In vivo detection of yeast alpha mating pheromone receptor Ste2p homodimerization by FRET

Thesis Type: Postgraduate

Institution Of The Thesis: Orta Doğu Teknik Üniversitesi, Faculty of Arts and Sciences, Department of Biology, Turkey

Approval Date: 2014




Ste2p is an alpha type pheromone sensing receptor of ‘a’ type Saccharomyces cerevisiae cells. Yeast life cycle could be haploid or diploid due to the signal sensed by Ste2p. This receptor is a G protein coupled receptor (GPCR). GPCRs are one of the most important drug targets because they are playing key roles in cell signaling. They have seven transmembrane domains and linked with a G protein in the cytosol. FRET is a method that is used for detecting protein-protein interactions by using the resonance energy transfer between fluorophores. The fluorophores are named as donor and acceptor. When two fluorophores are in close proximity to each other FRET signal is generated and this signal can be quantified to determine the distance between the donor and the acceptor. For this purpose Ste2p is labeled with two different fluorophores EGFP and mCherry, green and red, respectively. During the study two different positions of receptor was labeled with the two fluorophores. Yeast cells were transfected with these vectors bearing the fusion gene of receptor and fluorophores. After transfection of the two labeled receptors, the presence of FRET signal was observed under confocal microscope. viii FRET provides chance of observing live cells without causing harm. In this study in addition to detection of homodimerization we studied effect of agonist on receptor dimerization and fluorescent protein position on FRET efficiency.