Influence of copper depletion on iron uptake mediated by SFT, a stimulator of Fe transport

We recently identified a novel factor involved in cellular iron assimilation called SFT or Stimulator of Fe Transport (Gutierrez, J. A., Yu, J., Rivera, S., and Wessling-Resnick, M. (1997) J. Cell Biol. 149, 895-905). When stably expressed in HeLa cells, SFT was found to stimulate the uptake of both transferrin-and nontransferrin-bound Fe (iron), Assimilation of nontransferrin-bound Fe by HeLa cells stably expressing SFT was time- and temperature-dependent; both the rate and extent of uptake was enhanced relative to the activity of control nontransfected cells, Although the apparent K-m for Fe uptake was unaffected by expression of SFT (5.6 versus 5.1 mu M measured for control), the V-max of transport was increased from 7.0 to 14.7 pmol/min/mg protein, Transport mediated by SFT was inhibitable by diethylenetriaminepentaacetic acid and ferrozine, Fe3+- and Fe2+-specific chelators, Because cellular copper status is known to influence Fe assimilation, we investigated the effects of Cu (copper) depletion on SFT function, After 4 days of culture in Cu-deficient media, HeLa cell Cu,Zn superoxide dismutase activity was reduced by more than 60%, Both control cells and cells stably expressing SFT displayed reduced Fe uptake as well; levels of transferrin-mediated import fell by similar to 80%, whereas levels of nontransferrin-bound Fe uptake were similar to 50% that of Cu-replete cells, The failure of SFT expression to stimulate Fe uptake above basal levels in Cu-depleted cells suggests a critical role for Cu in SFT function, A current model for both transferrin-and nontransferrin-bound Pe uptake involves the function of a ferrireductase that acts to reduce Fe3+ to Fe2+, with subsequent transport of the divalent cation across the membrane bilayer, SFT expression did not enhance levels of HeLa cell surface reductase activity; however, Cu depletion was found to reduce endogenous activity by 60%, suggesting impaired ferrireductase function may account for the influence of Cu depletion on SPT-mediated Fe uptake, To test this hypothesis, the ability of SFT to directly mediate Fe2+ import was examined, Although expression of SMT enhanced Fe2+ uptake by HeLa cells, Cu depletion did not significantly reduce this activity, Thus, we conclude that a ferrireductase activity is required for SFT function in Fe3+ transport and that Cu depletion reduces cellular iron assimilation by affecting this activity.