SUMO1/Smt3, a ubiquitin-like protein modifier, is known to conjugate to other proteins and modulate their functions in various important processes. Similar to the ubiquitin conjugation system, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme). In our previous report (Takahashi, Y., Toh-e, A., and Kikuchi, Y. (2001) Gene 275, 223-231), we showed that Siz1/Ull1 (YDR409w) of budding yeast, a member of the human PIAS family containing a RINGlike domain, is a strong candidate for SUMO1/Smt3 ligase because the SUMO1/Smt3 modification of septin components was abolished in the ull1 mutant and Ull1 associated with E2 (Ubc9) and the substrates (septin components) in immunoprecipitation experiments. Here we have developed an in vitro Smt3 conjugation system for a septin component (Cdc3) using purified recombinant proteins. In this system, Ull1 is additionally required as well as E1 (Sua1⅐Uba2 complex), E2 (Ubc9), and ATP. A cysteine residue of the RING-like domain was essential for the conjugation both in vivo and in vitro. Furthermore, a region containing the RING-like domain directly interacted with Ubc9 and Cdc3. Thus, this SUMO/Smt3 ligase functions as an adaptor between E2 and the target proteins.SUMO 1 (small ubiquitin-like modifier)/Smt3 is a member of a growing family of ubiquitin-related proteins and is known to be conjugated to RanGAP1, PML (promyelocytic leukemia), I B␣, p53, septins, etc. (1-4). Not only are the amino acid sequences and the three-dimensional structures similar between SUMO/ Smt3 and ubiquitin, but their conjugation systems and the enzymes involved are highly related (5-9). In the ubiquitin pathway a third enzyme, ubiquitin ligase (E3), is often required for the final transfer of this modifier and plays a crucial role by recognizing target proteins and by promoting their conjugation (10). It remains unknown, however, whether any SUMO1/Smt3 ligases (E3s) are involved in this conjugation pathway.In the ubiquitin pathway, some E3 components such as Apc11 of the anaphase-promoting complex and Rbx1 of the SCF (Skp1, cullin, F-box protein)⅐ubiquitin ligase complex contain a zinc-binding RING domain with an octet of ordered cysteine and histidine residues forming a cross-brace around two zinc atoms (11,12). This type of ubiquitin ligases (E3s) has to interact with E2 and the substrate at the same time because apparently they do not form the thioester bond with ubiquitin. In the case of c-Cbl proto-oncoprotein, its RING domain interacts with UbcH7 (E2), and the tyrosine kinase binding domain, a region close to the RING domain, recognizes its substrate. Thus, c-Cbl proto-oncoprotein functions as a bridging molecule between E2 and its substrate (13).In budding yeast, Smt3 is the only member of the SUMO family, and the Smt3 conjugation system is essential for mitotic growth. The lethality of the smt3 deletion mutant can be suppressed by expressing human SUMO1, suggesting that SUMO1 is a functional homologue of yeast Smt...

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The Smt3 (SUMO) protein is conjugated to substrate proteins through a cascade of E1, E2, and E3 enzymes. In budding yeast, the E3 step in sumoylation is largely controlled by Siz1p and Siz2p. Analysis of Siz ÿ cells shows that SUMO E3 is required for minichromosome segregation and thus has a positive role in maintaining the fidelity of mitotic transmission of genetic information. Sumoylation of the carboxy-terminus of Top2p, a known SUMO target, is mediated by Siz1p and Siz2p both in vivo and in vitro. Sumoylation in vitro reveals that Top2p is an extremely potent substrate for Smt3p conjugation and that chromatin-bound Top2p can still be sumoylated, unlike many other SUMO substrates. By combining mutations in the TOP2 sumoylation sites and the SIZ1 and SIZ2 genes we demonstrate that the minichromosome segregation defect and dicentric minichromosome stabilization, both characteristic for Smt3p-E3-deficient cells, are mediated by the lack of Top2p sumoylation in these cells. A role for Smt3p-modification as a signal for Top2p targeting to pericentromeric regions was suggested by an analysis of Top2p-Smt3p fusion. We propose a model for the positive control of the centromeric pool of Top2p, required for high segregation fidelity, by Smt3p modification.

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