Anal Biochem. 2012 May 1;424(1):27-31. doi: 10.1016/j.ab.2012.02.005. Epub 2012 Feb 13.

Transplantation of the GAL regulon into G-protein signaling circuitry in yeast.

Ryo S¹, Ishii J, Iguchi Y, Fukuda N, Kondo A.

Author information

1: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan.

Abstract

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.