Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Dr. Bohr Gasse 9, Vienna A-1030, Austria.
Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Center for Molecular Biology, University of Vienna, Dr. Bohr Gasse 9, Vienna A-1030, Austria
Biochem J. 2018 Jan 2;475(1):61-74. doi: 10.1042/BCJ20170472.
The small ubiquitin-related modifier (SUMO) conjugation apparatus usually attaches single SUMO moieties to its substrates, but SUMO chains have also been identified. To better define the biochemical requirements and characteristics of SUMO chain formation, mutations in surface-exposed Lys residues of Arabidopsis SUMO-conjugating enzyme (SCE) were tested for activity. Lys-to-Arg changes in the amino-terminal region of SCE allowed SUMO acceptance from SUMO-activating enzyme and supported substrate mono-sumoylation, but these mutations had significant effects on SUMO chain assembly. We found no indication that SUMO modification of SCE promotes chain formation. A substrate was identified that is modified by SUMO chain addition, showing that SCE can distinguish substrates for either mono-sumoylation or SUMO chain attachment. It is also shown that SCE with active site Cys mutated to Ser can accept SUMO to form an oxyester, but cannot transfer this SUMO moiety onto substrates, explaining a previously known dominant negative effect of this mutation.
小泛素相关修饰物（SUMO）缀合装置通常将单个 SUMO 单元连接到其底物上，但也已经鉴定出 SUMO 链。为了更好地定义 SUMO 链形成的生化要求和特征，对拟南芥 SUMO 连接酶（SCE）表面暴露的赖氨酸残基的突变进行了活性测试。SCE 氨基末端区域的赖氨酸到精氨酸的变化允许 SUMO 从 SUMO 激活酶中接受，并支持底物单 SUMO 化修饰，但这些突变对 SUMO 链组装有显著影响。我们没有发现 SCE 的 SUMO 修饰促进链形成的迹象。鉴定到一种通过 SUMO 链添加进行修饰的底物，表明 SCE 可以区分用于单 SUMO 化或 SUMO 链连接的底物。还表明，具有活性位点半胱氨酸突变为丝氨酸的 SCE 可以接受 SUMO 形成氧酯，但不能将此 SUMO 单元转移到底物上，这解释了该突变先前已知的显性负效应。
