The first external loop of the metal ion transporter DCT1 is involved in metal ion binding and specificity

Abstract

The yeast null mutant smf1⌬ 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 D124A͞ Q126D partially restored it and changed the metal ion specificity in favor of Fe 2؉ . The maximal pre-steady-state currents at negatively imposed potentials shifted to a lower pH of Ϸ5. The triple mutant G119A͞D124A͞Q126D, which has no apparent transport activity, exhibited remarkable pre-steady-state currents at pH 7.5. Moreover, Zn 2؉ 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 Mn 2؉ 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.mutations ͉ uptake ͉ electrophysiology ͉ yeast ͉ oocytes In the last few years it has become apparent that, from bacteria to man, the family of natural resistance-associated macrophage protein (NRAMP) metal ion transporters plays a major role in metal ion homeostasis (1-4). This family is represented in yeast by three genes (SMF1, SMF2, and SMF3) and in mammals by NRAMP1 and NRAMP2 (DCT1). The family members function as general metal ion transporters and can transport not only Mn 2ϩ and Cu 2ϩ (5) but also Fe 2ϩ , Cd 2ϩ , Ni 2ϩ , and Co 2ϩ (6-8). Expression of DCT1 and Smf1p in Xenopus oocytes demonstrated that the driving force for the divalent metal ion transport is H ϩ -dependent (8, 9). In addition, in the expressing oocytes, a large H ϩ slip through DCT1 and Na ϩ slippage through Smf1p was observed (8, 10). The identity of the substrate and proton-binding sites on the transporters are not known, and the mechanism of metal ion transport is obscure. Few conserved amino acids were changed in Smf1p (11) and DCT1 (12, 13), but the results have not yet revealed many aspects of the action mechanism of these transporters. We developed a concerted approach for the study of Smf1p and DCT1. It uses yeast null mutants lacking the SMF metal ion transporters (14), and Xenopus oocytes, where those transporters are absent, as an expression medium for Smf1p and DCT1 in appropriate plasmids (10). The null mutant smf1⌬ is unable to grow in the presence of EGTA (5, 14). Expression of Smf1p or the...

The Mutation F227I Increases the Coupling of Metal Ion Transport in DCT1

“…Both use protons as their motive force to transport a broad-range of the same divalent metal ions and exhibit similar affinities for their various substrates (8,12,16). The mammalian DCT1 complements the phenotype of SMF1 null mutation in yeast (5,(17)(18)(19). The null mutant smf1⌬ is unable to grow in the presence of EGTA.…”

“…The differences between the two homologues have to be due to the non-conserved amino acids in both proteins. Consequently, to locate the sites on the transporters responsible for this phenomenon, we substituted amino acids in DCT1 for the corresponding ones residing in Smf1p (19). It has been found that a mutation in the conserved glycine at position 216 (G216R) in the putative TM4 of DCT1 causes microcytic anemia in mkϪ/Ϫ mice and Belgrade rats (20).…”

Solute Carrier 11 Cation Symport Requires Distinct Residues in Transmembrane Helices 1 and 6

“…The TMS1 Asp residue is part of a conserved DPGN motif that has been subjected to mutagenesis in studies using MntH or Nramp2 homologs, which showed loss of Me 2ϩ uptake caused by Gly exchange (11,47). The carboxyl end of Nramp2 TMS1 and adjacent extra loop were implicated in Me 2ϩ binding and coupling of Me 2ϩ uptake to the proton-motive force ((C/S/T)P(C/H)) that is conserved in the TMS6 of P 1B -type ATPases, which pump heavy metal cations using energy provided by ATP hydrolysis (48,49).…”

Regulation of Leucine Uptake by tor1+ in Schizosaccharomyces pombe Is Sensitive to Rapamycin

“…Measurement of leucine uptake: The method described in cued by a tor1 allele defective in rapamycin binding: If Karagiannis et al (1999) was followed with slight modifications adopted from Cohen et al (2003). rapamycin sensitivity in auxotrophs is the result of TOR or ⌬tor1 cells (TA390) were grown to log phase in minimal inhibition by FKBP12-rapamcin, it is expected that mumedium.…”