Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.
Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown.
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发表时间：
1983-07
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通讯作者：
A. Hershko;H. Heller;S. Elias;A. Ciechanover
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作者：
A. Hershko;H. Heller;S. Elias;A. Ciechanover
通过在泛素 - 琼脂糖上对粗制网织红细胞提取物进行亲和层析，已分离出三种将泛素与蛋白质结合所需的酶。一种是泛素激活酶（E1），它在ATP存在下与亲和柱共价连接，并且可以用AMP和焦磷酸盐特异性洗脱（Ciechanover，A.，Elias，S.，Heller，H.，和Hershko，A.（1982）J. Biol. Chem. 257，2537 - 2542）。第二种酶，命名为E2，当E1和ATP存在时结合到泛素柱上，并可用高浓度的巯基化合物洗脱。第三种酶，命名为E3，通过非共价相互作用吸附到亲和柱上，可以用高盐或升高的pH洗脱。125I - 泛素与蛋白质的结合绝对需要所有这三种酶。在泛素、ATP和亲和柱的未吸附部分存在的情况下，将125I - 白蛋白分解为酸溶性物质也需要所有这三种亲和纯化的酶。以下观察结果表明，E2的功能是以E2 - 泛素硫酯中间体的形式将活化的泛素转移到结合位点。（a）E2可被碘乙酰胺迅速灭活，但通过事先与E1、ATP和泛素一起孵育可防止其灭活。这表明E1介导的活化泛素转移到E2的对碘乙酰胺敏感的巯基位点。（b）上述E2与泛素柱结合的要求及其洗脱方式与E2和琼脂糖结合的泛素之间形成共价键的观点一致。（c）在125I - 泛素与E1和ATP一起孵育后，接着加入纯化的E2，活化的泛素从E1转移到几种低分子量形式的E2，如通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳所分析的。泛素与所有这些形式的连接具有硫酯键的特征。在进一步与E3和用于结合的蛋白质底物一起孵育时，活化的泛素从不同形式的E2 - 泛素转移到稳定的泛素 - 蛋白质结合物。因此，E3参与连接酶系统的最后一步。
By affinity chromatography of a crude reticulocyte extract on ubiquitin-Sepharose, three enzymes required for the conjugation of ubiquitin with proteins have been isolated. One is the ubiquitin-activating enzyme (E1), which is covalently linked to the affinity column in the presence of ATP and can be specifically eluted with AMP and pyrophosphate (Ciechanover, A., Elias, S., Heller, H., and Hershko, A. (1982) J. Biol. Chem. 257, 2537-2542). A second enzyme, designated E2, is bound to the ubiquitin column when E1 and ATP are present, and is eluted with a thiol compound at high concentration. The third enzyme, designated E3, is adsorbed to the affinity column by noncovalent interactions and can be eluted with high salt or increased pH. The presence of all three enzymes is absolutely required for the conjugation of 125I-ubiquitin with proteins. All three affinity-purified enzymes are also required for the breakdown of 125I-albumin to acid-soluble material in the presence of ubiquitin, ATP, and the unadsorbed fraction of the affinity column. The following observations indicate that the function of E2 is the transfer of activated ubiquitin to the site of conjugation in the form of an E2-ubiquitin thiol ester intermediate. (a) E2 is rapidly inactivated by iodoacetamide, but can be protected against inactivation by a prior incubation with E1, ATP, and ubiquitin. This suggests an E1-mediated transfer of activated ubiquitin to an iodoacetamide-sensitive thiol site of E2. (b) The requirements for the binding of E2 to the ubiquitin column and the mode of its elution, cited above, are consistent with the notion that a covalent linkage is formed between E2 and Sepharose-bound ubiquitin. (c) Upon the incubation of 125I-ubiquitin with E1 and ATP, followed by the addition of purified E2, activated ubiquitin is transferred from E1 to several low molecular weight forms of E2, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The linkage of ubiquitin to all these forms has the characteristics of a thiol ester bond. In a further incubation with E3 and a protein substrate for conjugation, activated ubiquitin was transferred from the different forms of E2-ubiquitin to stable ubiquitin-protein conjugates. Thus, E3 is involved in the last step of the ligase system.