Inhibition of the Ub-proteasome system increases the level of all non-K63 linked polyUb chains.
(A) Schematic of quantitative MS. In Method 1 (M1), isotope-labeled labeled peptides are used as standards that are added during trypsin digestion. In Method 2 (M2), labeled cells/proteins are spiked in after cell harvest, but before cell lysis, minimizing variations in sample processing. In addition, labeled proteins can be added in any steps between cell lysis and trypsin digestion as internal standards.
(B) Using labeled cells/proteins (M2, grey) instead of peptides (M1, black) reduced quantitative variations. Three yeast lysates with different amounts of polyUb chains (1x, 3x and 10x) were processed in parallel, and the data were normalized to the result of the 1× sample.
(C) Proteasome inhibitor treatment caused accumulation of Ub conjugates in a dose- and time-dependent manner. Strain JMP001 (pdr5Δ) expressing His-myc-Ub was treated and harvested for immunoblotting with myc antibodies.
(D) Distinct polyUb chain linkages were measured by MS, shown as mean ± SEM.
(E–F) Yeast strains with mutations in Ub-proteasome system raised K11 and K48 linkages but not K63-linked chains. Data are represented as mean and SEM.

Ub with K48 alone cannot support yeast viability and cumulative K to R substitutions lead to growth defects.
(A) The strategy for switching Ub expression in yeast.
(B) Expression of Ub-K48 as the only Ub source resulted in lethality (1X = ~100 cells).
(C) Growth curves of yeast strains expressing a single Ub gene under the P_CUP1 promoter YPD medium.
(D) Comparison of His-myc-Ub monomer and conjugated forms in yeast strains. Total cell lysates (10 μg) were blotted with anti-myc antibodies.
(E) Quantification of polyUb linkages in yeast strains by MS. All values are normalized according to the levels in the wild-type strain and shown as mean and SEM.

Validation of K11 linkage-specific substrates by virtual Western blots and protein turnover analyses.
(A–B) Representative isotope-labeled peptide pairs of Cdc48 and Ubc6 in the total cell lysate (TCL). The light and heavy labeled peptides were distinguished by different mass-to-charge ratios (m/z).
(C–D) Virtual Western blots reconstructed from proteomics data for Cdc48 and Ubc6, reflecting quantitative data in all gel bands in the TCL and the Ub-conjugates (UC). The protein abundance was represented by the darkness and thickness of the bands; and the molecular weight information was extracted from the 1D SDS gel.
(E–H) Protein half-life analyzed by cycloheximide chase and quantitative MS. The experiment was repeated, and the relative standard errors were under 10%.
(I–J) Protein half-life analysis in yeast strains expressing untagged WT or R11 Ub. The cells were treated with cycloheximide to inhibit translation. The degradation of HA-Ubc6 was examined by Western blotting (HA Ab).

Ub-K11 linkages function in the ER stress response.
(A) The Ub-K11R but not the K63R substitution affected cell growth under ER stress. The PDR5 gene was deleted from the strains to increase drug sensitivity (1X = ~30 cells). Cells were grown on YPD plates at 24°C, and recorded (control cells after 4 days; treated cells after 5 days).
(B) Under high concentration of DTT (30 mM), ubc6Δ or ubc7Δ strains had no growth defect.
(C) ER stress with DTT (30 mM) or tunicamycin (1 μg/ml) specifically raised the levels of K11 linkages (mean ± SEM) in the WT strain.
(D) The Ub-R11 strain had a higher level of UPR activation than wild-type and Ub-R63 cells. Yeast cells were transformed with a UPR reporter that expresses lacZ gene under the control of UPR element. The basal and induced (30 mM DTT) levels β-galactosidase activity were measured. Data are shown as mean and SEM, and the asterisks indicate p value < 0.01 (student t-test).
(E) Deletion of UPR gene (IRE1) had synthetic defects with the Ub-R11 mutation under low concentration of DTT (5 mM).

PolyUb chains with distinct linkages are processed by the 26S proteasome.
(A) Yeast DUB mutations have distinct effects on the composition of polyUb linkages in vivo. The data are normalized to the amount of linkages in isogenic wild-type strains and shown as mean ± SEM.
(B–C) Purified His-myc-Ub conjugates and 26S proteasome analyzed by gel electrophoresis.
(D) The Ub conjugates were not contaminated by active DUBs, shown by immunoblotting (myc Ab).
(E) The 26S (0.1 and 1 μg) deubiquitinated native Ub conjugates (0.3 μg) in a 2-h reaction.
(F) The proteasome-associated DUB activity was sensitive to high salt and MG132 (100 μM) treatment. The proteasome (0.3 μg) was incubated with the Ub conjugates (0.3 μg). For high-salt treatment, the proteasome was incubated for 30 min with 250 mM NaCl and then diluted to 50 mM NaCl before the reaction.
(G) The disassembly of polyUb linkages (2 μg of native Ub conjugates) by the 26S (2 μg), analyzed by MS using heavy isotope-labeled Ub conjugates as internal standards. Data are represented as mean and SEM.

Large-scale protein profiling of the wild-type and Ub-R11 strains to identify linkage-specific substrates.
(A) Outline of the SILAC method for comparing total cell lysate (TCL) and purified Ub conjugates (UC) in the two strains.
(B) Comparison of the TCL and the UC by SDS-PAGE. Both samples were resolved on a 6–12% gel, stained with Coomassie Blue, excised into ~50 gel bands, digested by trypsin, and analyzed by LC/MS/MS.
(C) Histograms of log abundance ratios of quantified proteins in the TCL (n = 1,576) and in the UC (n = 75).
(D) GO categories of biological processes of 91 proteins, the levels of which in the TCL were significantly altered by the Ub-K11R mutation.

Ubc6 and Doa10 contribute to the synthesis of K11 linkages.
(A) Purified GST-Ubc6 and a catalytically inactive mutant (GST-Ubc6m, C87S). When incubated with E1, and Ub for 2 h, Ubc6 was self-ubiquitinated. No free Ub polymers (e.g. dimers, trimers, and etc.) were observed.
(B) The Cys87 residue in Ubc6 is essential for its activity (immunoblotting with GST Ab).
(C) Time course of Ubc6 in vitro ubiquitination reaction.
(D) Comparison of Ubc6 self-ubiquitination (30 min) by WT or R11 Ub.
(E) MS measurement of polyUb linkages on Ubc6 (2 h reaction). The polyUb-Ubc6 was resolved on a SDS gel, excised and analyzed by MS using synthetic peptides as internal standards. Total amount of polyUb linkages was normalized to 100%. ND: not determined.
(F) MS analysis of Ub-R11-Ubc6 conjugates.
(G) Deletion of UBC6, DOA10, or both genes in yeast reduced the global level of K11 linkages. Data in panel E–G are represented as mean and SEM.