Question

Describe the role of APC/C in progression through the metaphase-to-anaphase transition. Be sure to include in your answer:a) What exactly does APC/C do enzymatically?b) How does it interact with cyclin and securing?c) The roles of separase and Cdc20

Answer 1

The anaphase-promoting complex (APC/C) is an RING finger E3-ubiquitin ligase involved in metaphase to anaphase transition. It targets proteins for degradation by the 26S proteasomal pathways. APC/C catalyzes the last step of the cascade that is involved in ubiquitinylation of substrates. E1, E2 and E3 enzymes are involved in ubiquitinylation. E1 enzyme is an ubiquitin-activating enzyme. E1 can bins ubiquitin, which is then transferred to the E2 enzyme. E2 along with E3 will transfer ubiquitin to lysine residue on target protein. APC/C is E3 ligase that acts with UbcH10/E2-C and UbcH5/Ubc4 (E2) via its RING zinc finger domain. However, substrate specificity and substrate modification is via APC/C. APC/C acts via two co-activators- Cdc20 and Cdh1. Cdc20 causes ubiquitinylation of cyclin involved in mitosis, securin etc. Cdh1 is required for degradation of mitotic cyclins. Both proteins have seven WD-40 C-terminal repeats that form a propeller structure required for protein-protein interaction. Cdh1 cannot bind to APC/C in S, G2 and M phases. This occurs because cyclin dependent kinase will phosphorylate Cdh1. APC/Ccdc20 is inhibited by Emi1 in S and G2 phases. Degradation of EM1 in prometaphase will activate APC/Ccdc20 in early mitotic phase.

APC/C is involved in separation of sister chromatids. It assembles small ubiquitin chains on B type cyclins. Cyclin B activates CDK1. APC/C also ubiquitinylates securin, which is an inhibitor of the sepharase protease. APC/C can also degrade S and M cyclins by promoting ubiquitinylation.

APC/C can recognize its targets via D box or KEN box sequences that are present in the N terminus of substrates. Cyclin A and Nek2A have excess APC/C binding sites. Cyclin A has higher affinity for APC/Ccdc20 as it can form a complex with Cdk. It also binds indirectly with APC/Ccdc20 via Cks1 mediated phosphorylation of Cdk. Securin also has two Cdk1 sites in yeast that can be phosphorylated. This phosphorylation prevents its ubiquitinylation. Securin can inactivate the cohesin-cleaving enzyme, separase in metaphase. As separase is inactive, there will be no sister chromatin separation in metaphase.

During transition to anaphase, when spindle assembly checkpoint (SAC) is activated, there is formation of Mad2-Cdc20 complexes. This leads to formation of mitotic check point complexes as cdc20, Mad2 and BubR1 associate. MCC is an effector of Sac, and binds APC/C. The MCC-APC/C complex will prevent ubiquitination of securin and cyclin B. Cyclin A and Nek2A are degraded as MCC-APC/C cannot inhibit them. SAC is turned off when the kinetochores attach to the spindle. As a result, there is disassembly of MCC. This causes APC/Ccdc20 to degrade securin. This causes sepharase to become active resulting in onset of anaphase.

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