Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Mol Cell. 2010 Nov 12;40(3):444-54. doi: 10.1016/j.molcel.2010.10.014.
Cse4 is a variant of histone H3 that is incorporated into a single nucleosome at each centromere in budding yeast. We have discovered an E3 ubiquitin ligase, called Psh1, which controls the cellular level of Cse4 via ubiquitylation and proteolysis. The activity of Psh1 is dependent on both its RING and zinc finger domains. We demonstrate the specificity of the ubiquitylation activity of Psh1 toward Cse4 in vitro and map the sites of ubiquitylation. Mutation of key lysines prevents ubiquitylation of Cse4 by Psh1 in vitro and stabilizes Cse4 in vivo. While deletion of Psh1 stabilizes Cse4, elimination of the Cse4-specific chaperone Scm3 destabilizes Cse4, and the addition of Scm3 to the Psh1-Cse4 ubiquitylation reaction prevents Cse4 ubiquitylation, together suggesting Scm3 may protect Cse4 from ubiquitylation. Without Psh1, Cse4 overexpression is toxic and Cse4 is found at ectopic locations. Our results suggest Psh1 functions to prevent the mislocalization of Cse4.
Cse4 是组蛋白 H3 的一种变体，在芽殖酵母的每个着丝粒中都被整合到一个单个核小体中。我们发现了一种 E3 泛素连接酶，称为 Psh1，它通过泛素化和蛋白水解来控制 Cse4 的细胞水平。Psh1 的活性依赖于其 RING 和锌指结构域。我们在体外证明了 Psh1 对 Cse4 的泛素化活性具有特异性，并绘制了泛素化位点。关键赖氨酸的突变可防止 Psh1 在体外对 Cse4 的泛素化，并在体内稳定 Cse4。虽然 Psh1 的缺失稳定了 Cse4，但 Cse4 特异性伴侣 Scm3 的消除使 Cse4 不稳定，并且将 Scm3 添加到 Psh1-Cse4 泛素化反应中可防止 Cse4 泛素化，这表明 Scm3 可能保护 Cse4 免受泛素化。没有 Psh1，Cse4 的过表达是有毒的，并且 Cse4 会出现在异位位置。我们的结果表明 Psh1 可防止 Cse4 的错误定位。
