The yeast null mutant smf1Δ cannot grow on medium containing EGTA. Expression of Smf1p or the mammalian transporter DCT1 (Slc11a2) suppresses the above-mentioned phenotype. Both can also be expressed in Xenopus oocytes, and the uptake activity and their electrophysiological properties can be studied. We used these systems to analyze the properties of mutations in the predicted external loop I of DCT1. The sensitivity of the transporter to amino acid substitutions in this region is manifested by the mutation G119A, which resulted in almost complete inhibition of the metal ion uptake activity and marked changes in the pre-steady-state currents in Xenopus oocytes. The mutation Q126D abolished the uptake and the electrophysiology, but the double mutant D124A/Q126D partially restored it and changed the metal ion specificity in favor of Fe2+. The maximal pre-steady-state currents at negatively imposed potentials shifted to a lower pH of ≈5. The triple mutant G119A/D124A/Q126D, which has no apparent transport activity, exhibited remarkable pre-steady-state currents at pH 7.5. Moreover, Zn2+ had a dual effect on this mutant; at pH 7.5 it eliminated the pre-steady state without generating steady-state currents, and at pH 5.5 it induced large pre-steady-state currents. The mutant D124A retained appreciable Fe 2+ uptake activity but exhibited very little Mn2+ uptake at pH 5.5 and was abolished at pH 6.5. The properties of the various mutants suggest that loop I is involved in the metal ion binding and its coupling to the proton-driving force.
|Number of pages||6|
|Journal||Proceedings of the National Academy of Sciences of the United States of America|
|State||Published - 16 Sep 2003|