Abstract

Fluoride is a ubiquitous environmental toxin with which all biological species must cope. A recently discovered family of fluoride export (FEX) proteins protects organisms from fluoride toxicity by removing it from the cell. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. Continuous expression is in contrast to many other toxin exporters in yeast, and this, along with the fact that two nearly duplicate proteins are encoded in the yeast genome, suggests that the threat posed by fluoride ions is frequent and detrimental. Structurally, eukaryotic FEX proteins consist of two homologous four-transmembrane helix domains folded into an antiparallel dimer, where the orientation of the two domains is fixed by a single transmembrane linker helix. Using phylogenetic sequence conservation as a guide, we have identified several functionally important residues. There is substantial functional asymmetry in the effect of mutation at corresponding sites in the two domains. Specifically, mutations to residues in the C-terminal domain proved significantly more detrimental to function than did similar mutations in the N-terminal domain. Our data suggest particular residues that may be important to anion specificity, most notably the necessity of a positive charge near the end of TMH1 in the C-terminal domain. It is possible that a cationic charge at this location may create an electrostatic well for fluoride ions entering the channel from the cytoplasm.

Highlights

  • Fluoride is broadly toxic, and organisms use fluoride export (FEX) proteins to expel it

  • We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane

  • FEX1 and FEX2 Produce Fluoride Conductance in the Plasma Membrane—With FEX1 and/or FEX2 proteins expressed at constitutive levels, whole-cell patch clamping of S. cerevisiae membranes demonstrated huge fluoride-ion currents consistent with both proteins functioning as fluoride channels

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Summary

Background

Organisms use fluoride export (FEX) proteins to expel it. We show here that FEX proteins in Saccharomyces cerevisiae function as ion channels that are selective for fluoride over chloride and that these proteins are constitutively expressed at the yeast plasma membrane. A major breakthrough in understanding fluoride biology came with the recent discovery of fluoride-responsive riboswitches in eubacteria and archaea These noncoding RNAs regulate the expression of a diverse set of genes, including many that encode membrane proteins thought to be involved in ion transport [8]. These have since been identified as fluoride exporters, and two distinct structural families are currently recognized. We define the transmembrane topology of the protein and identify conserved residues that are critical for function in the first and second transmembrane segments

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