Takahashi Yoshimitsu, Toh-E Akio, Kikuchi Yoshiko
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033.
J Biochem. 2003 Apr;133(4):415-22. doi: 10.1093/jb/mvg054.
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 lysine(15) 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.
SUMO/Smt3是一种类泛素修饰因子，已知其能与其他蛋白质结合并在多种过程中调节它们的功能。最近，Ull1/Siz1被发现是酵母中septin类泛素化所需的一种新型PIAS型E3。我们在此证明，第二种PIAS型Nfi1/Siz2也是一种SUMO连接酶。在双杂交系统中，它与Smt3、SUMO/Smt3结合酶Ubc9和一种septin成分Cdc3相互作用。包含Nfi1/Siz2类RING结构域的区域直接与Ubc9和Cdc3结合，但不与Smt3结合。Nfi1/Siz2在体外刺激Smt3与Cdc3结合。在这个体外系统中，在较高浓度的E1和E2酶存在下，Smt3形成多聚链。当Smt3的赖氨酸（15）残基被精氨酸取代时，Smt3链聚合被消除。使用这种多聚类泛素化缺陷型突变体Smt3，我们发现Cdc3和Nfi1/Siz2在多个位点被Smt3修饰。最后我们发现，Ull1/Siz1的C末端截短形式在体内定位错误，但在体外保留其SUMO连接酶活性。我们讨论了这些SUMO连接酶在体内和体外的调节。
