COMMUNICATION - PREFERENTIAL INTERACTION OF SENTRIN WITH A UBIQUITIN-CONJUGATING ENZYME, UBC9

Sentrin is a ubiquitin-like molecule that has been shown to interact w ith the death domains of Fas and tumor necrosis factor receptor 1 (TNF R1), PML, Rad51, Rad52, and RanGAP1. We have reported previously that sentrin can be conjugated to other proteins in a manner analogous to p rotein 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-h ybrid system was used to screen a human placenta cDNA library using se ntrin as bait. A strong positive interacting clone was found to contai n a cDNA insert encoding the ubiquitin-conjugating enzyme, Ubc9. The i nteraction between sentrin and Ubc9 required the ubiquitin domain and the C-terminal Gly-Gly residues of sentrin. This interaction appears t o be specific because sentrin could only interact weakly with UbcH5B, but could not interact with HHR6B, UbcH6 nor E2-EPF. In vitro translat ed sentrin could be precipitated by a GST-Ubc9 fusion protein, but not by glutathione S-transferase. A beta-mercaptoethanol-sensitive Ubc9-s entrin conjugate could also be identified in the in vitro binding assa y. Substitution of the conserved cysteine residue of Ubc9 by serine ab olished the formation of the Ubc9-sentrin conjugate. Taken together, U bc9 is a strong candidate to be the key conjugating enzyme in the sent rinization pathway.