Fig 1.
Identification of SGTA interacting DUBs.
A) Beads coupled to recombinant Thioredoxin-SGTA fusion proteins as indicated or Thioredoxin only (Control) were incubated with rabbit reticulocyte lysate for 16 hours at 4°C. The beads were washed as detailed in Materials and Methods. Twice the bead volume of SDS sample buffer was added, and samples were processed for SDS PAGE and Western blotting and probed with antibodies against Bag6, UBL4A and Hsp70 proteins using a fluorescence- based Odyssey ® Fc Imaging System (LICOR). B) Beads described in A, were incubated with HeLa lysate for 16 hours, washed and processed for Western blotting as detailed in A. Blots were probed with antibodies against USP5, USP14, USP9X and USP10 proteins using fluorescence- based detection on an Odyssey ® Fc Imaging System (LICOR).
Table 1.
SGTA interacting partners identified by mass spectrometry.
Pull-down experiments were performed as described in Materials and Methods. The beads were then analysed for bound proteins by mass spectrometry as detailed in the materials and methods followed by interrogating the reviewed human UniProt database for SGTA interacting partners. Identified proteins known to interact with SGTA are indicated together with the spectral counts. SGTA peptides detected reflect comparative efficiency for the coupling of SGTA or the mutants to beads.
Table 2.
Putative SGTA interacting DUBs identified by mass spectrometry.
Pull-down experiments were performed as described in Materials and Methods. The beads were then analysed for bound proteins by mass spectrometry as detailed in the materials and methods. All DUBs identified from the mass spectrometry are shown together with the spectral counts. The DUBs highlighted were prioritised for further studies based on high spectral counts and low background in the negative control.
Fig 2.
Knocking down USP5 reduces the steady state of a model MLP.
A) HeLa TRex Flp-In cells stably expressing OP91 after induction, were seeded at 50% confluence and transfected with 1 nM of siRNAi targeting the indicated DUBs. OP91 expression was induced 48 h later and cells grown for 20 hrs post induction. Total cell lysates were prepared, and products analysed by Western blotting with antibodies against OP91, SGTA, USP5, USP9X, USP14 and tubulin (loading control) using fluorescence- based detection (LICOR). The % knockdown efficiency is indicated for each DUB in lane 2, 3 and 4. B) OP91 signals were quantified using Odyssey 2.1 software and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to the NT control, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** C) HeLa TRex Flp-In cells stably expressing OP91 were transfected with non-targeting siRNAi or two independent siRNAi duplexes targeting USP5. Cells were treated as described in A, followed by blotting for OP91, USP5 and tubulin. D) OP91 signals were quantified using Odyssey ® Fc Imaging system and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to the NT control, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****.
Fig 3.
Loss of USP5 does not affect other substrates.
A) HeLa M cells were seeded at 50% confluence and transfected with 1 nM of siRNAi targeting the indicated DUBs. Ub-MGFP and Ub-RGFP were transfected 48 h later and cells grown for 20 hrs post-transfection. Total cell lysates were prepared and analysed by Western blotting using GFP antibodies and tubulin (loading control). B) Ub-RGFP signals were quantified using the Odyssey ® Fc imaging system and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to the NT control, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = ***. C) HeLa TRex Flp-In cells stably expressing OpD after induction were seeded at 50% confluence and transfected with 1 nM of siRNAi targeting the indicated DUBs. OpD expression was induced 48 h later, and cells grown for 20 hrs post induction. Total cell lysates were prepared and analysed by Western blotting using opsin antibodies to detect OpD, and tubulin (loading control). D) OP91 signals were quantified using Odyssey ® Fc imaging system and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to the NT control, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****.
Fig 4.
USP5 protects OP91 against proteasomal degradation.
A) HeLa T-REx Flp-In cells stably expressing OP91 were seeded at 50% confluence followed by transfection of oligos for knockdown of USP5 or a non-targeting oligo. Cells were grown for 48 hrs followed by induction of OP91 expression and growth for a further 20 hrs post. Cells were harvested and total RNA extracted and used in a qPCR reaction following standard qPCR protocol. B) Cells grown as described in A, inhibitors of the proteasome and autophagy were added 24 hours before cells were harvested. Samples were analysed by SDS-PAGE and Western blotting for OP91 and Tubulin loading control. C) OP91 signals were quantified using Odyssey ® Fc Imaging System and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****.
Fig 5.
Overexpression of USP5 stabilises OP91.
A) HeLa T-REx Flp-In cells stably expressing OP91 upon induction were transfected with V5-tagged Pex19 and SGTA or HA -tagged WT or C335A mutant of USP5. OP91 expression was induced 6 hours post-transfection. Cells were grown for a further 20 hours, post-induction. Total cell lysates were prepared and analysed by western blotting using antibodies against OP91, V5 tagged SGTA and Pex19, HA tagged USP5 and USP5C335A and tubulin (loading control). B) OP91 signals were quantified using Odyssey ® Fc Imaging System and normalised to the tubulin loading control, values show standard errors for n = 3. C) HeLa T-REx Flp-In cells stably expressing OP91 upon induction were transfected with plasmids encoding V5-tagged Pex19 and SGTA or HA -tagged WT USP5 or USP5C335A. OP91 expression was induced by addition of medium containing 1 μg/ml of tetracycline and grown for 20 hrs post induction. Prior to harvesting transfected cells were treated with 100 μg/ml cycloheximide (Sigma, Aldrich) to inhibit protein synthesis. Cells were lysed directly into SDS-PAGE sample buffer at indicated time-points followed by Western blotting with antibodies against opsin (OP91), HA (USP5 and USP5C335A), V5 (SGTA and Pex19) and tubulin (loading control) using fluorescence- based detection (LICOR). D) OP91 signal for each condition was quantified and plotted against the respective time points.
Fig 6.
USP5 specifically enhances SGTA activity.
A) HeLa T-REx Flp-In cells stably expressing OP91 after induction were seeded at 50% confluence and transfected with 1 nM of siRNAi targeting USP5 or a nontargeting control and incubated for 48 hours. After this, plasmids expressing V5 tagged SGTA or PEX19 were transfected followed by incubation for 6 hours before the medium was replaced with medium containing 1 mg/ml of tetracycline to induce OP91 expression. Cells were grown for 20 hrs post induction and total cell lysate were prepared and products analysed by western blotting using antibodies for opsin (OP91), V5 (SGTA and PEX19), and tubulin (loading control). B) OP91 signals were quantified using Odyssey ® Fc Imaging System and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to Pex19, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****. C) HeLa T-REx Flp-In cells were treated with siRNAi targeting USP5, USP9X, USP14 and a nontargeting control and analysed as detailed in A. D) OP91 signals were quantified using Odyssey ® Fc Imaging System and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons relative to Pex19, were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****.
Fig 7.
Association of SGTA and USP5 is enhanced in the presence of an MLP.
A) HeLa T-REx Flp-In cells stably expressing OP91 after induction were seeded at 50% confluence and induced to express OP91 in half of the samples. After growth, cells were harvested into non-denaturing buffer and subjected to co-immunoprecipitation using SGTA antibodies or IgG antibodies as negative control. Products were analysed by western blotting using antibodies against opsin (OP91), V5 (SGTA) and tubulin (loading control). B) HeLa TRex Flp-In cells stably expressing OP91 after induction were transfected with V5 tagged WT SGTA or SGTA-3xNNP/AAA C-terminal mutant (denoted here as SGTA C-term) followed by incubation for 6 hours before the medium was replaced with medium containing 1 mg/ml of tetracycline to induce OP91 expression. Cells were grown for 20 hrs post induction and total cell lysate were prepared and products analysed by western blotting using antibodies for opsin (OP91), V5 (SGTA and SGTA- C-term), and tubulin (loading control). C) Cells transfected with WT SGTA or the SGTA C-terminal mutant described in B were processed for immunoprecipitation using SGTA antibodies. Samples were analysed by western blotting using antibodies against SGTA, OP91 and USP5. D) USP5 signals in the IP lanes 5–8 were quantified using Odyssey ® Fc Imaging System and normalised to the tubulin loading control, values show standard errors for n = 3. Pairwise comparisons of the induced [+] relative to the uninduced [–], were performed using GraphPad Prism. Student t-test: P > 0.05 = ns, P ≤ 0.05 = *, P ≤ 0.01 = **, P ≤ 0.001 = *** and P ≤ 0.0001 = ****. E) HeLa TRex Flp-In cells stably expressing OpD upon induction were seeded at 50% confluence and grown to 80% confluency followed by induction of OpD expression in half of the samples. Cells were subjected to immunoprecipitation as detailed in A.