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Updated: Jun 7, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis
Annette Aichem1, Christiane Pelzer, Sebastian Lukasiak
1Biotechnology Institute Thurgau, University of Konstanz, Kreuzlingen CH-8280, Switzerland.
Abstract:
The ubiquitin-like modifier FAT10 targets proteins for degradation by the proteasome and is activated by the E1 enzyme UBA6. In this study, we identify the UBA6-specific E2 enzyme (USE1) as an interaction partner of FAT10. Activated FAT10 can be transferred from UBA6 onto USE1 in vitro, and endogenous USE1 and FAT10 can be coimmunoprecipitated from intact cells. Small interfering RNA-mediated downregulation of USE1 mRNA resulted in a strong reduction of FAT10 conjugate formation under endogenous conditions, suggesting that USE1 is a major E2 enzyme in the FAT10 conjugation cascade. Interestingly, USE1 is not only the first E2 enzyme but also the first known substrate of FAT10 conjugation, as it was efficiently auto-FAT10ylated in cis but not in trans.
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