Fig 1: FAT10 is unproductively activated by AOS1/UBA2. a No FAT10 conjugates are formed in HEK293-UBA6 knockout cells. FAT10 conjugate formation was investigated using crude cell lysates, prepared under denaturing conditions of FLAG-FAT10 expressing HEK293 wild-type (WT), or HEK293-UBA6 knockout (UBA6-ko) cells. FAT10 conjugates were visualized by western blotting using the antibodies indicated. Shown is one experiment out of three experiments with similar outcomes. b Reconstitution of UBA6-ko cells with overexpressed AOS1/UBA2 does not rescue FAT10 conjugate formation in these cells. HEK293-UBA6-ko cells were transiently transfected with expression plasmids for SUMO E1 subunits UBA2 and AOS1. Cells were treated with IFN-?/TNF-a to induce endogenous FAT10 expression. HEK293 wild-type (WT) cells were used as a control. Proteins were separated on 4–12% gradient gels (NuPage) and visualized by western blotting using the antibodies indicated. Shown is one experiment out of two experiments with similar outcomes. c No conjugation of FAT10 onto JunB under in vitro conditions in presence of the SUMOylation system instead of the FAT10ylation system after 60 min at 37 °C. In vitro JunB FAT10ylation assay was performed as shown in Fig. 2a. The exact recombinant protein amounts used are listed in the methods section. Shown is one experiment out of three experiments with similar outcomes. d Intra- and inter-protein crosslinks of AOS1/UBA2 and FAT10 in the absence of ATP. FAT10 (Sequence offset to UniProt sequence -1 aa due to tag) and the SUMO E1 subunits 6-His-AOS1 (Sequence offset to UniProt sequence: +23 aa) and UBA2 are depicted on the residue level. Lysine residues, as potential targets of the crosslinking agent used in this study, are indicated as black lines. Inter-protein crosslinks are shown in green and intra-protein crosslinks in purple. e Intra- and inter-protein crosslinks of AOS1/UBA2 and FAT10-AV in the absence of ATP, as described in d. f Schematic depiction of the mechanisms how FAT10 inhibits SUMO activation. (i) SUMO activation by AOS1/UBA2 in absence of FAT10. (ii) Inhibition of SUMO activation when FAT10 is non-covalently bound to the adenylation site of AOS1/UBA2, or when FAT10 is thioester bound to the active-site cysteine of the AOS1/UBA2. Source data are provided as a Source Data file
Fig 2: FAT10 can be activated by AOS1/UBA2. a In vitro experiment showing SUMO-1 or FAT10 activation by AOS1/UBA2. Activation was performed in presence or absence of ATP, as indicated, under in vitro conditions with recombinant proteins for 30 min at 37 °C. Proteins were separated on 4–12% gradient gels (NuPage) and activation was visualized by western blot analysis under non-reducing (non-red.) or reducing (red.) (4% 2-ME) conditions using the antibodies indicated. b Same as in a but additionally with the FAT10 diglycine mutants FAT10?GG and FAT10-AV. c Luminescence-based ATP consumption assay. Shown is the mean of five independent in vitro SUMO or FAT10 activation assays performed as shown in a. The residual ATP was measured by an ATP consuming luciferase reaction. The single recombinant proteins in the absence of ATP (w/o ATP) were used as control. d Same as in a but additionally with the AOS1/UBA2 active-site mutant AOS1/UBA2-C173A and UBC9. The exact recombinant protein amounts used in each experiment can be found in the methods section. Each western blot panel shows one experiment out of three experiments with similar outcomes. Source data are provided as a Source Data file
Fig 3: FAT10 inhibits SUMO activation by AOS1/UBA2. a In vitro co-immunoprecipitation experiment using recombinant AOS1/UBA2 and FAT10 variants. Immunoprecipitation was performed with a monoclonal FAT10-reactive antibody (clone 4F1 and Ref. 29) and subsequent western blot analysis with the antibodies indicated. b Analysis of in vitro SUMO-1 activation by AOS1/UBA2 in presence or absence of FAT10 and other UBL modifiers, as indicated. Proteins were incubated in in vitro buffer, supplemented with ATP for 30 min at 30 °C. Samples were analyzed under non-reducing (non-red.) or reducing (red.) (4% 2-ME) conditions on a western blot using an UBA2-reactive antibody. Shown is one experiment out of three experiments with similar outcomes. c In vitro SUMO-1 activation in presence or absence of FAT10, as described in b. S-F: pre-incubation of SUMO-1 and AOS1/UBA2 for 30 min at 30 °C before addition of FAT10; F-S: pre-incubation of FAT10 and AOS1/UBA2 for 30 min at 30° before addition of SUMO-1; S + F: addition of both modifiers at the same time. Samples were analyzed under non-reducing (non-red.) or reducing (red.) (4% 2-ME) conditions on a western blot with an UBA2-reactive antibody. d Analysis of UBC9 auto-SUMOylation in presence or absence of FAT10 under in vitro conditions, as described in b and c. A western blot using an UBC9-reactive antibody was performed under reducing conditions (10% 2-ME) to visualize auto-SUMOylation of UBC9. e FAT10 does not interfere with Ub activation. In vitro activation of His-tagged Ub by its cognate E1 enzyme UBE1, in the presence or absence of FAT10. Proteins were incubated in in vitro buffer for 30 min at 37 °C and reactions were stopped by addition of gel sample buffer, supplemented (red.) or not (non-red.) with 4% 2-ME and boiled. Activation was analyzed on 4–12% gradient gels (NuPage). f FAT10 does not interfere with ISG15 activation. In vitro activation of His-tagged ISG15 (His-ISG15) by its cognate E1 enzyme UBE1L in the presence or absence of FAT10 as described in e. The recombinant protein amounts used in each experiment can be found in the methods section. Each panel shows one experiment out of three experiments with similar outcomes. Source data are provided as a Source Data file
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