Access Type

Open Access Thesis

Date of Award

January 2013

Degree Type


Degree Name



Biological Sciences

First Advisor

Xiang-Dong Zhang


The covalent and reversible conjugation of small ubiquitin-like modifier (SUMO) proteins to hundreds of different cellular proteins is catalyzed by a cascade of enzymes including an E1-activating enzyme (SAE1/SAE2), an E2-conjugating enzyme (Ubc9) and multiple E3 ligases. As the only E2 enzyme for SUMO-conjugation, Ubc9 localizes mainly in the nucleus and plays an essential role in regulation of many cellular processes including cell cycle progression through mitosis, cell migration, genome stability, stress response, transcription, and nuclear transport in eukaryotic cells. It is hypothesized that the nuclear localization of Ubc9 is required for efficient sumoylation inside the nucleus because both the sole SUMO E1 enzyme and SUMO-conjugates are mainly in the nucleus. However, we still have a poor understanding of how Ubc9 is accumulated in the nucleus. Although the nuclear import receptor Importin 13 (Imp13) can mediate the nuclear import of Ubc9 using in vitro nuclear import assays, little is known about how Ubc9 nuclear localization is regulated in vivo. Here, we hypothesize that Imp13 is the major nuclear import receptor for Ubc9 and thus required for efficient global sumoylation in vivo. Consistent with this hypothesis, we found that knockdown of Imp13 by RNA interference (RNAi) causes a decrease of global sumoylation and also an increased cytoplasmic distribution of Ubc9. Furthermore, the Ubc9 mutant (R17E) with a defect in Imp13-interaction showed a significant increase of cytoplasmic distribution when compared to Ubc9 wild-type (WT). Moreover, overexpression of Imp13 greatly enhanced the nuclear localization of Ubc9-WT but not Ubc9-R17E mutant, whereas overexpression of Imp13 mutant (D426R) with a defect in Ubc9 binding could not promote the nuclear accumulation of Ubc9-WT. Lastly, we demonstrated that the Ubc9 mutants (R17E, R13A and H20D) with a defect in SUMO-binding have an elevated cytoplasmic distribution when compared to Ubc9-WT, suggesting that the non-covalent interaction between Ubc9 and SUMO is also important for Ubc9 nuclear accumulation. Hence, our results support a model that both Imp13-mediated nuclear import and the SUMO-binding activity of Ubc9 are critical for Ubc9 nuclear localization and efficient global sumoylation in mammalian cells.