Fig 2: The HECT E3-ligase HUWE1 is required for stress-induced NEDDylation. a Table showing the SILAC ratio for HUWE1 in the two replicate experiments performed for the aggregate composition upon HS. Heavy, HS; Light, untreated. b H1299 cells were either untreated (DMSO) or exposed to HS (2 h) or to overnight MG132 (5 μM) treatment. Cells were fixed and stained for HUWE1. c U2OS cells were transfected with siRNAs and treated as above. Cell extracts were used for western blotting. d U2OS cells were transfected with siRNAs before plasmid transfection of HA-RPL7 and His6-NEDD8 as indicated. Cells were exposed to HS (2 h) before Ni-NTA pull-down and western blotting. e, f H1299 cells were siRNA transfected as indicated and either heat-shocked (e) or MG132 (f) treated as before. Staining and quantitation of nuclear aggregates stained with NEDD8 or ubiquitin was performed as before. Scale bars, 10 µm. Values represent the average of three independent experiments ± SD

Fig 3: NEDDylation compromises proteasomal degradation of substrates. a U2OS cells were transfected with 3 μg of HA-RPL7 and 4 μg of His6-NEDD8 or His6-ubiquitin. Forty-eight hours post-transfection cycloheximide (CHX, 100 μg/ml) was added and cells were harvested at the indicated time points, before lysis and Ni-NTA pull-down. The signal in each lane within the length indicated by the bar was used for quantification. The graph represents the average values of three independent experiments ± SD. b His6-ubiquitin or His6-NEDD8 conjugates were isolated as described in Methods and used in vitro in a 26S proteasome processing/degradation assay. Western blotting was performed with the indicated antibodies (SE short exposure, LE long exposure)

Fig 4: RPL7 localises within the NEDD8/ubiquitin-stained aggregates. a, b H1299 cells were transfected with RPL7-GFP and 48 h later were treated or not with MG132 (5 µM-ON) and stained for either NEDD8 (a) or ubiquitin (b) (red). Enlarged insets represent the NEDD8/ubiquitin-stained structures observed upon MG132 surrounding RPL7. c, d Experiment performed as above and cells were stained with fibrillarin and NEDD8 (c) or nucleolin and ubiquitin (d). Nuclei were stained with DAPI. Scale bars, 10 µm

Fig 5: The stability and function of RPL40, but not RPS27a, are affected by the affinity tag. (A) Schematic representation of the organisation of the poly-ubiquitin (Ub) and ubiquitin–ribosomal protein fusion precursors. (B) Schematic representation of the ubiquitin (Ub)-ribosomal protein (RP) expression construct with N-terminal FLAG tag and C-terminal HA tag under the control of a tetracycline (tet)-regulated promoter. The bars and molecular masses above and below represent the expected fusion protein, and cleaved products, from the RPS27a and RPL40 cDNAs. Note that the HA-tagged ribosomal proteins appear slightly larger than expected (C), likely due to the basic nature of the amino acid sequence in each case and the gel-based mass for the HA-tagged ribosomal protein and expected mass for the fusion protein are shown in brackets. (C) U2OS cells stably expressing either RPS27a or RPL40 ubiquitin fusion precursor proteins under the control of a tetracycline-regulated promoter were incubated with 0–1000 ng/mL tetracycline (tet), as indicated above each lane. Proteins from these cells were harvested after 18 h and analysed by western blotting using antibodies that recognise the HA-tag, the FLAG-tag or karyopherin (Karyo; loading control), as indicated on the left of each panel. The positions of the various proteins and expected ubiquitin–ribosomal protein fusions are indicated on the right of the panels. * indicates a non-specific protein band detected by the anti-HA antibody. (D) U2OS cells stably expressing either the RPS27a or the RPL40 ubiquitin fusion precursor protein or the empty pcDNA5 vector were incubated for 18 h with 1000 ng/mL tetracycline in the absence (−) or presence (+) of 25 µM MG132. Proteins from these cells were analysed by Western blotting as described in panel (C). (E) The ribosomal protein-HA (RP-HA, green bars) and FLAG-Ub (black bars) Western blot signals from panel (D) were quantitated and normalised to karyopherin and plotted. Quantification is based on 3 independent repeats. Error bars indicate SEM. * < 0.05. (F) U2OS cells stably expressing either the RPS27a or the RPL40 ubiquitin fusion precursor protein or the empty pcDNA5 vector were analysed by immunofluorescence using antibodies that recognise the FLAG-tag (FLAG (Ub); red) or the HA-tag (HA (RP), green) or DAPI to visualise DNA (blue). (G) Whole-cell extracts from U2OS cells stably expressing either the RPS27a or the RPL40 ubiquitin fusion precursor protein were separated on 10–40% glycerol gradients. Fractions were analysed by western blotting, using an anti-HA antibody to detect the HA-tagged ribosomal protein and an anti-RPL7 antibody to visualise LSU complexes. Positions of free, non-RNP associated proteins (“Free”), 40S (SSU) and 60S (LSU) (pre-)ribosomal complexes are indicated.