Proteins modified by multiubiquitin chains are the preferred substrates of the proteasome. Ubiquitination involves a ubiquitin-activating enzyme, E1, a ubiquitin-conjugating enzyme, E2, and often a substrate-specific ubiquitin-protein ligase, E3. In Saccharomyces cerevisiae, efficient multiubiquitination needed for proteasomal targeting of a model substrate requires an additional conjugation factor, named E4. This protein, previously known as UFD2 in yeast, binds to the ubiquitin moieties of preformed conjugates and catalyzes ubiquitin chain assembly in conjunction with E1, E2, and E3. Intriguingly, E4 defines a novel protein family that includes homologs in human, Dictyostelium, fission yeast and in C. elegans. We are interested in the function of the E4 enzyme in multicellular organisms and therefore studying the function of the C. elegans homolog of UFD2, which we named UBO-2 (U-box domain protein-2). First we recombinantly expressed and purified UBO-2. Using an in vitro ubiquitination assay, we verified the binding of UBO-2 to a ubiquitinated model substrate as it was previously shown for yeast UFD2. Additionally UBO-2 is able to selfubiquitinate in vitro and therefore its C-terminal U-box domain is required. Furthermore we analyzed the expression pattern of
ubo-2 in transgenic animals using GFP fusion proteins driven by the endogenous promoter. Interestingly, UBO-2::GFP is expressed in all neurons of the worm, in some muscle cells of the pharynx and somatic muscle cells and is localized predominantly in the nucleus. Using the two-hybrid system we identified one specific binding partner. Interestingly, this interactor also bears a U-box domain like UBO-2; therefore we named it UBO-1 (U-box domain protein-1). UBO-1 shows selfubiquitination in vitro and thereby collaborates with the same E2-enzyme like UBO-2. In further biochemical studies we identified that both enzymes function in the same pathway and we identified one specific substrate. UBO-1 and UBO-2 form a trimeric complex with this substrate resulting in its multiubiquitination. To continue the analysis of both enzymes genetically, we generated deletion mutants of
ubo-1 and
ubo-2. We will present biochemical results including the in vitro ubiquitination assay and an epistasis analysis between our deletion mutants and conditional alleles of the substrate gene. Our data define a novel pathway and a novel mechanism involved in protein turnover during development.