Towards structural determination of eukaryotic fluoride export protein (FEX).

Abstract

Fluoride (F-) is ubiquitous in the environment. Exposure to high F- concentrations causes harmful effects to biological systems. To survive in an environment that contains F-, microorganisms have fluoride-specific exporters, fluoride ion channel (Fluc) in prokaryotes and fluoride exporter (FEX) in eukaryotes. These proteins export F- across the cell membrane and prevent F- accumulation in cells. The well-studied Fluc proteins are unusual fluoride-specific ion channels assembled as dual topology dimers. According to a hypothetical structural model, FEX has two homologous four-transmembrane domains with a transmembrane linker helix that enforces an antiparallel architecture. However, no structure of a FEX has been solved, and the transport mechanism of FEX proteins has not yet been determined, leaving a major gap in our understanding of fluoride export by eukaryotes. Here, we successfully purified the functional C. albicans FEX in S. cerevisiae. We observed that the fluoride transport rate of FEX from liposomes was ∼1000-fold less than the Fluc channels. To determine the molecular basis of F- transport by FEX, antibody complexation to an MPER epitope tag from the HIV gp41 will be utilized to increase the size of small membrane proteins for structural determination by single-particle Cryo-EM. Understanding the molecular basis of fluoride transport by C. albicans FEX will provide structural insights into the general mechanism of fluoride export by eukaryotes.