Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network.

数百种 E3 连接酶在识别泛素 (Ub) 修饰的特定底物方面发挥着关键作用。将 E3 的真正目标与 E3 交互器分开仍然是一个挑战。我们推出 BioE3，这是一种将底物与目标 Ub E3 连接酶相匹配的强大方法。使用 BirA-E3 连接酶融合体和 bioUb，对特定 E3 的 Ub 修饰底物进行位点特异性生物素化有助于蛋白质组鉴定。我们证明 BioE3 可以识别两种 RING 型 E3 连接酶的已知和新靶点：RNF4（DNA 损伤反应、PML 小体）和 MIB1（内吞作用、自噬、中心体动力学）。多功能 BioE3 可识别细胞器特异性 E3 (MARCH5) 和相对未表征的 E3 (RNF214) 的靶标。此外，BioE3 与 NEDD4（一种 HECT 型 E3）合作，识别与囊泡运输相关的新目标。BioE3 检测对化学物质反应的特异性改变，为靶向蛋白质降解开辟途径，并且可能适用于其他 Ub 样（UbL，例如 SUMO）和 E3 类型。BioE3 应用揭示了复杂的 UbL 网络的细胞调节。