The purpose of this investigation is to identify the ubiquitin conjugating enzyme that interacts with the anaphase promoting complex (APC) during the process of ubiquitination. APC is a multisubunit complex whose ubiquitin ligase activity is an essential component of cell cycle regulation. For example, securin degradation via APC ubiquitin ligase activity initiates metaphase to anaphase transition. APC mediated degradation of cyclin-B during anaphase enables progression into the G1 phase of cell cycling. Ubiquitination is the process by which the ubiquitin peptide is attached to a protein substrate via an amide isopeptide bond to the amino group of the lysine residue on the substrate. This pathway is a three-step process. Initially, the ubiquitin peptide is activated by the ubiquitin activating enzyme (E1). This is followed by the covalent conjugation of ubiquitin to the ubiquitin conjugating enzyme (E2). Subsequently, ubiquitin is attached onto a protein substrate via a complex interaction between E2, the protein substrate, and the protein-ligase enzyme (E3). The ubiquitination of proteins can result in either targeting of proteins to the 26S proteosome for degradation, or cellular localization. APC11 is a subunit of the APC complex that contains a RING-H2 domain. In an effort to identify the E2 ubiquitination conjugating enzymes that partner with F35G12.9, an APC11 homologue, we performed a RNAi screen of 13 of the E2 enzymes of C. elegans. Only
ubc-2 exhibited a meiotic-arrest phenotype. RNAi with F35G12.9 resulted in the same meiotic-arrest phenotype, similar to that seen for APC mutants (Davis et al. Genetics 160: 805-813). Based on this evidence, we suggest that
ubc-2 is the E2 enzyme that supports APC ligase activity in vivo, however this specificity is not seen in our purified system in vitro. In vitro studies of the ubiquitin ligase activity of F35G12.9 were performed using a biochemical assay with purified compounds. F35G12.9 together with UBC-2 demonstrates ubiquitination activity. In addition, F35G12.9 can engage in ubiquitin ligase activity in the presence of several C. elegans ubiquitin conjugating enzymes. We plan to further investigate determinants that confer E2 specificity for the APC complex. It is possible that this specificity might be effected by other subunits in the complex. Future studies will include mutational analysis of the F35G12.9 RING finger. We will also conduct further biochemical analysis of the F35G12.9 ligase activity in the presence of other subunits of the APC complex.