Mapping Functional Domains of Chloride Intracellular Channel (CLIC) Proteins in Vivo

Chloride intracellular channel (CLIC) proteins are small proteins distantly related to the omega family of glutathione S-transferases (GSTs). CLIC proteins are expressed in a wide variety of tissues in multicellular organisms and are targeted to specific cellular membranes. Members of this family are capable in vitro of changing conformation from a globular, soluble state to a membrane-inserted state in which they provide chloride conductance. The structural basis for in vivo CLIC protein function, however, is not well understood. We have mapped the functional domains of CLIC family members using an in vivo assay for membrane localization and function of CLIC proteins in the nematode Caenorhabditis elegans. A < 70 amino acid N-terminal domain is a key determinant of membrane localization and function of invertebrate CLIC proteins. This domain, which we term the ‘‘PTM‘‘ domain, named after an amphipathic putative transmembrane helix contained within it, directs distinct C. elegans CLIC homologs to distinct subcellular membranes. We find that within the PTM region, the cysteine residues required for GST-type activity are unnecessary for invertebrate CLIC function, but that specific residues within the proposed transmembrane helix are necessary for correct targeting and protein function. We find that among all tested invertebrate CLIC proteins, function appears to be completely conserved despite striking differences in the charged residues contained within the amphipathic helix. This indicates that these residues do not contribute to anion selectivity as previously suggested. We find that outside the PTM region, the remaining three-quarters of CLIC protein sequence is functionally equivalent not only among vertebrate and invertebrate CLIC proteins, but also among the more distantly related GST-omega and GST-sigma proteins. The PTM region thus provides both targeting information and CLIC functional specificity, possibly adapting GST-type proteins to function as ion channels.