The HECT domain of TRIP12 ubiquitinates substrates of the ubiquitin fusion degradation pathway.
The ubiquitin fusion degradation (UFD) pathway is a proteolytic system conserved in yeast and mammals in which an uncleavable ubiquitin moiety linked to the N terminus of a protein functions as a degradation signal of the fusion protein. Although key components of the UFD pathway in yeast have been identified, the E3 enzyme of the human UFD pathway has not been studied. In this work, we show that TRIP12 is the E3 enzyme of the human UFD pathway. Thus, TRIP12 catalyzes in vitro ubiquitination of UFD substrates in conjunction with E1, E2, and E4 enzymes. Knockdown of TRIP12 stabilizes not only artificial UFD substrates but a physiological substrate UBB+1. Moreover, TRIP12 knockdown reduces UBB+1-induced cell death in human neuroblastoma cells. Surprisingly, complementation of TRIP12 knockdown cells with the TRIP12 HECT domain mostly restores efficient degradation of UFD substrates, indicating that the TRIP12 HECT domain can act as the E3 enzyme for the UFD pathway in human cells. The TRIP12 HECT domain directs ubiquitination of UFD substrates in vitro and can be specifically cross-linked to the ubiquitin moiety of the substrates in vivo, suggesting that the TRIP12 HECT domain possesses a noncovalent ubiquitin-binding site. In addition, we demonstrate that UbDeltaGG, a mutant ubiquitin that cannot be conjugated to other proteins, is a substrate of the TRIP12 HECT domain both in vivo and in vitro, indicating that the C-terminal extension fused to the uncleavable ubiquitin is not required for substrate recognition in the UFD pathway. These results provide new insights into the mechanism of the mammalian UFD pathway and the functional nonequivalence of different HECT domains.
Park
Yoon
Y
Department of Biochemistry and Protein Network Research Center, Yonsei University, 134 Shinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Korea.
Yoon
Sungjoo Kim
SK
Yoon
Jong-Bok
JB
D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000502:physiology; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*
D002352:Carrier Proteins; D025801:Ubiquitin; C530813:TRIP12 protein, human; D044767:Ubiquitin-Protein Ligases