Biochemical characterization of Caenorhabditis elegans UBC-1: self-association and auto-ubiquitination of a RAD6-like ubiquitin-conjugating enzyme in vitro.
The Caenorhabditis elegans ubiquitin-conjugating enzyme UBC-1 is distinct from other RAD6 homologues in possessing a C-terminal tail 40 amino acid residues long [Leggett, 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 attachment and sporulation. In the present study we used mutagenesis in vitro to examine the role of the tail in specific aspects of UBC-1 structure and activity. Cross-linking experiments with purified recombinant UBC-1 reveal that it forms dimers and probably tetramers. The acidic tail of UBC-1 has an important role in this interaction because deletions of the tail significantly decrease, but do not abolish, this self-association. Ubiquitin conjugation assays show that, in addition to accepting a thiol-bound ubiquitin at its active site, UBC-1 is stably mono-ubiquitinated. Deletion analysis and site-directed mutagenesis localize the site of ubiquitination to Lys-162 in the tail. These findings demonstrate that the C-terminal tail of UBC-1 is important both for its quaternary structure and post-translational modification in vitro.
Leggett
D S
DS
Department of Biochemistry and Molecular Biology, University of British Columbia, 2146 Health Sciences Mall, Vancouver, Canada, V6T 1Z3.
Candido
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D001200:Ascaris / Q000201:enzymology; D002107:Caenorhabditis / Q000201:enzymology; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins; D003001:Cloning, Molecular; D016384:Consensus Sequence; D003432:Cross-Linking Reagents; D008025:Ligases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D044763:Ubiquitin-Conjugating Enzymes; D014452:Ubiquitins / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; C489359:ubc-1 protein, C elegans; D008025:Ligases