PMC

A) Schematic representation of ligase-DUb fusion proteins. The final step in the ubiquitination cascade involves transfer of Ub from an E2 conjugating enzyme to a substrate through the activity of an E3 ligase enzyme. Substrate deubiquitination is induced by expression of E3 ligase-DUb fusion proteins.
B) E3 ligases expressed in wild-type cells as fusions with a C-terminal DUb (UL36) enzyme and an HA epitope. 50 μM copper was added to the media to induce protein expression from the CUP1 promoter. Two transformants for each ligase-DUb fusion were validated; a black asterisk indicates particular fusions for which only one Ura⁺ clone expresses. Molecular weight markers are indicated.

A) Wild-type cells transformed with vector control, Pib1-DUb or Tul1-DUb were grown to mid-log phase before equivalent volumes harvested and spotted out in serial dilution (1:9) on SD-Ura-Arg media containing 50 μM copper chloride and varying concentrations of canavanine (upper). Experiments were repeated on media containing Arginine (middle), including a control set in which a catalytically dead version of Pib1-dub* was expressed (lower).
B) Cells transformed with either Ste3-GFP or Ste3-GFP-Ub alongside active and inactive versions of Pib1-DUb and Tul1-DUb were grown to mid-log phase before copper addition to induce expression of DUb-fusions. Cells were harvested and prepared for fluorescence microscopy after 2 hours. 5 μm scale bars are indicated.

A) Wild-type (SEY6210) yeast cells were grown to mid-log phase in SC media lacking uracil, harvested and a 10-fold serial dilution was spotted out on plates containing different solid media. Cells were plated on media containing 50 μM bathocupriosulfonic acid (BCSA) to repress gene expression. Cells were also plated on media lacking copper or plasmid expression was induced on plates containing 50 μM copper chloride. These plates were incubated at 30°C or 37°C. Cultures were also plated on copper containing media lacking glucose as a primary carbon source, which were instead supplied with oleate at 30°C and ethanol glycerol at 30°C and 37°C.
B) Wild-type yeast cells expressing labeled DUb fusions, or vector control, were grown to mid-log phase in standard SC-Ura media and prepared for immunoblot analysis using anti-HA and anti-Rsp5 antibodies. Cells were also grown to mid-log phase in minimal media and then labeled with MitoTracker for 1 hour, washed twice with water and then imaged by fluorescence microscopy. A Normarski image is included and 5 μm scale bars are indicated.

A) Schematic of ubiquitome purification from pdr5Δ ubi4Δ hbt1-CΔ cells transformed with vector as a control or Pib1-DUb-expressing cells, across 2 independent experiments. His-tagged Ub (His-Ub) attached to substrates (grey) facilitates binding to Ni-NTA. His-Ub conjugated to substrates of Pib1 (green) are predicted to be deubiquitinated by Pib1-DUb, thereby diminishing their recovery on Ni-NTA.
B) Ubiquitome purification from ubi4Δ pdr5Δ hbt1-CΔ cells not expressing His-Ub (Ø), expressing only His-Ub (vector control samples A and C) and cells co-expressing His-Ub and Pib1-DUb (samples B and D). Samples were analyzed by SDS-PAGE followed by silver staining (upper) and immunoblotting using anti-His antibodies (lower).
C) Experimental scheme for isolation and differential labeling of the ubiquitome from control and Pib1-DUb-expressing cells. Ubiquitomes were prepared from independent experiments (1 and 2), trypsinized, and subjected to differential di-methyl labeling (28 Da light label for vector control and 36 Da heavy label for Pib1-DUb samples). Samples were then mixed at a 1:1 ratio and subjected to LC-MS/MS analysis.
D) List of proteins that were detected only in control samples (both A and C) but not detected in ubiquitome purifications following expression of Pib1-DUb (left) and proteins that were detected only in Pib1-DUb samples (both B and D), right.
E) The variability between both light labeled control experiments (left) and both heavy labeled Pib1-DUb experiments (right) is shown as a ratio of peptide intensity (log₂ scale), with pink squares indicating candidate Pib1 substrates identified in (F).
F) The log₂ ratio of proteins between the (light) averaged control samples and the (heavy) averaged Pib1-DUb samples. Proteins reproducibly depleted upon Pib1-DUb expression are highlighted as pink squares.

A) Mutant prc1-1 (CPY*) cells expressing labeled ligase-DUb fusions were grown to mid-log phase before equivalent cells were harvested from each culture. Lysates were generated by treatment with 0.2 M NaOH, resuspension in Laemmli sample buffer containing 8 M urea, followed by SDS-PAGE and immunoblot analysis using antibodies that recognize CPY*, PGK and Dpm1. Different exposures of α-HA immunoblots have been separated by spaces/dotted lines. The steady state levels of CPY* were quantified by densitometry using Fiji software and normalized against loading control (right). Replicate number (n=) is indicated and the standard deviation shown with error bars.
B) prc1-1 cells transformed with vector, Hrd1-DUb and Roy1-DUb were grown to mid-log phase, treated with 200 μg/ml cycloheximide before samples were harvested for immunoblotting at 0, 30 and 60 minute time-points. Resolved lysates were probed with antibodies raised against HA, CPY* and PGK. The levels of CPY* were compared to the PGK loading control using densitometry and used to plot the degradation kinetics of CPY* over time, depicted in line graph (right). The standard deviation from three experiments is shown with error bars.
C) BY4741 cells stably expressing Cln2-TAP and expressing labeled ligase-DUb fusions from a plasmid were grown to mid-log phase an prepared for immunoblot analysis with anti-HA, anti-TAP, anti-CPY and anti-Rsp5 antibodies. Non-specific bands are labeled with an asterisks (*). Cln2-TAP levels were quantified as described in (A).

A) Schematic diagram showing how the cell surface localization of the high affinity tryptophan permease (Tat2) can be used to increase yeast cell survival when grown in restricted tryptophan conditions.
B) Growth assays were carried out in SC-Ura media containing 50 μM copper to induce expression of the DUb fusion library. An initial screen of all ligases in replete Trp (40 mg/L) and low Trp (2.5 mg/L) media was carried out. 20 ligase-DUb fusions that showed potentially increased viability in low Trp were subjected to a further series of assays on plates containing 40 mg/L, 2 mg/L, 1.5 mg/L, 1 mg/L and 0.5 mg/L Trp to establish growth enhancement more accurately. The growth advantage in low Trp was compared to growth in replete conditions and scored on an arbitrary scale between -6 (for greatest growth defect) and +4 (greatest growth enhancement) and 0 indicating no change in growth compared with a vector control from the same plate. Ligase-DUb fusions that confer a significant growth advantage (red dotted line) are labeled.
C) Representative experiments showing the three ligase-DUb fusions (Rsp5, Pib1 and Tul1) exhibiting a concentration dependent growth advantage in limited Trp.

A) Schematic diagram of CPY trafficking, where newly synthesized CPY traffics to the Golgi, where it is modified to a p2-CPY precursor form (black dots) before trafficking to late endosomes, packaging into luminal vesicles at the MVB and sorting to the vacuole where it is processed to the final mature mCPY form (black circular sector). This sorting is defective in vacuolar protein sorting pathway (vps) mutants and p2-CPY is instead secreted from the cell.
B) SEY6210 cells expressing both clones () of the DUb-fusion library were grown in the presence of copper and overlaid with a nitrocellulose membrane. Levels of secreted CPY were assessed by immunobloting the membranes with anti-CPY antibodies. All experiments contained vector controls of wild-type cells, as a negative control, and vps4Δ cells, which secrete >40% CPY, as a positive control.
C) Overlay experiments showing that pib1Δ and tul1Δ null cells do not secrete CPY, unlike vps4Δ and vps8Δ cells. However, expression of dominant anti-ligase versions of Pib1, Tul1 and Vps8 all secrete CPY, an effect that relies on the catalytic activity of the DUb fusion (dub* versions express a catalytically dead Cys>Ser mutant version).
D) Expression of DUb fusions, and catalytically dead (dub*) versions, was assessed by immunoblot analysis of lysates generated from cells incubated in media containing copper for 2 hours.
E) CPY secretion of wild-type, pib1Δ and tul1Δ cells transformed with an empty vector (left) or Rsp5-DUb (right) was assessed by immunoblotting the levels of CPY secreted onto an overlaid membrane during an overnight incubation.

A) Schematic showing interactions of Rsp5 with substrates, Sna3 and Cos5. The PPxY / PY motif of Sna3 is required for interaction with the WW domains of Rsp5 (upper). The N-terminal cytosolic portion of Cos5 is sufficient to confer a ubiqutination signal for vacuolar degradation. The C-terminal cytosolic portion of Cos5 can bind directly to Rsp5.
B) Wild-type cells expressing either Sna3-GFP or Cos5-GFP were grown to mid-log phase before addition of 50 μM CuCl₂ to induce expression of Rsp5-DUb from the CUP1 promoter. Cells were grown for a further 3 hours before harvesting and preparation for fluorescence microscopy. Control cells co-transformed with an empty vector were included (left) and 5 μm scale bars are indicated.
C) Wild-type cells expressing Sna3-HA or Cos5-HA were grown to mid-log phase before induction of Rsp5-DUb for 3 hours. Cells were then harvested, lysed and prepared for immunoblot analysis with anti-HA and anti-CPY antibodies. Three transformants for each condition were analyzed.
D) HA-tagged Rsp5 substrates (Sna3 and Cos5) were expressed in vps36Δ cells co-expressing Rsp5-DUb (+), or co-transformed with a vector control (-), before equivalent volumes were harvested and prepared for immunoblot analysis. Unmodified Sna3-HA (left) and Cos5-HA (right) are detected in addition to the mono-ubiquitinated (*) and di-ubiquitinated (**) species of each substrate. Contrast of ubiquitnated species (red box) was adjusted to allow densitometry analysis.
E) The intensity of each unmodified, mono-ubiquitinated (Ub) and di-ubiquitinated (Ub∼Ub) band for both Sna3-HA and Cos5-HA from (D) was measured using Fiji biological-image analysis software and used to ratio the level of ubiquitination / unmodified for each sample. The levels of mono- and di-Ub bands following expression of Rsp5-DUb are compared to vector control (100%; dotted line) and depicted as a histogram. Error bars indicate the standard deviation from 2 experiments.

A) Scheme for integrating the TEF1*-6xHis-ALINQERA-Ub cassette in place of the endogenous UBI4 gene.
B) Susceptibility of pdr5Δ ubi4Δ∷TEF1*-6xHis-ALINQERA-Ub to the proteasome inhibitor MG132 and the protein translation inhibitor cycloheximide. Cells were grown in liquid culture in the presence of the indicated concentrations of MG132. Growth was monitored by OD₆₀₀ and plotted relative to that of wild-type cells grown in the absence of drug (upper). Cells were serially diluted and plated onto SD media or SD media containing 0.2 μg/ml cycloheximide (lower).
C) Representative His-ALINQERA-Ub purification, with samples removed at each stage and prepared for analysis by silver stain and anti-His immunoblot.
D) Affinity Ni²⁺-NTA purification of lysates generated from ubi4Δ pdr5Δ hbt1-CΔ yeast cells not expressing His-tagged proteins. Contaminant proteins that bind strongly to the nickel column were eluted, visualized by silver stain and then identified by mass spectrometry (listed in table below).
E) Purifications of lysates generated from ubi4Δ pdr5Δ and ubi4Δ pdr5Δ hbt1-CΔ strains. The insert shows a zoomed in image of the purified Hbt1 band.
F) Yeast cells expressing His-ALINQERA-Ub were treated with MG-132, with a control untreated sample, before lysates were generated and samples were prepared for tandem MS/MS analysis. Triplicate experiments were performed for each condition. The number of unique peptides identified for the initial directed MS run (dark grey) or following recursive analyses (light grey) is depicted.
G) Proteins identified from analysis described in (F) were collated and differences following MG-132 treatment displayed as a Venn diagram (upper) and the change in protein levels following drug treatment shown (lower). Less than a 1.5 fold increase / decrease upon drug treatment was considered unaltered (grey).

A) Protein extracts were prepared by incubating harvested yeast cells in urea buffer (50 mM Tris pH 6.8, 10% glycerol, 8 M urea and bromophenol blue) either solely (-), with glass beads and vortexing (GB), or by addition of 3% SDS to buffer (SDS). Manipulations were also compared following a 3-minute incubation in 0.2 N NaOH. All lysates were analysed by SDS-PAGE and Coomassie staining.
B) Cells expressing His-HA-tagged ubiquitin were used to prepare lysates using the alkali and SDS method. Lysates were generated immediately (Ø) as in (B), or samples were treated with NaOH for 2 minutes (+) and an untreated control (-) prior to incubation in 50 mM NaN₃ for 30 minutes at room temperature. Lysates were then immunoblotted using anti- His tag and anti-PGK antibodies.
C) Protein extraction methods were compared from 10 ml, 100 ml and 1000 ml cultures, using anti-PGK, anti-CPY and anti-Dpm1 antibodies. The original lysates (Prep A) were stored in -20°C freezer and then analyzed alongside fresh lysates generated from cultures of the same volume (Prep B). An equivalent number of cells amongst the lysates generated from 10 ml, 100 ml, or 1000 ml cultures were analyzed.
D) Whole cell yeast lysates were generated using lysis buffer containing 3% SDS. SDS was then removed using centrifugal filtration devices (left) or by dialyzing against lysis buffer lacking SDS (right). Different protein amounts from each sample were analyzed by silver stain before (-) and after (+) detergent removal.
E) Left, wild-type cells expressing different his-tagged ubiquitin constructs were analyzed by silver stain and immunoblot using anti-His antibodies. Different linker regions (none, 2, 4, 6 and 8 amino acids) between the His₆-tag and Ubiquitin were compared. Right, cells expressing His-(no linker)-Ub and His-(8 amino acid linker: ALINQERA)-Ub cells were grown to mid-log phase and lysates were generated to compare original material. These lysates were then used to perform Ni²⁺-NTA affinity purifications, and the yield of protein for each transformant analyzed by silver staining and anti-His immunobloting.
F) Schematic diagram of two-step affinity purification of His-tagged ubiquitin.
G) His-Ub conjugates were affinity purified from parental control yeast cells (lacking His-Ub) and cells expressing His-ALINQERA-Ub expressed from the CUP1 promoter. An additional sample was prepared from His-ALINQERA-Ub expressing cells that were treated with 20 mM MG-132 for 45 minutes prior to harvesting and extraction. A sample of the initial lysate was analyzed by silver stain and immunoblotting using anti-His antibodies. Purifications were performed using a 1-step protocol (see methods), that involved binding to a nickel column and elution using low pH buffer, or a 2 step protocol that involved neutralizing the initial elution, rebinding to Ni-NTA and eluting with imidazole. Immunoblot analysis of the 2-step protocol was also performed using anti-Ub antibodies.