Here we present a successful transplantation of the GAL genetic regulatory circuitry into the G-protein signaling pathway in yeast. The GAL regulon represents a strictly regulated transcriptional mechanism that we have transplanted into yeast to create a highly robust induction system to assist the detection of on-off switching in G-protein signaling. In our system, we engineered yeast to drive the positive GAL regulatory gene in response to agonist-promoted G-protein signaling and to induce transcription of a green fluorescent protein (GFP) reporter gene under the control of the GAL structural gene promoter. Consequently, in response to agonist stimulation of G-protein-coupled receptors (GPCRs), the engineered yeast achieved more than a 150-fold increase in reporter intensity in up to 98% of cells, as determined by flow cytometric sorting. Surprisingly, agonist-stimulated induction of the GFP reporter gene was higher than that by galactose. Our approach to boost reporter gene induction could be applicable in establishing more efficient yeast-based flow cytometric screening systems for agonistic ligands for heterogeneous GPCRs.

• PMID: 22343189
• DOI full text
• PubMed

Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Ryo S, Ishii J, Iguchi Y, Fukuda N, Kondo A

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