Labeling and optimization of organelle markers for co-localization with yeast GPCR dimers

Süder, İlke
G-protein-coupled receptors, which are the most versatile chemical sensors, have prominent role in physiologically important cellular processes including cell growth and neurotransmission. Therefore, they are targeted by almost 50% of drugs in the market against diseases such as heart failure, neurological disorders and hypertension. It is well established phenomenon that GPCRs exist and function as dimers. Studies illustrate that dimerization may be favored for receptor activation, signal transduction, trafficking, cell surface mobility and ligand interactions. Even though evidences of homo- and hetero-dimerization accumulated there is no consensus on why GPCRs oligomerize. Since the pharmacological characteristics of the receptors may be altered when oligomerization occurs, the localization and reason of the phenomenon draws great attention. Comprehensive knowledge of the localization of a protein or biological process unravels the function of the protein or process. Yeast GPCRs, Ste2p, Ste3p and Gpr1p, serve as models for GPCR studies in vivo. Since Ste2p is known to form homodimer, dimerization studies have been focused on the receptor for years. Although subcellular fractionation data indicate that Ste2p dimers are found in the ER as many as in the plasma membrane, recent study conducted in our lab points out that the Ste2 dimer does not fluorescence in the ER when labeled with split EGFP. Hence, the aim of the study was to generate fluorescent organelle marker proteins which label the subcellular compartments on the trafficking route of membrane proteins. By co-localizing the markers with split EGFP tagged Ste2 dimer, where the dimerization occurs in living cells through a noninvasive approach could be identified. Therefore, using short targeting sequences, red ER marker fluorescent protein was constructed in the study. Using resident proteins of the late endosome, the Golgi apparatus and COPII vesicle, related full-length organelle marker proteins were prepared by tagging them at carboxy terminal with mCherry, a red FP. Furthermore, to be used for further studies in our lab, peroxisome markers using resident peroxisome protein were also generated. The visualized subcellular compartments showed characteristic morphologies consistent with previous descriptions. In order to assess the functionality of the organelle markers, they were co-localized with EGFP tagged Ste2p and Gpr1p. All the results were consistent with expectation based on knowledge on membrane protein trafficking. Therefore, it can be confidently suggest that all the markers are valuable resources for co-localization studies in live yeast cells. Moreover, they can serve for organelle marking in live cells without using expensive antibodies or harmful chemicals, identification of localization and thus function of unidentified proteins and monitoring the distribution and dynamics of organelles.


Optimization of internal tagging of inhibitory G-proteins for investigating their interactions with dopamine receptor D2 via fret method
Özcan, Gizem; Son, Çağdaş Devrim; Özçubukçu, Salih; Department of Biochemistry (2016)
G-Protein Coupled Receptors (GPCRs) constitute a large family of receptors which act by sensing the molecules outside the cell and start a signal transduction inside the cell through interacting with their associated G-proteins. This interaction results in activation or repression of related signaling pathways via associated secondary messengers. Dopamine receptor D2 (D2R) is a member of D2-like Dopamine Receptor group, which also belongs to the GPCR family. It is known that D2R has critical roles in emotio...
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...
Prediction of transmembrane regions of g protein-coupled receptors using machine learning techniques
Çınar, Muazzez Çelebi; Son, Çağdaş Devrim; Department of Molecular Biology and Genetics (2019)
G protein-coupled receptors (GPCRs) are one of the largest and the most significant membrane receptor families in eukaryotes. They transmit extracellular stimuli to the inside of the cell by undergoing conformational changes. GPCRs can recognize a diversity of extracellular ligands including hormones, neurotransmitters, odorants, photons, and ions. These receptors are associated with a variety of diseases in humans such as cancer and central nervous system disorders, and can be proclaimed as one of the most...
Investigation of activated sludge bioflocculation : influence of magnesium ions
Turtin, İpek; Sanin, Faika Dilek; Department of Environmental Engineering (2005)
Activated sludge systems are the most widely used biological wastewater treatment processes all over the world. The main working principles of an activated sludge system are the oxidation of biologically degradable wastes by microorganisms and the subsequent separation of the newly formed biomass from the treated effluent. Separation by settling is the most troublesome stage of an activated sludge process. A decrease in the efficiency of the separation of microbial biomass from the treated effluent causes a...
Parallelization of functional flow to predict protein functions
Akkoyun, Emrah; Can, Tolga; Department of Medical Informatics (2011)
Protein-protein interaction networks provide important information about what the biological function of proteins whose roles are unknown might be in a cell. These interaction networks were analyzed by a variety of approaches by running them on a single computer and the roles of the proteins identified were used to predict the function of the proteins unidentified. The functional flow is an approach that takes the network connectivity, distance effect, topology of the network with local and global views int...
Citation Formats
İ. Süder, “Labeling and optimization of organelle markers for co-localization with yeast GPCR dimers,” M.S. - Master of Science, Middle East Technical University, 2013.