The Study Of Substrate Specific Recognition By APC/C^Cdc20in S.Cerevisiae

The anaphase-promoting complex(APC/C)is a ubiquitin-protein ligase that plays an important role in the transition and smooth operation of the cell cycle at different phases.APC is a multisubunit complex,whose activity requires the binding of coactivator either of Cdc20 or Cdh1.The function of the coactivators is to recognize and recruit the APC substrates.Currently,it is believed that Cdc20 mainly works at the transition from metaphase to anaphase,whereas Cdh1 works in late mitosis and G1 phase.In vivo,the function of APC/CCdc20 is to degrade securin(Pds1 in S.cerevisiae)and some members of cyclin B family(Clb2 and Clb5 in S.cerevisiae).Securin is an inhibitor of separase.The cleavage of securing by Cdc20 leads to liberation of separase,which in turn cleaves the cohesin complexes that hold sister chromatids together and thereby initiates sister-chromatid separation.Cyclin B family members,plays an important role in cell cycle progression mainly in S and M phases.Through the degradation of these substrates,APC/CCdc20 regulates various cellular activities,including cell growth and division.In APC,the recognition of substrates by Cdc20 and Cdhl is through specific sequences in the substrate.Currently,the two well-known sequences are D-box and KEN-box.The degradation of Pdsl and Clb5 by Cdc20 are both dependent on the D-box in the substrates.The region of Cdc20 respon sible for recognition of these motifs is located on its carboxyl-terminal which contains a domain called WD40 repeats.The domain has been known to be important in interacting with various proteins.However,it is still not fully clear the exact sites of WD40 region of Cdc20 involved in the substrate recognition.In addition,whether the recognition of the D box in different substrates is through the same region in the WD40 region is not clear.In this study,we used Saccharomyces cerevisiae as the model and investigate the mechanism of Cdc20 recognition of its substrates.We found that Hsll is likely not a substrate of Cdc20,which is different from the published results.In addition,our study established some key sites of Cdc20 important for its interaction with Clb5,and demonstrated that Clb5 degradation is not required for cell cycle exit from mitosis.It may be that another cyclin,Clb2,plays an essential role in this process.Although the interactions of Cdc20 with the substrate Pdsl and Clb5 are similarly dependent on the D box,the results suggested that Cdc20 uses different regions/motifs in the WD40 region to participate these interactions.In summary,our results provide improved details about the interaction between Cdc20 and its substrates.This knowledge will be useful not only for our understanding of the mechanism that Cdc20 interacts with its substrates,but also for possible exploration of new Cdc20 substrates in future.