Abstract
The yeast Saccharomyces cerevisiae has been used extensively for ligand screening of human G-protein coupled receptors, due to its ease of genetic manipulation, low cost, rapid growth, and eukaryotic secretory pathway. Although the Caenorhabditis elegans genome was sequenced 13 years ago and encodes over 1,000 GPCRs, of which several hundred are believed to respond to volatile organic ligands, only one of these receptors, ODR-10, has been linked to a volatile ligand, 2,3-butanedione. ODR-3 is a G-protein a subunit believed to be involved in odorant detection and activated by ODR-10. Here we report the functional coupling of ODR-10 to the yeast pheromone signalling pathway using the yeast - C. elegans chimaeric Ga subunit (GPA-1:ODR-3). Interactions between ODR-10, ODR-3 and the chimaera were confirmed using the split ubiquitin yeast two-hybrid system. We also report the tailoring of a Saccharomyces cerevisiae strain for the analysis of C. elegans chemoreceptor function. In this study, a yeast gpa1D ste2D sst2D far1D quadruple mutant strain was constructed to efficiently couple nematode olfactory receptors with the yeast signalling pathway. We used two different reporters: green fluorescent protein and ß-galactosidase, to verify activation of the signal transduction pathway by ligand activated GPCR interactions. With this heterologously engineered yeast system, we aim to accelerate the de-orphaning of C. elegans GPCR proteins.