Max Delbrück-Center for Molecular Medicine in the Helmholtz Association, Berlin-Buch, Germany.
Department of Biochemistry, School of Medicine, University of Washington, Seattle, WA, USA.
EMBO J. 2020 Nov 16;39(22):e104863. doi: 10.15252/embj.2020104863. Epub 2020 Oct 5.
Protein modification with poly-ubiquitin chains is a crucial process involved in a myriad of cellular pathways. Chain synthesis requires two steps: substrate modification with ubiquitin (priming) followed by repetitive ubiquitin-to-ubiquitin attachment (elongation). RING-type E3 ligases catalyze both reactions in collaboration with specific priming and elongating E2 enzymes. We provide kinetic insight into poly-ubiquitylation during protein quality control by showing that priming is the rate-determining step in protein degradation as directed by the yeast ERAD RING E3 ligases, Hrd1 and Doa10. Doa10 cooperates with the dedicated priming E2, Ubc6, while both E3s use Ubc7 for elongation. Here, we provide direct evidence that Hrd1 uses Ubc7 also for priming. We found that Ubc6 has an unusually high basal activity that does not require strong stimulation from an E3. Doa10 exploits this property to pair with Ubc6 over Ubc7 during priming. Our work not only illuminates the mechanisms of specific E2/E3 interplay in ERAD, but also offers a basis to understand how RING E3s may have properties that are tailored to pair with their preferred E2s.
蛋白质与多聚泛素链的修饰是参与众多细胞途径的关键过程。链的合成需要两个步骤：泛素对底物的修饰（引发），然后是重复的泛素-泛素连接（延伸）。RING 型 E3 连接酶与特定的引发和延伸 E2 酶合作，催化这两个反应。我们通过显示在酵母 ERAD RING E3 连接酶 Hrd1 和 Doa10 指导的蛋白质降解过程中，引发是限速步骤，从而为蛋白质质量控制过程中的多泛素化提供了动力学见解。Doa10 与专用引发 E2 Ubc6 合作，而两个 E3 都使用 Ubc7 进行延伸。在这里，我们提供了直接证据表明 Hrd1 也使用 Ubc7 进行引发。我们发现 Ubc6 具有异常高的基础活性，不需要 E3 的强烈刺激。Doa10 利用这一特性在引发过程中与 Ubc6 配对，而不是与 Ubc7 配对。我们的工作不仅阐明了 ERAD 中特定 E2/E3 相互作用的机制，而且还为理解 RING E3 如何具有与其首选 E2 配对的特性提供了基础。
