The spindle assembly checkpoint (SAC):
- Evolutionarily conserved checkpoint mechanism to oversee fidelity of the mitotic chromosome separation process.
- Designed to stop the mitotically dividing cell, permitting chromosomes to completely attach to the bipolar spindle [1].
- Securin is a protein involved in controlling the transition of the cell cycle from metaphase to anaphase and anaphase onset.
- Basic function of SAC is to stop anaphase from occurring by inhibiting the anaphase-promoting complex/cyclosome (APC/C) mediated ubiquitin degradation of securin, and also of mitotic cyclin.
- SAC contains components identified to be responsible for detecting kinetochores not attached to chromatids and for propagating signals to the APC/C [2].
The Mad Proteins:
- Mad2 and Mad3 - mitotic spindle assembly checkpoint proteins - form the (MCC) of S. pombe. Mad2 and Mad3 mediate APC/C inhibition by associating with Cdc20.
- MCC - a complex of Mad2, Mad3 and Cdc20 binds to APC/C - prevents it from recognising its substrate for ubiquitylation.
- Mad2 and Mad3 antagonise Cdc20-dependent activation of the APC/C, but the interaction of Mad3 with Cdc20 only occurs after the formation of a complex between Mad2 and Cdc20.
- Means that signalling through the SAC only occurs once a Mad2-Cdc20 complex has formed – complex initiated by Mad1, a Mad2 receptor found at unattached kinetochores.
- Mad2 undergoes conversions between open (O-Mad2) and closed-Mad2 (C-Mad2) structural states - necessary for its association with Cdc20.
- The two states of Mad2 differ in orientation of a carboxy-terminal β-sheet - repositions in C-Mad2 to allow it to bind Mad1 and Cdc20.
- Model for SAC activation: Mad1 complexes with C-Mad2 - complex then recruits O-Mad2 through the C-Mad2 dimerisation interface. Mad1-bound C-Mad2 subunit then catalyses the binding of Mad2 to Cdc20, as the subunit induces conformational changes of O-Mad2 to C-Mad2 [2].
Activity of APC/C:
- APC/C activity and its ability for substrate depend on two co-activators: Cdc20 and Cdh1.
- Recognise substrates through two degrons (destruction motifs): the D (destruction) box and the KEN box.
- The D box has the consensus sequence RXXLXX(I/L)(S/T)N.
- Mad3 contains a KEN box (essential for MCC assembly) - could act as a pseudosubstrate to block substrate recognition by APC/C.
- Difference of opinion: other studies show degradation of Cdc20 by promotion of its ubiquitylation by the SAC depends on the KEN box of Mad3 [2].
- Mad2 and Mad3 inhibit Cdc20 and also gives suggestions as to how the MAD2L1-binding protein antagonises MCC assembly. The structure of Cdc20 in the context of the MCC - can visualise the recognition of degrons by co-activators for the first time.
- Mad2 and Mad3 interact so KEN box of Mad3 faces KEN box receptor on the WD40 domain of Cdc20.
- Structures also show a mimic of the D box on the c-terminal of Mad3 - D box binding site on Cdc20 demonstrates basis of D box recognition by co-activators [2].
