title: Yeast Fex1p Is a Constitutively Expressed Fluoride Channel with Functional Asymmetry of Its Two Homologous Domains.
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.
authors.affiliation: From the Departments of Molecular Biophysics and Biochemistry.
authors.forename: Kathryn D
authors.initials: KD
authors.lastname: Smith
authors.affiliation: From the Departments of Molecular Biophysics and Biochemistry.
authors.forename: Patricia B
authors.initials: PB
authors.lastname: Gordon
authors.affiliation: Cellular and Molecular Physiology.
authors.forename: Alberto
authors.initials: A
authors.lastname: Rivetta
authors.affiliation: Genetics, and.
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Allen
authors.affiliation: From the Departments of Molecular Biophysics and Biochemistry.
authors.forename: Tetyana
authors.initials: T
authors.lastname: Berbasova
authors.affiliation: Cellular and Molecular Physiology.
authors.forename: Clifford
authors.initials: C
authors.lastname: Slayman
authors.affiliation: From the Departments of Molecular Biophysics and Biochemistry, Chemistry, Yale University, New Haven, Connecticut 06520 scott.strobel@yale.edu.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D025141:Drug Resistance, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005459:Fluorides / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal; D016681:Genome, Fungal; D017136:Ion Transport; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010802:Phylogeny; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: C000602399:Fex1 protein, S cerevisiae; D008565:Membrane Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D005459:Fluorides
keywords: FEX; Fluc; anion transport; fluoride; ion channel; membrane transport; microscopy; patch clamp; topology