Date of Award

8-2010

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Sharon Ashworth

Third Committee Member

David J. Neivandt

Abstract

Guanine nucleotide binding proteins (G proteins) are the one of major proteins regulating wide range of signaling pathways. Due to the wide spectrum of G protein involvement in signal transduction and their oncogenic characters, intensive research has been conducted to elucidate the vital functions to invent more powerful yet harmless pharmaceutical drugs. Two prominent features of G proteins, de/activation cycle and shuttling of G proteins, has been conserved through evolution and mounting evidence suggests they are deeply connected to the functional integrity of the cell. Detailed analysis identified that lipid modifications are the key components mediating the spatiodynamics of G proteins. Moreover, several lines of evidence indicate that translocation of proteins between segregated domains on the plasma membrane and between the cytosol and plasma membrane is activity dependent. However, there is no established model integrating these observations and key features. Here, we identified Dictyostelium Gα2 subunit adapted two different activity dependent shuttling pathways. We showed that palmitoylation deficit Gα2 mutant can not form heterotrimer. Also, dissociation inhibited Gα2 mutant accumulated at the intracellular membrane. From these results, we speculate that protein depalmitoylation causes the subunits dissociation and translocation of proteins to cytosol. Then we found activation of G protein does not influence on shuttling process and while in the process the proteins do not undergo palmitoylation turnover. To further investigate the key event causing G protein shuttling in active state, we measure the membrane binding affinity of activated/inactivated and figure out whether de/activation cycle affect the trasnlocation of the protein between cytosl and the plasma membrane. Taken all data together, we concluded that one shuttling pathway is regulated by the careful balance of de/palmitoylation, led in G protein inactive state. On contrary, the other pathway is not regulated de/palmitoylation cycle. Activated G proteins laterally translocate to different domains and are released from the plasma membrane by GTPase-mediated G protein inactivation.

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