SUMO proteins are a conserved family of ubiquitin-related proteins that become conjugated to substrates in a manner similar to ubiquitin. Fission and budding yeast each contain a single SUMO family protein. These proteins have been implicated in the regulation of the cell cycle in both organisms. In mammals, there are three SUMO isoforms. The conjugation pathway for all isoforms is similar to the ubiquitin conjugation pathway: SUMO proteins must be processed to yield a C-terminal di-glycine motif. After processing, the first step in the SUMO conjugation pathway is the ATP-dependent formation of a thioester bond between SUMO proteins and their activating (E1) enzyme. The second step is the formation of a thioester bond between SUMO proteins and their conjugating (E2) enzyme, Ubc9. In the last step, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). We are examining SUMO conjugation targets and SUMO pathway enzymes in a number of ways, in order to understand the biological role(s) of this pathway, particularly with respect to mitosis and cell cycle progression. We used Xenopus egg extracts to examine cell cycle-dependent changes in SUMO-conjugated proteins. We found a set of high molecular weight, chromatin-dependent mitotic SUMO-containing species, which protein sequencing revealed to be SUMO-conjugated Topoisomerase-II. Topoisomerase-II is modified exclusively by SUMO-2/3 during mitosis under normal circumstances; this modification is maximal in metaphase, followed by rapid deconjugation during anaphase. The differential extraction properties of modified and unmodified Topoisomerase-II suggest that SUMO-2/3 conjugation may mobilize Topoisomerase-II from mitotic chromatin in a manner that is important for chromosome segregation. Analysis using dominant negative forms of Ubc9 and Topoisomerase-II inhibitors are further consistent with this idea. Together, our findings indicate that SUMO-2/3 conjugation of Topoisomerase-II is important for remodeling of mitotic chromosomes at the metaphase-anaphase transition, and that failure of such remodeling could be expected to cause high levels of chromosome mis-segregation in vivo. Ran-GTP has an important role in regulating the organization of the cell during both interphase and mitosis (see Z01 HD008740-02). Given this role, knowledge of the distribution of Ran regulators will be essential for understanding the control and function of this pathway. In metazoans, RanGAP1 is conjugated with SUMO-1. SUMO-1 modification causes RanGAP1 to associate with Ubc9 and RanBP2, a large nuclear pore protein with Ran-GTP binding domains and a SUMO E3 ligase domain. In previous studies, we examined the behavior of RanGAP1 during mitosis, and found that RanGAP1 associates with kinetochores through a SUMO-1 dependent mechanism. RanBP2 co-localized with RanGAP1 on spindles, suggesting that a complex between these two proteins may be involved in mitotic targeting of RanGAP1. Recently, we have examined the structural requirements for targeting RanGAP1 and RanBP2, as well as their function in mitosis. We find that RNAi against RanBP2 displaced RanGAP1 from kinetochores, supporting the notion that these proteins target to kinetochores as part of a single complex. Both proteins were displaced after RNAi of integral kinetochore components, suggesting that they require intact kinetochore structures to localize appropriately. By contrast, peripheral kinetochore proteins were not essential for correct targeting of either protein. Cells depleted of RanBP2 show abnormalities in both spindle formation and mitotic progression, substantiating the importance of their function during mitosis. SUMO proteases catalyze the processing of SUMO-1 prior to conjugation and the deconjugation of SUMO-1 from other proteins. Budding yeast as two SUMO proteases, Ulp1p and Ulp2p/Smt4p. Ulp1p is concentrated near the nuclear periphery and interacts with nuclear pore components in two-hybrid assays. ULP1 is an essential gene, and temperature sensitive Ulp1p mutants arrest at the G2/M transition of the cell cycle. In mammals, there are at least seven SUMO protease family members. We are systematically investigating these enzymes. We have previously shown that one mammalian SUMO protease (SENP2) associates to the nuclear pore through an interaction between its N-terminus and Nup153. More recently, our analysis of SENP protease suggests that others also associate to the nuclear pore using different binding sites. Together with earlier findings regarding association of conjugating enzymes to the nuclear pore (Ubc9 and RanBP2), our results confirm that the nuclear pore is a critical site of SUMO pathway regulation in the cell.