Johnson E S, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021, USA.
J Biol Chem. 1997 Oct 24;272(43):26799-802. doi: 10.1074/jbc.272.43.26799.
At least one essential function of Smt3p, a Saccharomyces cerevisiae ubiquitin-like protein similar to the mammalian protein SUMO-1, involves its posttranslational covalent attachment to other proteins. Using Smt3p affinity chromatography, we have isolated the second enzyme of the Smt3p conjugation pathway and have found that it is identical to Ubc9p, a previously identified protein that has extensive sequence similarity to the ubiquitin-conjugating enzymes (E2s) and that is required for yeast to progress through mitosis. A hallmark of E2s is the ability to form a thioester bond-containing covalent intermediate with ubiquitin (Ub). While we were unable to detect formation of a Ub approximately Ubc9p thioester, Ubc9p was found to form a thioester with Smt3p, indicating that Ubc9p is the functional analog of E2s in the Smt3p pathway and that this step is distinct from the ubiquitin pathway. Ubc9p is required for attachment of Smt3p to other proteins in vitro, suggesting that it is the only such enzyme in S. cerevisiae. These results suggest that, like ubiquitination, Smt3p conjugation may be a critical modification in cell cycle regulation.
Smt3p是一种与哺乳动物蛋白质SUMO-1相似的酿酒酵母泛素样蛋白，其至少一种基本功能涉及其在翻译后与其他蛋白质的共价连接。利用Smt3p亲和层析，我们分离出了Smt3p缀合途径的第二种酶，发现它与Ubc9p相同，Ubc9p是一种先前鉴定的蛋白质，与泛素缀合酶（E2s）具有广泛的序列相似性，并且是酵母进行有丝分裂所必需的。E2s的一个标志是能够与泛素（Ub）形成含硫酯键的共价中间体。虽然我们无法检测到Ub与Ubc9p硫酯的形成，但发现Ubc9p与Smt3p形成硫酯，这表明Ubc9p是Smt3p途径中E2s的功能类似物，并且这一步骤与泛素途径不同。Ubc9p在体外是Smt3p与其他蛋白质连接所必需的，这表明它是酿酒酵母中唯一的此类酶。这些结果表明，与泛素化一样，Smt3p缀合可能是细胞周期调控中的一种关键修饰。
