Ubiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profiling in vivo. We used quantitative proteomics to compare the seven lysine-linked ubiquitin chains between wild-type yeast and its 20 DUB-deletion strains, which may reflect the linkage specificity of DUBs in vivo. Utilizing the specificity and ubiquitination heterogeneity, we developed a method termed DUB-mediated identification of linkage-specific ubiquitinated substrates (DILUS) to screen the ubiquitinated lysine residues on substrates modified with certain chains and regulated by specific DUB. Then we were able to identify 166 Ubp2-regulating substrates with 244 sites potentially modified with K63-linked chains. Among these substrates, we further demonstrated that cyclophilin A (Cpr1) modified with K63-linked chain on K151 site was regulated by Ubp2 and mediated the nuclear translocation of zinc finger protein Zpr1. The K48-linked chains at non-K151 sites of Cpr1 were mainly regulated by Ubp3 and served as canonical signals for proteasome-mediated degradation.

泛素链特异性已被描述为一些去泛素酶 (DUB)，但缺乏全面的体内分析。我们使用定量蛋白质组学比较了野生型酵母与其 20 个 DUB 缺失菌株之间的 7 个赖氨酸连接的泛素链，这可能反映了 DUB在体内的连锁特异性. 利用特异性和泛素化异质性，我们开发了一种称为 DUB 介导的连锁特异性泛素化底物鉴定 (DILUS) 的方法，以筛选用某些链修饰并受特定 DUB 调节的底物上的泛素化赖氨酸残基。然后，我们能够识别出 166 个 Ubp2 调节底物，其中 244 个位点可能被 K63 连接链修饰。在这些底物中，我们进一步证明了在 K151 位点上用 K63 连接链修饰的亲环蛋白 A (Cpr1) 受 Ubp2 调节并介导锌指蛋白 Zpr1 的核转位。Cpr1 的非 K151 位点的 K48 连接链主要受 Ubp3 调节，并作为蛋白酶体介导的降解的典型信号。