Quantitative and dynamic analyses of G protein-coupled receptor signaling in yeast using Fus1, enhanced green fluorescence protein (EGFP), and His3 fusion protein.
The mechanism of G protein-coupled receptor (GPCR) signaling in yeasts is similar to that in mammalian cells. Therefore, yeasts can be used in GPCR assays, and several ligand detection systems using a pheromone signaling pathway in yeasts have been developed by employing yeasts with disrupted chromosomal genes that code for proteins producing specific effects. In this study, the construction of yeast strains that can detect ligand binding mediated by interactions between the G protein and GPCR using either fluorescence or auxotrophic selectivity is demonstrated. The strain was constructed by integrating the fusion gene of pheromone-responsive protein (FUS1), enhanced green fluorescence protein (EGFP), and auxotrophic marker protein (HIS3) into the FUS1 locus. Moreover, the influence of gene disruptions on the yeast signal transduction cascade is closely investigated with respect to both quantitative and dynamic aspects to further develop a high-throughput screening system for the GPCR assay using yeasts. Yeast strains with a disrupted SST2 gene, which is a member of the RGS (regulator of G protein signaling) family, and a disrupted FAR1 gene, which mediates cell cycle arrest in response to a pheromone, were monitored by measuring their fluorescence and growth rate. This method will be applicable to other comprehensive GPCR ligand screening methods.
Ishii
Jun
J
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Nada-ku, Japan.
Matsumura
Shizuka
S
Kimura
Sakurako
S
Tatematsu
Kenji
K
Kuroda
Shun'ichi
S
Fukuda
Hideki
H
Kondo
Akihiko
A
D001681:Biological Assay; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D005800:Genes, Fungal / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D000072235:Mating Factor; D008565:Membrane Proteins; D030781:Organisms, Genetically Modified; D010455:Peptides / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013997:Time Factors
C490024:FUS1 protein, S cerevisiae; D008565:Membrane Proteins; D010455:Peptides; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D000072235:Mating Factor