The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships - PubMed (original) (raw)
The Ftr1p iron permease in the yeast plasma membrane: orientation, topology and structure-function relationships
Scott Severance et al. Biochem J. 2004.
Abstract
Ftr1p is the permease component of the Fet3p-Ftr1p high affinity iron-uptake complex, in the plasma membrane of Saccharomyces cerevisiae, that transports the Fe3+ produced by the Fet3p ferroxidase into the cell. In this study we show that Ftr1p probably has seven transmembrane domains with an orientation of N-terminal outside, and C-terminal inside the cell. Within the context of this topology of the Fet3p-Ftr1p complex, we have identified several sequence elements in Ftr1p that are required for wild-type uptake function. First to be identified were two REXLE (Arg-Glu-Xaa-Leu-Glu) motifs in transmembrane domains 1 and 4. Alanine substitutions at any one of these combined six arginine or glutamic acid residues inactivated Ftr1p in iron uptake, indicating that both motifs were essential to iron permeation. R-->K and E-->D substitutions in these two motifs led to a variable loss of activity, suggesting that while all six residues were essential, their contributions to uptake were quantitatively and/or mechanistically distinct. The terminal glutamate in an EDLWE89 element, associated with transmembrane domain 3, and a DASE motif, located in extracellular loop 6, were also required. The double substitution to AASA in the latter, inactivated Ftr1p in iron uptake while the Ftr1p(E89A) mutant had only 20% of wild-type activity. The two REXLE and the EDLWE and DASE motifs are strongly conserved among fungal Ftr1p homologues, suggesting that these motifs are essential to iron permeation. Finally another important residue, Ile369, was identified in the Ftr1p cytoplasmic C-terminal domain. Deletion or substitution of this residue led to a 70% loss of iron-uptake activity. Ile369 was the only residue identified in this domain that made such a major contribution to iron uptake by the Fet3p-Ftr1p complex.
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