The 26S proteasome degrades K63-linked polyubiquitinated-Sic1^PY. (A) The purified 26S proteasomes from RPN11-3xFLAG strain (WT) or Δrpn10 RPN11-3xFLAG strain (Δrpn10) and the mock purified materials from the parent wild-type strain (no-tag) were subjected to SDS–PAGE followed by staining with CBB (left) or analysed by western blot with the indicated antibodies (right). (B) Degradation of the ubiquitinated-Sic1^PY by the wild-type 26S proteasome. Sic1^PY was ubiquitinated by Rsp5 with wild-type Ub (Ubn-Sic1^PY, resulting K63-linked polyUb chain), with methylated Ub (mUb, multiple mono-ubiquitination), or with Ub mutants; UbK48R (K63-linked polyUb chains), UbK63R (K48-linked polyUb chains), or UbK48R K63R (short Ub chains, probably contains K33-linkage). The Ub-omitted reaction was also tested (Sic1^PY) as a control. Each substrate (200 nM) was incubated with 25 to 100 nM of the wild-type 26S proteasome at 25°C for 10 min. The reaction was terminated by the addition of SDS-loading buffer and was analysed by western blotting with T7-antibody. (C) Kinetic analysis of Sic1^PY degradation. Each substrate (200 nM) was incubated with the wild-type 26S proteasome (50 nM) at 25°C. The reaction was terminated at the indicated time points and analysed as in (B). (D) Degradation assay of Sic1^PY Ub conjugates with lysine-less (K0), single-lysine only (K48 or K63) and double-lysines (K48+K63) Ub mutants. Each substrate (200 nM) was incubated with the wild-type 26S proteasome (50 nM) at 25°C for 10 min and analysed as in (B).

Mga2-p120 is modified with mainly K63-linked ubiquitin chains in vivo. (A) SDS–PAGE analysis of the Mga2 Ub conjugates from MG132-treated cells. Wild-type cells (YYS1246, Δpdr5 Δlys2 background) carrying P_GAL1-^FLAGMGA2 (^FlagMga2) and P_TDH3-Ub plasmids were cultured in SRaf medium. Mga2 expression was induced by 1% galactose for 2 h in the presence of 100 μM MG132 or DMSO. Mga2 and its Ub conjugates were affinity-purified by anti-Flag M2 agarose, and subjected to SDS–PAGE analysis followed by CBB staining. (B) MS spectrum of the ubiquitinated-Mga2. The gel portion of the Mga2-p120 Ub conjugates from MG132-treated cells, indicated by a blanket in (A), was excised and subjected to in-gel digestion with trypsin. The resulting peptides were analysed by MALDI-TOF MS. Peaks corresponding to K48- and K63-linkages are indicated in red and blue text, respectively. (C) Detection of the heavy isotope-labeled Ub chains of the Mga2 Ub conjugates using SILAC. The cells (YYS1301) carrying a control plasmid grew in light medium and the cells (YYS1303) carrying the P_GAL1-^FLAGMGA2 (^FlagMga2) grew in heavy medium. After the addition of 1% galactose for 2 h in the presence of 100 μM MG132, the two cultures were mixed and analysed as in (A). MS spectra of the SILAC ion pairs are presented: a linear Ub peptide (1–6 amino acids), Ub K48-, and K63-linkages. (D) Stabilizations of both the K48- and K63-linked Ub chains on the Mga2 by proteasome-inhibition. The cells (YYS1303) were grown in heavy or light medium. Mga2 expression was induced by 1% galactose for 2 h in the presence of 100 μM MG132 for heavy culture or DMSO for light culture. Then, the two cultures were mixed and analysed as in (C). (E, F) Absolute quantitation of the Ub chains on the Mga2. The tryptic digests of light Ub chains (a 1:1 mixture of K48- and K63-linked di-Ubs) were used as internal standards. Note that the mixture of di-Ubs generates a Ub (1–6 amino acid) and each linkages at a 4:1 ratio. The heavy lysine-labeled Mga2 Ub conjugates were prepared as in (C). Then, the peptides were mixed and the relative ion intensities were analysed by MS. The amount of the total Ub, K48-linkage and K63-linkage were calculated. See also, Supplementary Figure S13.

Quantitation of the proteasome-bound Ub chains. (A) Identification of the K63-linked Ub chains from the purified 26S proteasome. The RPN11-3xFLAG cells (YYS1320) grew in heavy medium and the control cells (YYS1314) grew in light medium were treated with 100 μM MG132 for 2.5 h. Then, the 26S proteasome was purified under a mild condition and analysed as in Figure 4C. MS spectra of SILAC pair ions are presented: a Ub peptide (11-27 amino acid), Ub K48-, and K63-linkages. (B) Both the K48- and K63-linkages were accumulated by proteasome-inhibition. The RPN11-3xFLAG cells (YYS1320) were grown in heavy or light medium. The heavy culture was treated with 100 μM MG132 for 3 h, whereas the light culture was treated with DMSO for 2.5 h. The proteasomes were purified and relative ion intensities were measured as in Figure 4D. (C, D) Absolute quantitation of the Ub chains of the proteasome-bound ubiquitinated proteins. Tryptic digests of the heavy lysine-labeled Ub conjugates in (A) were mixed with the internal standard Ub peptides and analysed by MS. The amount of the total Ub, K48-linkage, and K63-linkage were calculated. See also, Supplementary Figure S15 and S16.

Rsp5 assembles K63-linked ubiquitin chains on Sic1^PY. (A) Multiple types of Ub chains can be formed on Sic1^PY when using Ub mutants. T7-Sic1^PY-His₆ (Sic1^PY, left) or its single lysine construct (Sic1K36^PY, right) was ubiquitinated by E1, E2 and Rsp5, with the indicated Ub mutants and analysed by western blotting with anti-T7 antibody. (B) Purification of the ubiquitinated-Sic1^PY. After ubiquitination as in (A), the samples were denatured with 6 M urea and the Sic1^PY Ub conjugates were pulled down with TALON resin. As a control, Ub-omitted reaction was conducted. Gels were stained with Coomassie brilliant blue (CBB). (C) MS analysis of the purified ubiquitinated-Sic1^PY. Gel regions of the ubiquitinated-Sic1^PY (Ubn-Sic1^PY in A) were excised and subjected to in gel-digestion with trypsin. The resulting peptides were analysed by MALDI-TOF mass spectrometry (MS). The major and specific peaks are labeled. The peak corresponding to K63- and K48-linkages are indicated in blue and red, respectively. The Ub fragments derived from the mutants are indicated by Ub^* in green. The ideal masses (m/z) of all seven specific ubiquitin linkages are indicated by the triangles.

The 26S proteasome and its ubiquitin receptors bind K63-linked polyubiquitin chains. (A) Self-ubiquitination of GST-Rsp5 with Ub mutants. GST-Rsp5 was self-ubiquitinated with a series of Ub mutants. After the reactions, a portion was analysed by SDS–PAGE. The topologies of ubiquitination on Rsp5 are as follow K63-linked chains with wild-type Ub (WT), with UbK48R (K48R), with UbK63 (K63) or with UbK48+K63 (K48 K63); K48-linked Ub chains with UbK63R (K63R) or with UbK48 (K48); short Ub chains that probably contain K33-linkages with UbK48R K63R (K48R K63R); multiple mono-Ub with methylated-Ub (m) or with lysine-less Ub (K0). The degradation products of GST-Rsp5 are indicated by the asterisk. (B) Association of the 26S proteasome and self-ubiquitinated Rsp5 monitored by gel shift assay. The 26S proteasome was preincubated with the self-ubiquitinated GST-Rsp5 produced as in (A), then, the mixtures were subjected to 3.5% native-PAGE followed by in-gel activity assay using suc-LLVY-amc. As a control, Ub-omitted reaction (−) was also tested. Doubly capped and singly capped species of the 26S proteasomes are indicated by 26S^d and 26S^s, respectively. (C) SDS–PAGE analysis of GST-fusion proteins. GST-fusion proteins (1 μg) were subjected to SDS–PAGE followed by CBB-staining. GST-rpn10N5 is an Ub-interacting motif mutant, in which LAMAL sequence was mutated to NNNNN. GST-UbL^Rad23 is a deletion mutant of C-terminal Ub-associated domains. (D) GST pull-down assays of free K48- and K63-linked Ub chains with the proteasomal Ub receptor proteins. Free K48-linked (left) or K63-linked (right) Ub chains were preincubated with the indicated GST-fusion proteins, then, GST-fusion proteins were pulled down with glutathione-immobilized agarose. The bound materials were eluted with SDS-loading buffer and analysed by western blot with anti-Ub antibody. GST alone, GST-rpn10N5 and GST-UbL^Rad23 were used as the control.

K63-linked Ub chains promote Mga2-p120 processing in vivo. (A) Steady-state levels of Mga2 in the Ub mutant-expressed cells. The cells expressing ^FlagMga2 and respective Ubs (YYS1301-1306) were cultured in SRaf medium. Mga2 was expressed by 1% galactose for 3 h in the presence or in the absence of 100 μM MG132. The total cell extracts were analysed by western blot with anti-Flag antibody for the detection of both Mga2-p120 and Mga2-p90 (upper) and anti-Pgk1 antibody as a loading control (lower). The protein levels of p120 and p90 are shown with mean±s.d. values of three experiments in the right graph. (B, C) Cycloheximide-chase analysis of Mga2. The respective cells as in (A) were cultured in SRaf medium. After Mga2 expression by 1% galactose for 1 h, translation was inhibited by cycloheximide at a final concentration of 0.4 mg/ml. Aliquots were taken at the indicated time points after cycloheximide addition. The total cell extracts were analysed by western blot with anti-Flag antibody for the detection of both Mga2-p120 and Mga2-p90 (upper) and anti-Pgk1 antibody as a loading control (lower). To inhibit proteasome activity, cells were treated with MG132 (100 μM) upon the Mga2 expression. The protein levels of p120 are shown with mean+SD values of three experiments in the graph. (D) Expression levels of wild type and mutant Ubs. Cell extracts were analysed by western blot with anti-Ub. (E) Absolute quantitation of the Ub chains on the Mga2 from the mutant Ub-expressed cells. The Mga2 Ub conjugates were purified from the MG132-treated cells cultured in SILAC medium. The ubiquitination of Mga2 was confirmed by Sypro Orange-staining and was quantified by MS as in Figure 4E. See also Supplementary Figure S14. (F) The 26S proteasome binds the ubiquitinated Mga2 in vivo. Mga2 and its Ub conjugates were immunoprecipitated under a mild condition and were analysed by western blot analysis with anti-Rpn11 and anti-Cdc48 antibodies. The ubiquitination levels of Mga2 were monitored by Sypro Orange-staining.