Biochemistry, Department of



A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Biochemistry, Under the Supervision of Professor Donald P. Weeks. Lincoln, Nebraska: May, 2012

Copyright (c) 2012 Amy Rose Ann Knobbe


The eukaryotic protein post-translational modification by SUMOylation is involved in a diverse array of cellular processes, including various stress responses. A fully functional SUMOylation system is present in the unicellular green alga Chlamydomonas reinhardtii, and SUMOylation of multiple high molecular weight proteins is induced in response to abiotic stress in this organism. We report here the characterization of a SUMO E2 conjugase deletion mutant in C. reinhardtii, mut5. SUMO E2 conjugase enzymes are responsible for the conjugation of the protein SUMO to a lysine residue within a target protein. C. reinhardtii mutants in which the SUMO E2 conjugase CrUBC9 has been deleted (mut5) fail to modify proteins with SUMO in response to multiple stress conditions, and this failure to SUMOylate generally results in a reduced tolerance to a given stress. Complementation of CrUBC9 mutants with the deleted gene demonstrates that CrUBC9 is solely responsible for SUMOylation under stress conditions, and that it predominantly localizes to the nucleus.

In addition, we identify the likely presence of two distinct SUMO E2 conjugase proteins in the C. reinhardtii genome. This is in marked contrast to virtually all other organisms studied to date, in which a single essential SUMO E2 conjugase has been identified. Bioinformatic analyses allowed the identification of the likeliest candidate for this second SUMO E2 conjugase, CrUBC3. Comparison of CrUBC3 and CrUBC9 reveals that they have distinct sequence features that distinguish them from other known SUMO E2 conjugases. In addition, we show that the transcripts encoding these proteins are regulated in a distinct and opposite manner in response to stress, consistent with separate functions for these proteins within the cell. Expression of these proteins in a heterologous yeast system allowed the examination of the functionality of these two proteins as SUMO E2 conjugases, and suggests that the two likely function in distinct, non-overlapping capacities within C. reinhardtii.

Advisor: Donald P. Weeks