Preferential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9.
Sentrin is a ubiquitin-like molecule that has been shown to interact with the death domains of Fas and tumor necrosis factor receptor 1 (TNFR1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to protein ubiquitination (Kamitani, T., Nguyen, H. P., and Yeh, E. T. H. (1997) J. Biol. Chem. 272, 14001-14004). Furthermore, the conserved C-terminal Gly-Gly residues are required for sentrinization to occur. To identify enzymes which play a role in sentrinization, the yeast two-hybrid system was used to screen a human placenta cDNA library using sentrin as bait. A strong positive interacting clone was found to contain a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The interaction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears to be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translated sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-sentrin conjugate could also be identified in the in vitro binding assay. Substitution of the conserved cysteine residue of Ubc9 by serine abolished the formation of the Ubc9-sentrin conjugate. Taken together, Ubc9 is a strong candidate to be the key conjugating enzyme in the sentrinization pathway.
Gong
L
L
Research Center for Cardiovascular Diseases, Institute of Molecular Medicine for the Prevention of Human Diseases, University of Texas-Houston Health Science Center, Houston, Texas 77030, USA.
Kamitani
T
T
Fujise
K
K
Caskey
L S
LS
Yeh
E T
ET
D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D015723:Gene Library; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008623:Mercaptoethanol / Q000494:pharmacology; D011485:Protein Binding; D025842:SUMO-1 Protein; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; C092317:RANGAP1 protein, human; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D008623:Mercaptoethanol; C082461:UBC6 protein, S cerevisiae; C471546:UBE2B protein, human; C097309:UBE2D2 protein, human; C489967:UBE2J1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D008025:Ligases; D000098843:Ubiquitin-Conjugating Enzyme UBC9