Volume 66, Issue 4, Pages 488-502.e7 (May 2017) In Vivo Ubiquitin Linkage-type Analysis Reveals that the Cdc48-Rad23/Dsk2 Axis Contributes to K48-Linked Chain Specificity of the Proteasome  Hikaru Tsuchiya, Fumiaki Ohtake, Naoko Arai, Ai Kaiho, Sayaka Yasuda, Keiji Tanaka, Yasushi Saeki  Molecular Cell  Volume 66, Issue 4, Pages 488-502.e7 (May 2017) DOI: 10.1016/j.molcel.2017.04.024 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Yeast Ub-Binding Effector Proteins Analyzed in This Study (A) Domain architectures of 14 yeast Ub-binding proteins analyzed in this study. Known Ub-binding domains (UBDs) are indicated by thick-bordered boxes. (B) Co-immunoprecipitation of ubiquitylated proteins with 3xFLAG-tagged UBD proteins. 3xFLAG-tagged UBD protein complexes were immunoprecipitated from chromosomally 3xFLAG-tagged UBD cells with anti-FLAG antibody, and were analyzed by immunoblot with indicated antibodies. See also Figure S1 and Table S1. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Ub Chain-Type Selectivity of Ub-Binding Proteins In Vivo (A) Abundances of Ub linkages in yeast lysate, as a percentage of total Ub linkages. Yeast lysate prepared in a buffer containing 1% Triton X-100 was analyzed by Ub-AQUA/PRM. (B) Schematic representation for preparing UBD protein-associating ubiquitylated proteins for Ub-AQUA/PRM. The UBD protein complex was immunoprecipitated with anti-FLAG antibody via 3xFLAG (IP). UBD-associated ubiquitylated proteins were further isolated by transferring to biotinylated TR-TUBE (TUBE transfer). Samples from two preparations were analyzed by Ub-AQUA/PRM. (C) Ub-AQUA/PRM analysis of UBD protein complexes. Immunoprecipitated UBD protein complexes (Figure 1B) were analyzed by Ub-AQUA/PRM. Data are normalized against the levels of immunoprecipitated 3xFLAG-tagged UBD proteins, and are represented as means ± SEM of three biologically independent experiments. Total amount of Ub (i), amounts of eight Ub linkages across the UBD proteins (ii), percentages of total linkages to total Ub (iii), and proportion of each linkage as a percentage of total linkages for the indicated UBD proteins (iv) are shown. (D) Ub-AQUA/PRM analysis of BIOTUBE-captured ubiquitylated proteins. UBD-associated ubiquitylated proteins were transferred to BIOTUBE, followed by subjection to Ub-AQUA/PRM analysis. Data represented as in (C). See also Figure S2 and Table S2. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Ub-AQUA/PRM Analysis of Ent2- and Vps27-Bound Ub Conjugates in Mutants (A) Two-step affinity purification of Ent2-bound Ub conjugates from abp1Δ and sla1Δ mutants. Lysate, anti-FLAG immunoprecipitates, and BIOTUBE-captured Ub conjugates were analyzed by immunoblotting with the indicated antibodies. (B) Ub-AQUA/PRM analysis of Ent2-bound Ub conjugates in abp1Δ and sla1Δ mutants. Data are represented as in Figure 2D. (C) Two-step affinity purification of Vps27-bound Ub conjugates from hse1Δ and vps4Δ mutants. Lysate, anti-FLAG immunoprecipitates, and BIOTUBE-captured Ub conjugates were analyzed by immunoblotting with the indicated antibodies. Relative abundances of the immunoprecipitated Vps273xFLAG are shown in the anti-FLAG blot. (D) Ub-AQUA/PRM analysis of Vps27-bound Ub conjugates in hse1Δ and vps4Δ mutants. After normalization to Vps273xFLAG, data are represented as in (B). See also Figure S3 and Table S3. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Perturbation of Ub Network by Proteasome Inhibitor (A) Changes in Ub linkage compositions after addition of the proteasome inhibitor bortezomib (BTZ). Lysate from pdr5Δ cells treated with 50 μM BTZ for the indicated times was analyzed by Ub-AQUA/PRM. (B–F) Changes of the UBD protein-bound Ub linkages after BTZ treatment. The 3xFLAG-tagged UBD cells in the pdr5Δ background were treated with 50 μM BTZ. Ubiquitylated proteins that bound to Rad233xFLAG (B), Ufd13xFLAG (C), Ent23xFLAG (D), Vps273xFLAG (E), and Cue53xFLAG (F) were isolated by two-step purification and subjected to Ub-AQUA/PRM analysis. Upper panels represent the absolute amounts of eight Ub linkages after the BTZ treatment. Lower panels represent the means ± SEM (n = 3 independent experiments) for total amount of Ub, abundances of Ub linkages as a percentage of total Ub, and four major Ub linkages. See also Figure S4 and Table S4. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Ub Chain-Type Selectivity of Ub-Binding Proteins In Vitro (A) Ub chain-binding assay of proteasomal Ub receptors. GST-tagged full-length Rpn10, Rad23, and Dsk2 were immobilized to glutathione-Sepharose beads and incubated with K48- or K63-linked unanchored Ub chains. After washing, the proteins were eluted with LDS-loading buffer and analyzed by immunoblotting with anti-Ub antibody (upper panels). Blot membranes were also stained with Ponceau S (lower panels). Positions of Ub chains of different lengths are numbered. Bands corresponding to cyclic Ub and ubiquitylated E2 are indicated by asterisks (Satoh et al., 2010). (B) Ub chain-binding assay of Cdc48 cofactors. Full-length Ufd1, Npl4, Shp1, and Ufd3 were analyzed as in (A). (C) Ub chain-binding assay of endocytic adaptors. Full-length Ent1 and Ent2, synthesized in a wheat germ cell-free system, were analyzed as in (A). (D) Ub chain-binding assay of ESCRT-0 proteins. Full-length Vps27, Hse1, and Vps27-Hse1 complex (V/H) were analyzed as in (A). (E) Ub chain-binding assay of Cue5. Cue5 (1–190 amino acid residues) containing the CUE domain was analyzed as in (A). See also Figure S5 and Table S5. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Yeast Npl4 Is Highly Specific for Long K48-Linked Ub Chains (A) Comparison of Ub binding by Npl4 and the Ufd1-Npl4 complex. GST-tagged Rpn10, Ufd1, Npl4, and GST-Ufd1-Npl4 complex (U/N) were used in GST pull-down assay to determine binding to K48- or K63-linked unanchored Ub chains. Anti-Ub immunoblots (upper) and Ponceau S protein staining (lower) are shown. (B) GST pull-down assays under saturated conditions. In this experiment, GST-tagged proteins were present in excess relative to unanchored Ub chains. Fifty percent input, unbound Ub chains (U), and bound Ub chains (B) were analyzed by immunoblotting with anti-Ub antibodies. (C) Comparisons of domain structures of Npl4 and NPLOC4. Domain structures and disordered regions (>0.5) were determined using HHpred (Alva et al., 2016) and DISOPRED2 (Ward et al., 2004), respectively. (D) Domain analysis of Npl4 and NPLOC4 to determine the regions responsible for Ub binding. Mutant Npl4 and NPLOC4 truncated at indicated amino acid residues were expressed in E. coli as GST-tagged proteins and analyzed as in (A). See also Figure S6. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 The Cdc48-Rad23/Dsk2 Axis Is a Major Route to the Proteasome (A) Proteasome-bound Ub conjugates in wild-type and dsk2Δ rad23Δ cells. RPN113xFLAG pdr5Δ, RPN113xFLAG pdr5Δ dsk2Δ rad23Δ, and control cells were treated with 50 μM bortezomib (BTZ) for 1 hr. Proteasome complexes were immunoprecipitated with anti-FLAG antibody and eluted with LDS-loading buffer. Lysate and eluates were analyzed by immunoblotting with the indicated antibodies. (B–D) Ub-AQUA/PRM analysis of proteasome-bound Ub conjugates. Pie chart of eight Ub linkages in BTZ-treated proteasomes from wild-type cells (B). Absolute amount of eight Ub linkages in wild-type (WT) and dsk2Δ rad23Δ cells (C). Total amount of Ub, linkage percentage, and amounts of three abundant Ub linkages are shown (D). Data represent means ± SEM from three biological replicates. (E) Proteasome-bound Ub conjugates in cdc48-3 and cdc48-3 dsk2Δ rad23Δ cells. RPN113xFLAG pdr5Δ cdc48-3, RPN113xFLAG pdr5Δ dsk2Δ rad23Δ cdc48-3, and control cells were grown at 28°C and treated with 50 μM BTZ for 1 hr, and then proteasome complexes were analyzed as in (A). (F–H) Ub-AQUA/PRM analysis of proteasome-bound Ub conjugates in cdc48-3 and cdc48-3 dsk2Δ rad23Δ cells. Absolute amounts of eight Ub linkages in proteasome complexes from the indicated cells (F). Total amount of Ub, linkage percentage, and amounts of five abundant Ub linkages (G). Proportions of K48 and K63 linkages to the linkage sum (H). Data represent means ± SEM of three biological replicates. (I) A model for the major route for degradation of proteasomal substrates (see text for details). See also Figure S7 and Table S6. Molecular Cell 2017 66, 488-502.e7DOI: (10.1016/j.molcel.2017.04.024) Copyright © 2017 Elsevier Inc. Terms and Conditions