IDENTIFICATION OF A NOVEL UBIQUITIN-CONJUGATING ENZYME INVOLVED IN MITOTIC CYCLIN DEGRADATION

Background: The destruction of cyclin B is required for exit from mito sis, and is mediated by the ubiquitin pathway. Recently, a 20S complex , termed the anaphase-promoting complex (APC) or the cyclosome, has be en genetically and biochemically identified as the cyclin-specific ubi quitin ligase (E3). In addition, a ubiquitin-conjugating enzyme (E2), UBC4, was shown to be involved in cyclin ubiquitination in Xenopus egg extracts. Another E2 activity, designated UBCx, can independently sup port cyclin ubiquitination in Xenopus. A similar activity (E2-C) has a lso been observed in clams. However, the molecular identity of Xenopus UBCx or clam E2-C has not been established. Results: We have purified and cloned Xenopus UBCx. Sequence comparisons with known E2s reveal t hat UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction b ox-dependent cyclin ubiquitination. UBCx and UBC4 are active in a simi lar concentration range and with similar kinetics. At saturating enzym e concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC 4. Conclusions: UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyz ed by UBCx is dependent on both the destruction box and the APC, sugge sting that these E2s function through a similar mechanism. However, as the patterns of conjugates generated by these E2s are distinct, these enzymes may play different roles in promoting cyclin proteolysis in m itosis.