The E2/E3 enzyme UBE2O ubiquitylates diverse clients to mediate important processes, including targeting unassembled ‘orphan’ proteins for quality control and clearing ribosomes during erythropoiesis. How quality-control factors, such as UBE2O, select clients on the basis of heterogeneous features is largely unknown. Here, we show that UBE2O client selection is regulated by ubiquitin binding and a cofactor, NAP1L1. Attaching a single ubiquitin onto a client enhances UBE2O binding and multi-mono-ubiquitylation. UBE2O also repurposes the histone chaperone NAP1L1 as an adapter to recruit a subset of clients. Cryo-EM structures of human UBE2O in complex with NAP1L1 reveal a malleable client recruitment interface that is autoinhibited by the intrinsically reactive UBC domain. Adding a ubiquitylated client identifies a distinct ubiquitin-binding SH3-like domain required for client selection. Our findings reveal how multivalency and a feed-forward mechanism drive the selection of protein quality-control clients.

E2/E3 酶 UBE2O 泛素化不同的客户以介导重要过程，包括针对未组装的“孤儿”蛋白进行质量控制和在红细胞生成过程中清除核糖体。UBE2O 等质量控制因素如何根据异质性特征选择客户在很大程度上是未知的。在这里，我们表明 UBE2O 客户选择受泛素结合和辅助因子 NAP1L1 的调节。将单个泛素附加到客户端可增强 UBE2O 结合和多单泛素化。UBE2O 还将组蛋白伴侣 NAP1L1 重新用作适配器以招募一部分客户。与 NAP1L1 复合的人类 UBE2O 的低温电磁结构揭示了一个可塑的客户招募界面，该界面被本质上反应性的 UBC 域自动抑制。添加泛素化客户端可识别客户端选择所需的不同泛素结合类 SH3 域。我们的研究结果揭示了多价性和前馈机制如何驱动蛋白质质量控​​制客户的选择。