Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding of branched Ub function in cell signaling has been stunted by the absence of accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins and devise approaches to probe K48–K63-branched Ub function. We establish a method to monitor cleavage of linkages within complex Ub chains and unveil ATXN3 and MINDY as debranching enzymes. We engineer a K48–K63 branch-specific nanobody and reveal the molecular basis of its specificity in crystal structures of nanobody-branched Ub chain complexes. Using this nanobody, we detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition and after DNA damage. Together with our discovery that multiple VCP/p97-associated proteins bind to or debranch K48–K63-linked Ub, these results suggest a function for K48–K63-branched chains in VCP/p97-related processes.

支链泛素 (Ub) 链构成了人体细胞中 Ub 聚合物的很大一部分。尽管它们丰富，但由于缺乏可用的方法和工具，我们对细胞信号传导中分支 Ub 功能的理解受到了阻碍。在这里，我们鉴定了细胞支链特异性结合蛋白，并设计了探测 K48-K63 支链 Ub 功能的方法。我们建立了一种监测复杂 Ub 链内连接断裂的方法，并揭示了 ATXN3 和 MINDY 作为脱支酶。我们设计了 K48-K63 分支特异性纳米抗体，并揭示了纳米抗体分支 Ub 链复合物晶体结构中其特异性的分子基础。使用这种纳米抗体，我们检测到含缬洛新蛋白 (VCP)/p97 抑制和 DNA 损伤后 K48-K63-Ub 分支增加。连同我们发现多个 VCP/p97 相关蛋白与 K48-K63 连接的 Ub 结合或脱支，这些结果表明 K48-K63 支链在 VCP/p97 相关过程中的功能。