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. Human SUMO paralogues 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 Sentrin specific proteases/Ubiquitin like proteases (SENP/Ulps) 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 SUMO proteases, so it is likely that SUMO modification is highly dynamic in vivo. Like processing, SUMO deconjugation is mediated by SENP/Ulps. [unreadable] We are interested in the roles of individual vertebrate SUMO paralogues. Using live imaging and photobleaching methods, we have shown that mammalian SUMO paralogues show discrete temporal and spatial patterns of utilization, arguing that they are functionally distinct and specifically regulated in vivo. One biochemical difference that may be important for unique paralogue behaviors is the capacity to form linked chains. Like ubiquitin, Smt3p, SUMO-2 and -3 can form conjugated chains in vitro and in vivo, although the prevalence and physiological role of SUMO chains have not been established. We have examined the localization, biological function and enzymatic specificity of SUSP1, the largest human SENP/Ulp. We found that SUSP1 localizes to the nucleoplasm. SUSP1 depletion within cell lines expressing green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused re-distribution of EGFP-SUMO-2 and -3, particularly into PML bodies. PML bodies are nuclear structures of undefined function that contain the Promyelocytic Leukemia Protein (PML), a major SUMO conjugation substrate. Notably, both the size and number of PML bodies increased under these circumstances. Further analysis suggested that this change resulted primarily from a deficit of SUMO-2/3 deconjugation activity. We did not observe a comparable re-distribution of EGFP-SUMO-1. We have investigated the specificity of SUSP1 using vinyl sulfone (VS) inhibitors and model substrates. We found that SUSP1 has a strong paralogue preference for SUMO-2/3, and that it acts preferentially on substrates containing three or more SUMO-2/3 moieties. Together, our findings argue that SUSP1 may play a very specialized role in dismantling highly conjugated SUMO-2 and -3 species that is critical for PML body maintenance.[unreadable] Notably, results of other experiments suggest that the PML protein may not only be an important substrate for SUMO conjugation, but also a regulator of this pathway. PML was first identified through its fusion to the retinoic acid receptor alpha (RAR-alpha) in acute promyelocytic leukemia (APL) patients. In APL cell lines that express PML:RAR-alpha, we observed that a subset of proteins showed retinoic acid-dependent changes in their SUMO modification. Interestingly, PML and PML:RAR-alpha expression in yeast both stimulated hSUMO-1 modification, but differentially complemented yeast SUMO pathway mutants. Together with additional findings, our data indicate that PML and PML:RAR-alpha may regulate SUMO conjugation, and suggest that fusion of RAR-alpha to PML may affect this activity. It is obviously of interest to speculate that such changes in SUMO modification patterns may contribute toward the onset of APL.[unreadable] Finally, we have examined SUMO-2/3-specific modification of Topoisomerase-II in mitotic Xenopus egg extracts, and found that the SUMO ligase PIASy is specifically required for this conjugation. Moreover, PIASy depletion from extracts eliminated essentially all chromosomal accumulation of SUMO-2-conjugated species, suggesting that it is the primary ligase for mitotic chromosomal substrates of SUMO-2. PIASy-dependent SUMO-2-conjugated species concentrated on the inner centromere, and inhibition of PIASy blocked anaphase sister chromatid segregation. Taken together, our observations suggest that PIASy is a critical regulator of mitotic SUMO-2 conjugation, and that its activity may have particular relevance for centromeric functions required for proper chromosome segregation.