title: Transplantation of the GAL regulon into G-protein signaling circuitry in yeast.
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.
authors.affiliation: Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Nada, Kobe 657-8501, Japan.
authors.forename: Shintaro
authors.initials: S
authors.lastname: Ryo
authors.forename: Jun
authors.initials: J
authors.lastname: Ishii
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Iguchi
authors.forename: Nobuo
authors.initials: N
authors.lastname: Fukuda
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
mesh_terms: D019204:GTP-Binding Proteins / Q000378:metabolism*; D005690:Galactose / Q000235:genetics*; D005800:Genes, Fungal / Q000235:genetics*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D018085:Regulon / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D019204:GTP-Binding Proteins; D005690:Galactose