摘要：SummaryUbiquitin chain specificity has been described for some deubiquitinases (DUBs) but lacks a comprehensive profilingin 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 DUBsin 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.Graphical AbstractDisplay OmittedHighlights•Quantitative proteomics is used to reflect DUB's linkage specificityin vivo•DILUS is developed to decode the ubiquitin code on the level of ubiquitinated sites•K63 chains on K151 of Cpr1 regulated by Ubp2/Ubp3 mediates Zpr1's nuclear transition•K48 chains on non-K151 of Cpr1 regulated by Ubp3 controls its proteasomal proteolysisMolecular Biology; Omics
