Figure 2. USP14 cleaves arrayed tetraubiquitin chains proximally, stopping at the last chain.

a, K48-linked Ub₄ conjugates of NCB1 were prepared, resulting in one or more tetraubiquitin modifications. ADP-Ptsm (3 nM) was incubated with multi-K48Ub₄-NCB1 (~90 nM) in the presence or absence of USP14 (60 nM). Arrow indicates K48Ub₄-NCB1 species modified with a single chain and derived by USP14 cleavage. b, Assays as in a, except in the presence of ATP. c, K48Ub₄-multichain conjugate of NCB1 was purified, and assays were performed as in a, except that a control was added in which the pan-specific deubiquitinating enzyme USP21 (0.5 μM) was used (10 min incubation). Arrowheads indicate free Ub₄ chains released by USP14 (K48Ub₄) and products of USP21 activity such as free Ub. Samples were analysed by SDS–PAGE/immunoblotting for HA (a and b) or Ub (c). d, Tandem Mass Tag (TMT) labeling and quantitative mass spectrometry. Purified K48Ub₄-multi-chain NCB1 conjugates were assayed in biological duplicates for each condition as in a. The reaction was then quenched and subjected to trypsin digestion, TMT labeling, and tMS² analysis in a Q-Exactive for quantification of K48-linked Ub peptides. e, Proposed en bloc mode of deubiquitination by USP14. f, Modeling of free ubiquitin chain cleavage by proteasome-activated Ubp6. Access of a K11-linked di-ubiquitin chain to the active site of Ubp6 appears occluded. Structures were obtained from the PDB database: Ubp6 and proteasome (5A5B)⁵ and Ub (1UBQ). C118 contains the active site nucleophile.