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 RING-like 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 (Sua1.Uba2 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.