tailieunhanh - Báo cáo khoa học: Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening

The functional expression of olfactory receptors (ORs) is a primary require-ment to examine the molecular mechanisms of odorant perception and cod-ing. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization meth-ods. | iFEBS Journal Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening Jasmina Minic1 Marie-Annick Persuy1 Elodie Godel1 Josiane Aioun1 Ian Connerton2 Roland Salesse1 and Edith Pajot-Augy1 1 INRA Neurobiologie de 1 Olfaction et de la Prise Alimentaire Recepteurs et Communication Clinique Jouy-en-Josas France 2 Division of Food Sciences Schoolof Biosciences University of Nottingham Loughborough Leicestershire UK Keywords Golf odorant screening olfactory receptors Saccharomyces cerevisiae yeast bioassay Correspondence E. Pajot-Augy INRA Neurobiologie de l Olfaction et de la Prise Alimentaire Domaine de Vilvert 78352 Jouy-en-Josas Cedex France Fax 33 1 34 65 22 41 Tel 33 1 34 65 25 63 E-mail Received 13 July 2004 revised 1 October 2004 accepted 18 November 2004 doi The functional expression of olfactory receptors ORs is a primary requirement to examine the molecular mechanisms of odorant perception and coding. Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae. The membrane expression of the receptor was shown by Western blotting and immunolocalization methods. Moreover we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter. Yeasts were engineered to coexpress I7 OR and mammalian Ga subunit to compensate for the lack of endogenous Gpa1 subunit so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis. The sensitivity of the bioassay was significantly enhanced using mammalian Golf compared to the G. 15 subunit resulting in dose-dependent responses of the .