In vivo detection of yeast alpha mating pheromone receptor ste2p homodimerization by FRET

Bulut, Giray
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.


In Vivo detection of GPCR dimerizations in saccharomyces cerevisiae using FRET and BIFC
Üstünkaya, Beren; Son, Çağdaş Devrim; Department of Biology (2014)
G protein-coupled receptors (GPCRs) are a class of membrane proteins that are composed of seven transmembrane domain and mediate physiological response to a diverse array of stimuli. In eukaryotic microorganisms, GPCRs regulate cell growth, development, morphogenesis, motility, and life span. In higher eukaryotic organisms as humans, they mediate the action of hundreds of peptide hormones, sensory stimuli, odorants, neurotransmitters, and chemokine. Due to their wide spectrum of action mechanisms, GPCRs are...
Detecting g-protein coupled receptor interactions using enhanced green fluorescent protein reassembly
Kumaş, Gözde; Son, Çağdaş Devrim; Yanık, Tülin; Department of Biotechnology (2012)
The largest class of cell surface receptors in mammalian genomes is the superfamily of G protein-coupled receptors (GPCRs) which are activated by a wide range of extracellular responses such as hormones, pheromones, odorants, and neurotransmitters. Drugs which have therapeutic effects on a wide range of diseases are act on GPCRs. In contrast to traditional idea, it is recently getting accepted that G-protein coupled receptors can form homo- and hetero-dimers and this interaction could have important role on...
Balkan, Seyda Tuğçe; Son, Çağdaş Devrim; Küçük Baloğlu, Fatma; Department of Biochemistry (2021-8-11)
GPCR’s are seven-transmembrane receptors that transmit external signals to the intracellular environment via secondary messenger systems through heterotrimeric G proteins. Heterotrimeric G proteins consist of α and β-γ subunits. Until recent years, scientists thought that GPCR signal transduction occurs between one GPCR and one heterotrimeric G protein; however, recently, it has been shown that GPCR’s can make oligomers. Oligomerization of GPCR allows cells to tune the intensity of the signal and respond ap...
In vitro activity of panomycocin, a novel exo-beta-1,3-glucanase isolated from Pichia anomala NCYC 434, against dermatophytes
Izgu, Fatih; Altinbay, Demet; Tureli, Akif Emre (2007-01-01)
Killer proteins that are produced and secreted into the environment by certain yeast strains are considered as promising antifungal agents. In this study, in vitro activity of Pichia anomala NCYC 434 (K5) killer protein, panomycocin, which is a 49 kDa glycoprotein with an exo-beta-1,3-glucanase activity was tested against 41 isolates of dermatophytes. Minimum inhibitory concentrations (MICs) were determined by a broth microdilution method based on the reference document M38-A of Clinical and Laboratory Stan...
Analysis of ligand-receptor cross-linked fragments by mass spectrometry
Son, Çağdaş Devrim; Hurst, GB; Naider, F; Becker, JM (Wiley, 2005-03-01)
G-protein coupled receptors (GPCRs) are a class of integral membrane receptor proteins that are characterized by a signature seven-transmembrane (7-TM) configuration. The alpha-factor receptor (Ste2p) from Saccharomyces cerevisiae is a GPCR that, upon binding of a peptide ligand, transduces a signal to initiate a cascade of events leading to the mating of haploid yeast cells. This study summarizes the application of affinity purification and of matrix-assisted laser-desorption ionization time-of-flight (MAL...
Citation Formats
G. Bulut, “In vivo detection of yeast alpha mating pheromone receptor ste2p homodimerization by FRET,” M.S. - Master of Science, Middle East Technical University, 2014.