Conjugation of the ubiquitin activating enzyme UBE1 with the ubiquitin-like modifier FAT10 targets it for proteasomal degradation

PLoS One. 2015 Mar 13;10(3):e0120329. doi: 10.1371/journal.pone.0120329. eCollection 2015.

Abstract

The ubiquitin-like modifier HLA-F adjacent transcript 10 (FAT10) directly targets its substrates for proteasomal degradation by becoming covalently attached via its C-terminal diglycine motif to internal lysine residues of its substrate proteins. The conjugation machinery consists of the bispecific E1 activating enzyme Ubiquitin-like modifier activating enzyme 6 (UBA6), the likewise bispecific E2 conjugating enzyme UBA6-specific E2 enzyme 1 (USE1), and possibly E3 ligases. By mass spectrometry analysis the ubiquitin E1 activating enzyme ubiquitin-activating enzyme 1 (UBE1) was identified as putative substrate of FAT10. Here, we confirm that UBE1 and FAT10 form a stable non-reducible conjugate under overexpression as well as under endogenous conditions after induction of endogenous FAT10 expression with proinflammatory cytokines. FAT10ylation of UBE1 depends on the diglycine motif of FAT10. By specifically downregulating FAT10, UBA6 or USE1 with siRNAs, we show that UBE1 modification depends on the FAT10 conjugation pathway. Furthermore, we confirm that UBE1 does not act as a second E1 activating enzyme for FAT10 but that FAT10ylation of UBE1 leads to its proteasomal degradation, implying a putative regulatory role of FAT10 in the ubiquitin conjugation pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Humans
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Binding
  • Proteolysis*
  • Ubiquitin-Activating Enzymes / chemistry
  • Ubiquitin-Activating Enzymes / metabolism*
  • Ubiquitination
  • Ubiquitins / chemistry
  • Ubiquitins / metabolism*

Substances

  • UBA1 protein, human
  • UBD protein, human
  • Ubiquitins
  • Proteasome Endopeptidase Complex
  • Ubiquitin-Activating Enzymes

Grants and funding

JB was funded by the Konstanz Research School "Chemical Biology" at the University of Konstanz; This work was supported by the German Research Foundation (DFG) Collaborative Research Centre SFB969 (Project C01), the DFG Priority Program "SPP1365 Ubiquitin Family Network" (grant GR1517/10-1), the Velux Foundation (grant 855), the Thurgauische Stiftung für Wissenschaft und Forschung, the Swiss State Secretariat for Education and Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.