BIOCHEMICAL-CHARACTERIZATION OF CAENORHABDITIS-ELEGANS UBC-1 - SELF-ASSOCIATION AND AUTOUBIQUITINATION OF A RAD6-LIKE UBIQUITIN-CONJUGATINGENZYME IN-VITRO

The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is disti nct from other RAD6 homologues in possessing a C-terminal tail 40 amin o acid residues long [Legett, Jones and Candido (1995) DNA Cell Biol. 14, 883-891]. Such extensions from the core catalytic domain have been found in a subset of known conjugating enzymes, where they have been shown to have diverse roles including target recognition, membrane att achment and sporulation. In the present study we used mutagenesis in v itro to examine the role of the tail in specific aspects of UBC-1 stru cture and activity. Cross-linking experiments with purified recombinan t UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because delet ions of the tail significantly decrease, but do not abolish, this self -association. Ubiquitin conjugation assays show that, in addition to a ccepting a thiol-bound ubiquitin at its active site, UBC-1 is stably m ono-ubiquitinated. Deletion analysis and site-directed mutagenesis loc alize the site of ubiquitination to Lys-162 in the tail. These finding s demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.