Preliminary Study On APC/C-mediated Degradation Of Num1

During the asymmetric mitosis of budding yeast,in order to move the spindle along the cytoplasmic microtubules,dynein must be anchored to the cell membrane while being attached to the microtubule.The anchoring of dynein at the cell membrane relies on Num1.Our sequence analysis of Num1 reveals that it contains several destructin degrons of the anaphase-promoting complex?APC?,including the D box and KEN motif that can bind to the APC activators,namely Cdh1 and Cdc20.In addition,subunit of APC was among the proteins interacting with Num1 identified through the CC/PA domain of Num1.Therefore,it is speculated that APC may mediate the degradation of Num1.Regulated protein synthesis and degradation as well as protein phosphorylation are the themes of cell cycle regulation.However,as one of the key events during the cell cycle,the regulating mechanism of spindle orientation is still unclear.It is therefore necessary to study the function of APC in Num1's degradation and reveal how it affects the cell cycle.We set out to study the function of APC in Num1's degradation from three aspects:1)whether the deletion of APC's activators changes the accumulation of Num1 protein during the cell cycle;2)whether Num1 interacts with APC's activitors Cdh1 and Cdc20;3)whether inactivation of APC affects Num1's degradation and when?The major results are as follows:First,the cdh1?NUM1-GFP strain was constructed,and the accumulation of Num1-GFP protein was compared between the cdh1?and the wild type cells.Preliminary data showed that deletion of CDH1 might delay the degradation of Num1protein.In addition,the pds1?clb5?double mutant necessary for deleting CDC20was constructed.Second,two groups of strains to test the interaction of Num1 with Cdh1 were constructed,and co-immunoprecipitation experiment was carried out by using the first group of strains.It was determined that the second group of strains including GAL1-3HA-CDH1,GAL1-3HA-CDH1 GAL1-NUM1-YFP and GAL1-NUM1-YFP would be more suitable for testing the interaction of Num1 and Cdh1.In addition,the plasmid pRS306:CDC20^5'-UTR-3HA-CDC20¹-700-700 fortaggingCDC20 on the chromosomal locus with 3HA was constructed.Last,the cdc16-1 NUM1-GFP strain for testing the effect of APC inactivation on the accumulation of Num1 protein was constructed.The cdc20-1 NUM1-GFP strain for testing the effect of APC inactivition at certain stage on the accumulation of Num1protein was also constructed.Additionaly,the cdc15-2 NUM1-GFP strain for testing the effect of blocking mitotic exit on the accumulation of Num1 protein was constructed,which also lays the foundation for comparing the level of Num1 protein at different stages of the cell cycle,including G1 phase,S phase,G2/M phase and M phase.