At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.

在蛋白质泛素化级联反应的核心，泛素结合酶 (E2s) 形成反应性泛素-硫酯中间体，以实现泛素向细胞底物的有效转移。因此，E2s 的精确调节对于细胞稳态至关重要，并且它们的失调通常与肿瘤发生有关。除了与泛素连接酶 (E3) 一起驱动底物泛素化外，许多 E2 还可以自身泛素化，从而促进它们自身的蛋白酶体周转。为了研究平衡这些不同活动的机制，我们剖析了 UBE2S 的调节动态，这是一种人类 APC/C 相关的 E2，可确保细胞周期调节剂在有丝分裂过程中的忠实泛素化。我们发现了 UBE2S 的二聚体状态，它通过阻断催化关键的泛素结合位点来实现自身抑制。二聚化受到 UBE2S 富含赖氨酸的羧基末端延伸的刺激，这也是将该 E2 募集到 APC/C 所必需的，并且随着底物丰度的限制而自动泛素化。与这种机制一致，我们发现二聚化缺陷的 UBE2S 在细胞中翻转得更快，并且在药物诱导的有丝分裂停滞期间不会促进有丝分裂滑移。我们建议当 APC/C 不完全活跃时，二聚化会减弱自泛素化诱导的 UBE2S 周转。更广泛地，