Evolution of a Yeast Receptor for Non-Native Ligands Using a Novel High-Throughput Screening Method

This research aims to develop novel yeast biosensors that take advantage of the yeast pheromone mating pathway, one of the best understood eukaryotic signaling pathways. Through this pathway, Saccharomyces cerevisiae detects mating pheromone with the Ste2 G-protein coupled membrane receptor (GPCR). Though S. cerevisiae has been used as a system to express heterologous mutagenized GPCRs, this research takes advantage of the native signaling machinery in yeast and eliminates the need for extensive pathway engineering that is often necessary for functional expression of heterologous receptors.
The objective of this experiment was to determine if the Ste2 receptor could be mutated to yield novel detection capabilities. Error-prone polymerase chain reaction was used to create a library of mutagenized Ste2 receptors. We searched this library for receptors that showed activity for variants of the native pheromone ligand. These variants differ from the native ligand by one amino acid, and the native Ste2 receptor shows little activity for these pheromone variants at physiological concentrations.
Our results indicate that we were successful in identifying mutated receptors that respond to the pheromone variants. The library of mutant Ste2 receptors was sorted with fluorescence activated cell sorting (FACS). Developing a sorting protocol based on Bayesian statistics allowed us to minimize the amounts of false positive and false negative receptors in the mutant population. Receptor activation was indicated by green fluorescent protein (GFP) expression. The GFP value was normalized by red fluorescent protein (RFP), which was constitutively expressed. After FACS, individual mutant receptors were isolated from the population for characterization. Dose response curves were obtained by treating individual receptors with varying amounts of pheromone variant. EC50 values, the concentration of ligand at which half of the maximal response is obtained, were calculated for each isolated receptor.
For mutant receptors, the pheromone variant EC50 values obtained were significantly lower than those obtained with the native Ste2 receptor, implying that the isolated receptors are more sensitive than the native Ste2 receptor is for the pheromone variants. These results lay the foundation for future work for the directed evolution of the Ste2 receptor to detect non-native ligands.