The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis.
The ubiquitin-mediated degradation of mitotic cyclins is required for cells to exit from mitosis. Previous work with cell-free systems has revealed four components required for cyclin-ubiquitin ligation and proteolysis: a nonspecific ubiquitin-activating enzyme E1, a soluble fraction containing a ubiquitin carrier protein activity called E2-C, a crude particulate fraction containing a ubiquitin ligase (E3) activity that is activated during M-phase, and a constitutively active 26S proteasome that degrades ubiquitinated proteins. Here, we identify a novel approximately 1500-kDa complex, termed the cyclosome, which contains a cyclin-selective ubiquitin ligase activity, E3-C. E3-C is present but inactive during interphase; it can be activated in vitro by the addition of cdc2, enabling the transfer of ubiquitin from E2-C to cyclin. The kinetics of E3-C activation suggest the existence of one or more intermediates between cdc2 and E3-C. Cyclosome-associated E3-C acts on both cyclin A and B, and requires the presence of wild-type N-terminal destruction box motifs in each cyclin. Ubiquitinated cyclins are then rapidly recognized and degraded by the proteasome. These results identify the cyclosome-associated E3-C as the component of the cyclin destruction machinery whose activity is ultimately regulated by cdc2 and, as such, the element directly responsible for setting mitotic cyclin levels during early embryonic cell cycles.
Sudakin
V
V
Unit of Biochemistry, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa.
Ganoth
D
D
Dahan
A
A
Heller
H
H
Hershko
J
J
Luca
F C
FC
Ruderman
J V
JV
Hershko
A
A
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D016213:Cyclins / Q000096:biosynthesis / Q000378:metabolism*; D017931:DNA Primers; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology*; D005260:Female; D006801:Humans; D007700:Kinetics; D008025:Ligases / Q000097:blood / Q000302:isolation & purification / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D015388:Organelles / Q000201:enzymology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012617:Sea Urchins; D017384:Sequence Deletion; D013379:Substrate Specificity; D044764:Ubiquitin-Activating Enzymes; D044767:Ubiquitin-Protein Ligases
D016213:Cyclins; D017931:DNA Primers; D011994:Recombinant Proteins; D044767:Ubiquitin-Protein Ligases; D016203:CDC2 Protein Kinase; D008025:Ligases; D044764:Ubiquitin-Activating Enzymes