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Characterization of UBC-1, a UBC2/RAD6-like ubiquitin conjugating enzyme from the nematode Caenorhabditis elegans

University of British Columbia

The Caenorhabditis elegans gene ubc-1, encoding a homologue of the Saccharomyces cerevisiae ubiquitin conjugating enzyme UBC2/RAD6, was cloned. The biochemical properties of UBC-1 were examined as well as its expression throughout the development of the organism. The ubc-1 gene encodes a 192 amino acid ubiquitin conjugating enzyme that shows considerable sequence identity with the Saccharomyces cerevisiae UBC2/RAD6 (66%), as well as with homologues from Schizosaccharomyces pombe (70%), Drosophila melanogaster (83%) and human (84%). However, UBC-1 is distinct in being the only RAD6 homologue, other than RAD6 itself, with a carboxy-terminal extension. Analysis of ubc-1 homologues from Caenorhabditis briggsae and Ascaris suum showed that the carboxy-terminal extension is conserved, suggesting that it plays an important role in nematodes. When constitutively expressed from the yeast promoter ADH1, ubc-1 complements the DNA repair and growth deficiencies in a S. cerevisiae rad6A mutant, demonstrating that ubc-1 is a functional homologue of RAD6. Northern blot analysis demonstrated that ubc-1 is transcribed as a 1.7 kb mRNA which is constitutively expressed at similar levels, in all life stages. The ubc-1 mRNA transcript is trans-spliced by SL2, suggesting it may be transcribed as part of a polycistronic message. However, no detectable signal on Northern blots was seen using probes spanning 2 kb upstream of ubc-1. Also, when the upstream 2 kb and first intron of ubc-1 were fused in frame with LacZ and injected into C. elegans, the resultant stable lines showed strong expression in the gut, suggesting the presence of a promoter within this region. Therefore, although ubc-1 mRNA is spliced by SL2, no evidence was found that it is transcribed as part of a polycistronic messager RNA. Biochemical analysis of bacterially expressed recombinant UBC-1 showed that it is capable of forming a thiol ester bond with ubiquitin, demonstrating that it is indeed a ubiquitin conjugating enzyme. Surprisingly, in addition to forming a thiol ester with ubiquitin, UBC-1 also stably monoubiquitinated itself on lysine 162 in its C-terminal tail. Inactive UBC-1 lacking the active site cysteine was not monoubiquitinated at lysine 162, even in the presence of equimolar functional UBC-1, demonstrating that UBC-1 likely ubiquitinates itself via an intramolecular reaction. Cross-linking experiments using purified recombinant UBC-1 revealed that it self-associates in vitro, forming dimers and probably tetramers. The tail of UBC-1 plays an important role in this interaction, as deletions of this region significantly decreased, but did not abolish the ability of UBC-1 to self-associate. These findings demonstrate that the tail of UBC-1 is important both for its quaternary structure and its post-translational modification.

Thesis/Dissertation

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2009-03-30

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http://hdl.handle.net/2429/6656

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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