Functional coupling of a nematode chemoreceptor to the yeast pheromone response pathway

Abstract

Sequencing of the Caenorhabditis elegans genome revealed sequences encoding more than 1,000 G-protein coupled receptors, hundreds of which may respond to volatile organic ligands. To understand how the worm's simple olfactory system can sense its chemical environment there is a need to characterise a representative selection of these receptors but only very few receptors have been linked to a specific volatile ligand. We therefore set out to design a yeast expression system for assigning ligands to nematode chemoreceptors. We showed that while a model receptor ODR-10 binds to C. elegans Ga subunits ODR-3 and GPA-3 it cannot bind to yeast Ga. However, chimaeras between the nematode and yeast Ga subunits bound to both ODR-10 and the yeast Gbc subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Ga subunit (GPA1) of the Saccharomyces cerevisiae (ste2D sst2D far1D) triple mutant ("Cyb") with a Gpa1/ODR-3 chimaera and introduced ODR-10 as a model nematode GPCR. This strain showed concentration-dependent activation of the yeast MAP kinase pathway in the presence of diacetyl, the first time that the native form of a nematode chemoreceptor has been functionally expressed in yeast. This is an important step towards en masse de-orphaning of C. elegans chemoreceptors.