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

Thesis Type: Postgraduate

Institution Of The Thesis: Middle East Technical University, Turkey

Approval Date: 2013

Thesis Language: English

Student: İlke Süder



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.