with intracellular iron and demonstrate that iron transport mediated by SFT is itself an iron-dependent process.