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Journal of Biochemistry Advance Access originally published online on February 14, 2008
Journal of Biochemistry 2008 143(5):667-674; doi:10.1093/jb/mvn018
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© 2008 The Japanese Biochemical Society.

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

Jun Ishii1, Tsutomu Tanaka2, Shizuka Matsumura3, Kenji Tatematsu4, Shun’ichi Kuroda4, Chiaki Ogino3, Hideki Fukuda2 and Akihiko Kondo3,*

1Department of Molecular Science and Material Engineering, Graduate School of Science and Technology; 2Organization of Advanced Science and Technology; 3Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University; and 4Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Japan

*To whom correspondence should be addressed. Tel: +81 78 803 6196, Fax: +81 78 803 6196, E-mail: akondo{at}kobe-u.ac.jp

Received January 2, 2008; Accepted February 3, 2008


  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.

Key Words: G protein-coupled receptor, yeast, enhanced green fluorescent protein, signalling, flow cytometer

Abbreviations: EGFP, enhanced green fluorescent protein; FCM, flow cytometer; GPCR, G protein-coupled receptor; G-proteins, guanine nucleotide-binding proteins; HTS, high-throughput screening; RGS, regulator of G-protein signalling


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