J. Biochem, 2003, Vol. 133, No. 4 415-422
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Comparative Analysis of Yeast PIAS-Type SUMO Ligases In Vivo and In Vitro
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033
SUMO/Smt3, a ubiquitin-like modifier, is known to conjugate other proteins and modulate their functions in various processes. Recently, Ull1/Siz1 was discovered as a novel PIAS-type E3 required for septin sumoylation in yeast. We demonstrate here that the second PIAS-type Nfi1/Siz2 is also a SUMO ligase. It interacted with Smt3, SUMO/Smt3 conjugating enzyme Ubc9 and a septin component Cdc3 in the two-hybrid system. The region containing the RING-like domain of Nfi1/Siz2 bound directly to Ubc9 and Cdc3, but not to Smt3. Nfi1/Siz2 stimulated Smt3 conjugation to Cdc3 in vitro. In this in vitro system, Smt3 formed polymeric chains in the presence of higher concentrations of E1 and E2 enzymes. When the lysine15 residue of Smt3 was substituted with arginine, Smt3 chain-polymerization was abolished. Using this polysumoylation-deficient mutant Smt3, we found that Cdc3 and Nfi1/Siz2 were modified with Smt3 at multiple sites. Finally we found that the C-terminal truncated form of Ull1/Siz1 was mis-localized in vivo, but retained its SUMO ligase activity in vitro. We discuss the regulation of these SUMO ligases in vivo and in vitro.
+ To whom correspondence should be addressed. Tel/Fax: +81-3-5684-9420, E-mail: kikuchi{at}biol.s.u-tokyo.ac.jp
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