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） 链在人体细胞中构成了 Ub 聚合物的相当大一部分。尽管它们丰富，但由于缺乏可访问的方法和工具，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 链内键裂解的方法，并揭示了 ATXN3 和 MINDY 作为去分支酶。我们设计了一种 K48-K63 分支特异性纳米抗体，并揭示了其在纳米抗体支链 Ub 链复合物的晶体结构中特异性的分子基础。使用这种纳米抗体，我们检测到含缬氨酸蛋白 （VCP）/p97 抑制后和 DNA 损伤后 K48-K63-Ub 分支的增加。结合我们发现多个 VCP/p97 相关蛋白与 K48-K63 连接的 Ub 结合或分叉，这些结果表明 K48-K63 支链在 VCP/p97 相关过程中发挥作用。