SUMO proteins are a family of ubiquitin-related proteins that become covalently linked to other cellular proteins. While budding yeast has a single SUMO, called Smt3p, there are three commonly expressed mammalian SUMO paralogues, called SUMO-1, -2 and -3. SUMO-2 and -3 are 96% identical, while SUMO-1 is roughly 45% identical to either SUMO-2 or -3. In this report, SUMO-2 and -3 will be collectively called SUMO-2/3 under circumstances where they cannot be distinguished from each other. SUMO-1 is less abundant than SUMO-2/3, and it is conjugated to a distinct spectrum of targets. SUMO-1 also has different in vivo dynamics and responses to physiological stress such as heat shock. Unlike SUMO-2/3, SUMO-1 is concentrated at nuclear pore complexes (NPCs), the primary conduit of nucleocytoplasmic trafficking, reflecting the fact that it is the preferred conjugation partner of RanGAP1. RanGAP1 is the GTPase activator for Ran, a small GTPase that controls nuclear transport. SUMO-1 conjugation of RanGAP1 promotes its stable association to the NPCs through binding to a large nucleoporin, Nup358/RanBP2. Human SUMO proteins have been implicated in a variety of cell functions, including nuclear trafficking, chromosome segregation, chromatin organization, transcription and RNA metabolism. The conjugation pathway for SUMO proteins is similar to the ubiquitin conjugation pathway: SUMO proteins are processed by Ubiquitin like proteases/Sentrin specific proteases (Ulps/SENPs) to reveal a di-glycine motif at their C-termini. After processing, SUMO proteins undergo ATP-dependent formation of a thioester bond to their activating (E1) enzyme, Aos1/Uba2. The activated SUMO proteins are transferred to form a thioester linkage with their conjugating (E2) enzyme, Ubc9. Finally, an isopeptide bond is formed between SUMO proteins and substrates through the cooperative action of Ubc9 and protein ligases (E3). The linkage of SUMO proteins to their substrates can be severed by Ulps/SENPs, so it is likely that SUMO modification is highly dynamic in vivo. Ulp/SENPs play an important role in determination of the spectrum of conjugated species because they directly regulate the production of free, conjugatable SUMO proteins and the half-life of conjugated species. There are six members of the Ulp/SENP family in mammals and five in amphibians (Xenopus laevis). We are systematically evaluating the physiological roles and regulation of these enzymes. To investigate how Ulp/SENPs are regulated in a developmental context, we isolated and characterized all Ulp/SENPs in Xenopus laevis. Xenopus possess homologues of mammalian SENP3, 5, 6 and 7. All of these enzymes reacted with HA-tagged vinyl sulfone derivatives of SUMO-2 (HA-SU2-VS) but not SUMO-1 (HA-SU1-VS), suggesting that they act primarily on SUMO-2 and -3. In contrast, Xenopus possess a single member of the SENP1/SENP2 subfamily of Ulp/SENPs, most closely related to mammalian SENP1. Xenopus SENP1 reacted with HA-SU1-VS and HA-SU2-VS, suggesting that it acts on all SUMO paralogues. We analyzed the mRNA and protein levels for each of the Ulp/SENPs through development;we found that they show distinct patterns of expression that may involve both transcriptional and post-transcriptional regulation. Finally, we have characterized the developmental function of the most abundant Ulp/SENP found within Xenopus eggs, SENP3. Depletion of SENP3 using morpholino antisense oligonucleotides (morpholinos) caused accumulation of high molecular weight SUMO-2/3 conjugated species, defects in developing embryos and changes in the expression of some genes regulated by the transforming growth factor beta pathway. These findings collectively indicate that SUMO proteases are both highly regulated and essential for normal development. We have analyzed SENP3 and the highly-related SENP5 in mammalian cells. We have found that SENP3 and SENP5 localize within the granular component of the nucleolus, a sub-nucleolar compartment that contains B23/Nucleophosmin. B23/Nucleophosmin is an abundant shuttling phosphoprotein, which plays important roles in ribosome biogenesis, and which has been strongly implicated in hematopoietic malignancies. Moreover, we found that B23/Nucleophosmin binds SENP3 and SENP5 in Xenopus egg extracts, and that B23/Nucleophosmin promotes the stability of SENP3 and SENP5 in mammalian tissue culture cells. After either co-depletion of SENP3 and SENP5 or depletion of B23/Nucleophosmin, we observe accumulation of SUMO proteins within nucleoli. Finally, depletion of these Ulp/SENPs causes defects in ribosome biogenesis reminiscent of phenotypes observed in the absence of B23/Nucleophosmin. Together, these results suggest that regulation of SUMO deconjugation may be a major facet of B23/Nucleophosmin function in vivo. We are currently working to understand how SUMOylation contributes toward ribosome biogenesis through examination of SENP3 and SENP5 regulation, as well as through the identification of ribosomal SUMOylation targets. We had previously shown that SENP6 (also called SUSP1) localizes within the nucleoplasm, where it plays a specialized role in dismantling highly conjugated SUMO-2 and -3 species. This function is similar to the chain-editing activity of SENP6s closest relative in budding yeast, Ulp2. It has recently been shown that poly-SUMO-2/3 conjugated species are frequently degraded through the action of RNF4, an ubiquitin ligase that targets them for proteasomal degradation. We have examined the role of SENP6 in chromosome segregation, a process that requires Ulp2p in yeast. We found that cells depleted of SENP6 by RNA interference showed defects in spindle assembly and metaphase chromosome congression. Systematic analysis of kinetochore composition in these cells revealed that a subset of proteins became undetectable on kinetochores after SENP6 depletion. We further found SENP6 antagonizes RNF4 in regulation of the stability of these kinetochore components, revealing a novel mechanism whereby the finely balanced activities of SENP6 and RNF4 control vertebrate kinetochore assembly through SUMO-targeted destabilization of inner plate components.