Engineered yeasts as reporter systems for odorant detection.
The functional expression of olfactory receptors (ORs) is a primary requirement to utilize olfactory detection systems. We have taken advantage of the functional similarities between signal transduction cascades in the budding yeast Saccharomyces cerevisiae and mammalian cells. The yeast pheromone response pathway has been adapted to allow ligand-dependent signaling of heterologous expressed G-protein coupled receptors (GPCRs) via mammalian or chimeric yeast/mammalian Galpha proteins. Two different strategies are reported here which offer a positive screen for functional pairs. The OR and Galpha protein are introduced into the modified yeast cells such that they hijack the pheromone response pathway usually resulting in cell cycle arrest. The first strategy utilizes ligand-induced expression of a FUS1-HIS3 reporter gene to permit growth on a selective medium lacking histidine; the second to induce ligand-dependent expression of a FUSI-Hph reporter gene, conferring resistance to hygromycin. Validation of the systems was performed using the rat 17 receptor response to a range of aldehyde odorants previously characterized as functional ligands. Of these only heptanal produced a positive growth response in the concentration range 5 x 10(-8) to 5 x 10(-6) M. Induction conditions appear to be critical for functional expression, and the solvents of odorants have a toxic effect for the highest odorant concentrations. The preference of rat 17 receptor for the ligand heptanal in yeast has to be compared to concurrent results obtained with mammalian expression systems.
Pajot-Augy
Edith
E
INRA, Biologie Cellulaire et Moléculaire, Equipe Récepteurs et Communication Cellulaire, Jouy-en-Josas, France. pajot@jouy.inra.fr
Crowe
Mark
M
Levasseur
Grégoire
G
Salesse
Roland
R
Connerton
Ian
I
D000818:Animals; D017930:Genes, Reporter; D006639:Histidine / Q000378:metabolism; D008024:Ligands; D018034:Olfactory Receptor Neurons / Q000166:cytology / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D018035:Receptors, Odorant / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015290:Second Messenger Systems / Q000502:physiology*; D012997:Solvents
D008024:Ligands; D010675:Pheromones; D021122:Protein Subunits; D018035:Receptors, Odorant; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D006639:Histidine