Kirkpatrick Donald S, Hathaway Nathaniel A, Hanna John, Elsasser Suzanne, Rush John, Finley Daniel, King Randall W, Gygi Steven P
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Nat Cell Biol. 2006 Jul;8(7):700-10. doi: 10.1038/ncb1436. Epub 2006 Jun 25.
Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.
蛋白质泛素化调节许多细胞过程，包括蛋白质降解、信号转导、DNA修复和细胞分裂。在经典模型中，通过赖氨酸48连接的均匀多聚泛素链是26S蛋白酶体识别和降解所必需的。在此，我们使用重组系统和定量质谱法证明，细胞周期蛋白B1是由复杂拓扑结构的泛素链修饰的，而不是由均匀的赖氨酸48连接的链修饰的。发现后期促进复合物将单泛素连接到细胞周期蛋白B1上的多个赖氨酸残基上，随后通过泛素的多个赖氨酸残基（赖氨酸63、赖氨酸11和赖氨酸48）进行多聚泛素链延伸。这些异质泛素链足以与泛素受体结合，也足以被26S蛋白酶体降解，即使它们是用缺乏赖氨酸48的突变泛素合成的。总之，我们的观察结果扩展了可被视为充分降解信号的范围，并为底物泛素化机制提供了独特的见解。
