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The cytokine-inducible ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) targets its substrates for degradation by the proteasome. FAT10 is conjugated to its substrates via the bispecific, ubiquitin-activating and FAT10-activating enzyme UBA6, the likewise bispecific conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By MS analysis, we found that USE1 undergoes self-FAT10ylation in cis, mainly at Lys323. Mutation of Lys323 to an arginine did not abolish auto-FAT10ylation of USE1, but every other lysine could instead be modified with FAT10. Similarly to bulk FAT10 substrates, FAT10ylation of USE1 accelerated its proteasomal degradation. Interestingly, the USE1–FAT10 conjugate continued to be an active E2 enzyme, because both FAT10 and ubiquitin could still be thioester-linked to the USE1–FAT10 conjugate. We therefore suggest that the major function of USE1 auto-FAT10ylation is to serve as a negative feedback mechanism to limit the conjugation of FAT10 upon its cytokine-mediated induction by reducing the amount of USE1 through proteasomal degradation of the USE1–FAT10 conjugate.•USE1physically interacts with FAT10 by anti bait coimmunoprecipitation (View interaction)•USE1physically interacts with FAT10 by anti tag coimmunoprecipitation (View interaction)The ubiquitin-like modifier FAT10 is conjugated to its substrates via the bispecific, ubiquitin- and FAT10-activating enzyme UBA6, the bi-specific conjugating enzyme USE1, and possibly E3 ligases. USE1 auto-FAT10ylation leads to its proteasomal degradation and serves as a negative feedback mechanism to limit the FAT10 conjugation pathway. Interestingly, it does not inactivate USE1 concerning ubiquitin or FAT10 conjugation.