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 Gα subunits ODR-3 and GPA-3 it cannot bind to yeast Gα. However, chimaeras between the nematode and yeast Gα subunits bound to both ODR-10 and the yeast Gβγ subunits. FIG2 was shown to be a superior MAP-dependent promoter for reporter expression. We replaced the endogenous Gα subunit (GPA1) of the Saccharomyces cerevisiae (ste2Δ sst2Δ far1Δ) 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.

Figure 1. Characterisation of yeast/C. elegans Gα chimaeras.

A. Sequences of the five C-terminal amino acids of Gα proteins used in this study. B. Complementation of growth arrest due to Gpa-1 t^s mutation with Gpa1/C. elegans Gα chimaeras at 24°C. All constructs grow at 34°C as shown in Figure S3. In the panel: 1. Endogenous yeast Gpa1 used as a positive control. 2. C. elegans GPA-3 fails to rescue the growth at 24°C. 3. C. elegans ODR-3 fails to rescue the growth at 24°C. 4. Gpa1/GPA-3 chimaera complements the Gpa-1 t^s mutation. 5. Gpa1/ODR-3 chimaera complements the Gpa-1 t^s mutation. 6. Vector-alone used as a negative control.

Figure 2. ODR-10 interaction with Gpa1/ODR-3 chimaera and Gpa1/GPA-3 chimaera in the split-ubiquitin yeast two-hybrid system.

(a) ODR-10 does not interact non-specifically with NubG. (b) ODR-10 does not interact with yeast Gpa1. (c) and (d) ODR-3 couples to ODR-10 either NubG attached to N- or C-terminus. (e) and (f) GPA-3 couples to ODR-10 either NubG attached to N- or C-terminus. (g) and (h) Gpa1/ODR-3 chimaera couples to ODR-10 either NubG attached to N- or C-terminus. (i) and (j) Gpa1/GPA-3 chimaera couples to ODR-10 either NubG attached to N- or C-terminus. Yeast transformants contained both a Cub fusion and a NubG fusion construct were grown on drop out media (SD -Leu and -Trp) (Figure S4) and selective medium lacking histidine (SD -Leu, -Trp and -His) containing 35 mM 3-Amino-1,2,4-triazole (3-AT). β-galactosidase assays were performed to verify interactions. Cells were spotted as one-tenth dilutions starting at Abs_{600 nm} 1.

Figure 3. Expression of GFP² tagged olfactory receptor ODR-10 in the Cyb yeast mutant.

From left to right, Cyb cells stained with Evans Blue– a plasma membrane specific dye; GFP² signal and plasma membrane localisation; overlay of two previous images. Scale bar = 5 µm.

Figure 4. Comparison of efficiency and signal to noise ratio of FUS1 and FIG2 promoters in driving β-galactosidase reporter expression.

α-factor sensing using the lacZ reporter gene assay in yeast cells (ste2Δfar1Δsst2Δ triple deletion alleles in the Cyb yeast strain) expressing the PGK1: STE2. β-galactosidase activity was measured in the yeast strains by stimulating with different concentrations of α-factor. PGK1: STE2 and FUS1: lacZ (grey bars); PGK1: STE2 and FIG2: lacZ (black bars). The mean pseudo Z-factor value calculated for FUS1 promoter was 0.366 and for FIG2 promoter 0.88 based on three biological repeats. Values are mean and standard deviations of three biological repeats.

Figure 5. Schematic illustration of engineered yeast pheromone signalling pathway for analysing nematode ORs.

In this pathway, the endogenous yeast GPCR, Ste2 was knocked out and replaced with a nematode OR (ODR-10), which induces signalling when activated by its ligand diacetyl. As nematode Gα subunits (ODR-3 and GPA-3) have low affinity for the yeast Gβγ, Gpa1/ODR-3 and Gpa1/GPA-3 chimaeras have been developed to incorporate receptor-binding properties of nematode subunits into the Gpa1 subunit that interacts with the yeast signalling machinery. Gene disruption was used to knock out SST2 (to enhance signalling) and FAR1 (to prevent cell cycle arrest). Signal activation was monitored by transforming lacZ reporter under FIG2 promoter.

Figure 6. Concentration-response profile for odorant stimulated reporter gene expression.

A. Expression of lacZ reporter gene driven by the FIG2 promoter in Cyb yeast mutant containing different chimaeric Gα subunits. Values represent means ± SD of experiments performed on three independent transformants. The significance of the Cyb GPA1/odr-3 chimaera PGK1:odr-10 response was tested using a student t-test at the 100 µM concentration by direct comparison with the Cyb GPA1/odr-3 chimaera PGK1:odr-10 H110Y mutant. * p<0.01 B. Expression of lacZ reporter gene driven by the FIG2 promoter in Cyb GPA1/odr-3 chimaera yeast mutant in 96 well plate. Values represent means ± SD of experiments performed on three independent transformants. The significance of the Cyb GPA1/odr-3 chimaera PGK1:odr-10 response was tested using a student t-test at the 500 µM concentration by direct comparison with the Cyb GPA1/odr-3 chimaera PGK1:odr-10 H110Y mutant. * p<0.01 C. Change in abundance of diacetyl in the Cyb GPA1/odr-3 chimaera yeast mutant containing receptor construct PGK1:odr-10 and FIG2: lacZ reporter at different incubation times. Starting concentration of diacetyl in culture was 700 µM. Error bars are the standard error of three measurements.