Fig. 7

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-γ/TNF-α 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