Identification of a novel ubiquitin-conjugating enzyme involved in mitotic cyclin degradation.
BACKGROUND
The destruction of cyclin B is required for exit from mitosis, and is mediated by the ubiquitin pathway. Recently, a 20S complex, termed the anaphase-promoting complex (APC) or the cyclosome, has been genetically and biochemically identified as the cyclin-specific ubiquitin 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 support cyclin ubiquitination in Xenopus. A similar activity (E2-C) has also 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 that UBCx is a novel ubiquitin-conjugating enzyme. Purified recombinant UBCx is sufficient to complement purified APC and E1 in destruction box-dependent cyclin ubiquitination. UBCx and UBC4 are active in a similar concentration range and with similar kinetics. At saturating enzyme concentrations, UBCx converts twice as much substrate into ubiquitin conjugates, but generates conjugates of lower molecular mass than UBC4.


CONCLUSIONS
UBCx is a novel ubiquitin-conjugating enzyme involved in cyclin ubiquitination in Xenopus. Like UBC4, ubiquitination catalyzed by UBCx is dependent on both the destruction box and the APC, suggesting 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 mitosis.
Yu
H
H
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
King
R W
RW
Peters
J M
JM
Kirschner
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016213:Cyclins / Q000378:metabolism*; D018076:DNA, Complementary; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008938:Mitosis; D008969:Molecular Sequence Data; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D016213:Cyclins; D018076:DNA, Complementary; D014452:Ubiquitins; D029867:Xenopus Proteins; D044763:Ubiquitin-Conjugating Enzymes; C513378:ube2c protein, Xenopus; C063109:ubiquitin-conjugating enzyme UBC4; D044767:Ubiquitin-Protein Ligases; D008025:Ligases