Fig 2: Endogenous FAT10 conjugation is not impaired in USE1/E2 double-knockout or USE1/E2 knockdown cells.(A) HEK293 WT (293), USE1-ko cells, or USE1/UBE2D3, UBE2O, UBE2G2, or UBE2C double-knockout cells were treated for 24 h with IFN? and TNF to induce endogenous FAT10 expression. Cells were lysed under denaturing conditions and subjected to immunoprecipitation using a FAT10-reactive monoclonal antibody (clone 4F1). Endogenous FAT10 conjugates were visualized with a polyclonal FAT10-reactive antibody. ß-Actin was used as a loading control. Asterisks mark the heavy and light chain of the antibody used for immunoprecipitation. (B) Same experiment as described in (A), with the difference that USE1-ko cells were treated with 40 nM specific siRNA directed against UBE2A, UBE2B, or UBE2D1, to specifically knock down these E2 conjugating enzymes. The mean of the siRNA knockdown efficiency of three independent experiments is shown in Fig 7A (the experiments were performed in each case in parallel to the crude lysate experiments shown in Fig 7A). Asterisks mark the heavy and light chain of the antibody used for immunoprecipitation, as well as a stripping leftover in the Western blot against ß-actin. Shown is one representative experiment out of three experiments with similar outcomes in (A, B), respectively.Source data are available for this figure.

Fig 3: UBE2G1 and UBE2D3 sequentially catalyze the in vitro ubiquitination of IKZF1 and GSPT1 in the presence of pomalidomide and CC-885, respectively.(A– D) In vitro ubiquitination of IKZF1 (A and C) and GSPT1 (B and D) MBP fusion proteins by recombinant CRL4CRBN complex. Recombinant protein products as indicated were incubated with or without 80 µM POM (A and C) or 80 µM CC-885 (B and D) in the ubiquitination assay buffer containing 80 mM ATP at 30°C for 2 hr, and then analyzed by immunoblotting. (E) Sequential in vitro ubiquitination of GSPT1 by recombinant CRL4CRBN complex. MBP-GSPT1 recombination protein was incubated with Ube1, UBE2D3, Cul4-Rbx1, DDB1-cereblon, Ubiquitin, ATP and CC-885 in the ubiquitination assay at 30°C for 4 hr. After purification over size-exclusion chromatography, pre-ubiquitinated MBP-GSPT1 protein was then incubated with Ube1, DDB1-cereblon, Ubiquitin, ATP and UBE2G1 with or without CC-885 or Cul4A-Rbx1 in the ubiquitination assay at 30°C for 2 hr, followed by immunoblot analysis. (F) Schematic showing the sequential ubiquitination of CRBN neomorphic substrates by UBE2D3 and UBE2G1. Results shown in (A–E) are representative of three independent experiments.

Fig 4: UBE2G1 and UBE2D3 cooperatively promote the in vivo ubiquitination of IKZF1.(A and B) 293T parental and UBE2G1-/-;UBE2D3-/- (clone 4) cells were transiently transfected with plasmids expressing cereblon, V5-tagged IKZF1 and 8xHis-Ub with or without UBE2G1, UBE2D3 or both. (C) 293T parental and UBE2G1-/- (clone 13) cells were transiently transfected with plasmids expressing cereblon, IKZF1-V5, 8xHis-Ub with or without UBE2G1 wild-type or C90S mutant. In (A), (B) and (C), 48 hr after transfection, cells were treated with MG-132 (10 µM) and POM at the indicated concentrations for additional 8 hr. Ubiquitinated protein products enriched with magnetic nickel sepharose were subjected to immunoblot analysis. Immunoblot analysis of whole cell extracts showing equal input proteins is shown in Figure 5—figure supplement 1A-C. All results shown in this figure are representative of three independent experiments.

Fig 5: UBE2D3 and UBE2O accept FAT10 onto their active site cysteine.(A) In vitro FAT10 loading experiment. Recombinant 6His-tagged UBE2D3 (His-UBE2D3) or its active site cysteine mutant His-UBE2D3-C85A was incubated with FLAG-UBA6 and HA-FAT10 C0 (C134L) in in vitro buffer in the presence or absence of ATP, as indicated. Proteins were incubated for 30 min at 37°C, and reactions were stopped by the addition of 5x gel sample buffer and subsequent boiling. Reactions were applied to SDS–PAGE and Western blot analysis using HA- or His-reactive antibodies, under non-reducing (non-red.) or reducing (4% 2-ME, red.) conditions. (B) HEK293-USE1-ko cells were transiently transfected with expression constructs for HA-UBE2D3, HA-UBE2D3-C85A, FLAG-FAT10, or its conjugation-incompetent mutant FLAG-FAT10-AV, as indicated. After 24 h, cells were lysed and cleared lysates were subjected to immunoprecipitation (IP) using HA-reactive antibodies. UBE2D3-FAT10 conjugates were analyzed under non-reducing (non-red.) or reducing (4% 2-ME, red.) conditions, using the antibodies indicated. ß-Actin was used as a loading control. (C) HEK293 WT, USE1-ko, UBE2D3-ko, USE1/UBE2D3-ko, UBE2O-ko, or USE1/UBE2O-ko cells were transiently transfected with an expression construct for FLAG-FAT10. Crude lysates were prepared under denaturing conditions, and proteins were subjected to SDS–PAGE and Western blot analysis, using the antibodies indicated. ß-Actin was used as a loading control. All experiments were performed at least three times with similar outcomes. (D) Cartoon shows the domain structure of UBE2O with the conserved regions 1–3 (CR1-3 domains), coiled-coil domain (CC), the ubiquitin core catalytic domain (UBC), and two putative nuclear localization signals, as described in Hormaechea-Agulla et al (2018). The Western blot shows a semi–in vitro FAT10 loading experiment. Truncated FLAG-tagged UBE2O WT (FLAG-UBE2O trunc), as indicated in the cartoon, or its active site cysteine mutant FLAG-UBE2O trunc-C1040A cells were purified from transiently transfected HEK293-UBA6/USE1/UBE2O triple-knockout cells by immunoprecipitation, using FLAG-reactive antibodies. Beads were washed intensively, and proteins were left bound to the beads. Recombinant proteins were added as indicated, and reactions were incubated for 30 min at 37°C. Reactions were stopped by the addition of 5x gel sample buffer and subsequent boiling. Proteins were subjected to SDS–PAGE and Western blot analysis under non-reducing conditions using the antibodies indicated. An asterisk marks stripping leftover from the Western blot, shown in the upper panel (IB: HA). (E) HEK293-USE1/UBE2O double-knockout cells were transiently transfected with expression constructs for the proteins indicated. 24 h later, cells were lysed and cleared lysates were subjected to immunoprecipitation using FLAG-reactive antibodies. Loading of FAT10 onto UBE2O variants was analyzed under non-reducing (non-red.) or reducing (4% 2-ME, red.) conditions with the antibodies indicated. ß-Actin was used as a loading control.Source data are available for this figure.