Yeast-Based Fluorescence Reporter Assay of G Protein-coupled Receptor Signalling for Flow Cytometric Screening: FAR1-Disruption Recovers Loss of Episomal Plasmid Caused by Signalling in Yeast

Abstract

Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.

Functional Coupling of a Nematode Chemoreceptor to the Yeast Pheromone Response Pathway

“…GFP and luciferase have both been used to detect signalling with mammalian GPCRs [16]–[18], [29]. In our hands, GFP was inconsistent and unreliable for detecting nematode OR activation (data not shown).…”

“…The STE2 mutation is important because the presence of an endogenous pheromone receptor can diminish the ability to detect signalling from foreign GPCRs, apparently by sequestering G proteins into refractory pre-activation complexes [28]. The FAR1 gene was deleted to avoid cell death due to overstimulation of this transduction pathway [14], [29]–[31]. The ability to detect signalling was further enhanced by deleting the regulator of G-protein (RGS), SST2 , which results in increased gain in the MAP kinase transduction cascade [14],[32].…”

“…The FUS1 promoter has been used frequently to detect signal transduction by heterologously expressed GPCRs [14], [18], [29], [51] whereas the FIG2 promoter had not been used in this system before. Both strains ( FUS1:lacZ , PGK1:STE2 and FIG2:lacZ , PGK1:STE2 ) showed dose-dependent expression of the lacZ reporter after activation with the Ste2 ligand, α-mating peptide (Figure 4).…”

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

“…We therefore tested the ability of the other active somatostatin isoform S-28 [26] and other secretion signal sequences (pre-region of α-factor and signal sequences derived from S. cerevisiae Suc2p and Rhizopus oryzae glucoamylase) to mediate signal transduction in the IMG-50 yeast strain. This strain has a slightly different genetic background to IMFD-70 ( FAR1 -intact strain [28], the description of the far1 Δ allele can be found in Materials and Methods; Table 1), but the expression profiles of the GFP reporter genes remained essentially unchanged (Figure S3). Also, the insertion of GS linkers (GGGGS and GGGSGGGGS) between the S-14 peptide and Flag–Flo42 did not improve GFP expression (Figure S4).…”

Fluorescent proteins in microbial biotechnology—new proteins and new applications

“…A variation on the theme of using GFP as a reporter of molecular interactions was developed by Ishii et al (2008); a promoter::gfp fusion activated by a MAP kinase signalling cascade was used to monitor G-protein coupled receptor (GPCR) signalling in S. cerevisiae. This technique could possibly be used to screen heterologously-expressed GPCRs, which represent potential drug targets.…”