Hücre yüzeyi proteinlerinin analizi için yeni bir metod geliştirilmesi

Plasma membrane is an evolutionary conserved cellular compartment for all cells. It directly or indirectly participates in vital pathways in the cell such as nutrition uptake, signal transduction, cell to cell communication, migration, adhesion, and cell division. Therefore, to elucidate molecular mechanisms of the cell or regulation of these pathways, we need to understand how plasma membrane components interact with inner part of the cell and their specific roles in these pathways. One unbiased way to address these questions is to take proteomics approach. Yet, unique structure of the plasma membrane and low abundancy of the cell membrane proteins make it challenging to study plasma membrane proteins with traditional proteomics approach. Therefore, there is a need for more effective approaches for identification of plasma membrane proteins. In the first part of my thesis, I developed a novel approach for plasma membrane protein enrichment. In this method, I used centrifugation and BioID enrichment methods subsequently. To that extent, I modified biotin ligase (BirA) enzyme to target and biotinylate cell membrane proteins. Then, by using subsequent physical and affinity-based enrichment methods, plasma membrane protein levels increased in the sample. Proteins then identified by using mass spectrometry. In the second part of my thesis, I focused on two members of Chloride Intracellular Channel (CLIC) proteins, CLIC1 and CLIC4. In a recent quantitative proteomics study from our laboratory showed that CLIC1 and CLIC4 enriched at the plasma membrane during cell division Currently, CLICs are not associated with any cell cycle dependent function. In the second part of my thesis, I focused on the characterization of the CLIC1 and CLIC4 proteins in cell cycle dependent manner. To that extent, I generated CRISPR-Cas9 mediated knockout cell lines and investigated their effects on cytokinesis fidelity. CLIC1 and CLIC4 knockout cells increased cytokinesis abnormalities indicating their role at the plasma membrane during cell division.