Date of Award
Clancy, Mary; Burt-Utley, Kathleen
Differences have been shown in the steady-state accumulation and half-lives between Brassica FAD3 (BF3) and tung FAD3 (TF3) proteins expressed in yeast cells cultured at 30°C. TF3 has a greater steady-state accumulation and longer half-life than BF3. These differences are attributed to post-translational modification and have been shown to be controlled by an Nterminal element. I attempted to determine specific amino acids important for regulation, and further characterize the mechanism contributing to the differences. Through site-directed mutagenesis, it was shown that replacing lysine residues with asparagines in the BF3 and TF3 Ntermini increased protein stability, while replacing an asparagine with lysine in the TF3 Nterminus decreased its stability. Furthermore, I showed that the TF3 polyglutamic region (six consecutive glutamic acid residues) is primarily responsible for the higher steady-state amount of TF3 in comparison to BF3. This negatively charged region likely acts as an electrostatic shield protecting the protein from degradation.
Bourassa, Linda, "Post-translational Regulation of Plant Fatty Acid Desaturases as Expressed in Saccharomyces cerevisiae" (2008). University of New Orleans Theses and Dissertations. 828.