Abstract
The fluoride export protein (FEX) in yeast and other fungi provides tolerance to fluoride (F-), an environmentally ubiquitous anion. FEX efficiently eliminates intracellular fluoride that otherwise would accumulate at toxic concentrations. The FEX homolog in bacteria, Fluc, is a ‘double-barreled’ channel formed by dimerization of two identical or similar subunits. FEX in yeast and other eukaryotes is a monomer resulting from covalent fusion of the two subunits. As a result, both potential fluoride pores are created from different parts of the same protein. Here we identify FEX proteins from two multicellular eukaryotes, a plant Arabidopsis thaliana and an animal Amphimedon queenslandica, by demonstrating significant fluoride tolerance when these proteins are heterologously expressed in the yeast Saccharomyces cerevisiae. Residues important for eukaryotic FEX function were determined by phylogenetic sequence alignment and functional analysis using a yeast growth assay. Key residues of the fluoride channel are conserved in only one of the two potential fluoride-transporting pores. FEX activity is abolished upon mutation of residues in this conserved pore, suggesting that only one of the pores is functional. The same topology is conserved for the newly identified FEX proteins from plant and animal. These data suggest that FEX family of fluoride channels in eukaryotes are ‘single-barreled’ transporters containing one functional pore and a second non-functional vestigial remnant of a homologous gene fusion event.
Highlights
Fluoride is a toxin that is widespread in nature
fluoride export protein (FEX) proteins are important for fluoride tolerance in eukaryotes [16]
Baker’s yeast S. cerevisiae deficient in FEX are sensitive to low micromolar fluoride ion, a concentration that is common in the natural environment and many municipal water supplies
Summary
Fluoride is a toxin that is widespread in nature. The average fluoride concentration in the ocean is 70 μM [1], but much higher fluoride levels (>100 mM) can be detected near active volcanoes [2,3,4]. Each protein consists of two homologous domains joined into a single protein by a transmembrane linker (Fig 1D) that forces an antiparallel orientation of the N-terminal and C-terminal domains [18] This domain arrangement is analogous to the engineered fusion constructs that were created for the study of Fluc [15]. There is a G (A)xxxR sequence in the first helix and an N in the middle of the second helix of each FEX domain or Fluc monomer Various mutations of these conserved residues within FEX suggested that the residues in the C-terminal domain have greater effects on yeast fluoride tolerance than the equivalent residues in the N-terminal domain, though the cause of this asymmetric effect on protein activity was not clear. The crystal structures of Fluc, combined with our earlier mutagenesis study, inspired the investigation of the asymmetrical nature of the two pores in the yeast FEX protein. These results suggest that among eukaryotic fluoride channels, Pore II is functional for fluoride export while Pore I has lost its ancestral function through evolutionary drift, resulting in a vestigial pore
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