n vitro microtubule MedChemExpress Relebactam binding properties of both Sli15-20D and of phosphorylated wild-type Sli15 in comparison with the nonphosphorylated protein or Sli15-20A support the contention that Sli15-20D is mimicking the constitutively phosphorylated form of the protein. Given that neither sli15-20A nor sli15-20D confer a significant chromosome biorientation defect, it is likely that the elevated chromosome loss rates observed in each mutant result from altered behavior of the spindle microtubules resulting from inappropriate levels of CPC association, although we cannot exclude the possibility of a small defect in bi-orientation that was below the limit of detection in our biorientation assay. Since it is likely that Sli15-20A remains phosphorylated on its Cdk sites, Cdk phosphorylation on its own may be insufficient to block spindle association of the CPC. However, since Sli15-20D retains some ability to interact with the anaphase spindle, albeit at a reduced level, Ipl1 phosphorylation also appears insufficient to block spindle association of the CPC in anaphase when the Cdk sites have been dephosphorylated. Thus our data and those of Nakajima et al. support the notion of combinatorial regulation of CPC localization by both Ipl1 and Cdk phosphorylation. While phosphorylation at the Cdk sites in Sli15 is clearly regulated, it is not clear to what extent phosphorylation of the Ipl1-dependent sites may also be controlled. Sli15 bound to Ipl1 within the CPC might be constitutively phosphorylated on its Ipl1 sites, providing a constant level of phosphorylation to tune the basal microtubule binding affinity of the complex and against which changes in phosphorylation of the Cdk sites can push the affinity for microtubules in one direction or the other. Ipl1 is clearly active throughout anaphase, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19631915 supporting the idea that Sli15 may remain phosphorylated on its Ipl1 sites at this stage, but protein PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19630005 phosphatases such as PP1, which is known to counteract Ipl1 function, may provide a means to antagonize CPC autophosphorylation. Association of the CPC with spindle microtubules is regulated in multiple ways: binding via the interaction between Ipl1 and Bim1 is inhibited by Cdk phosphorylation on Ser-50 and Ser-76, while both Cdk and Ipl1-dependent phosphorylation of Sli15 antagonize its interaction with microtubules as shown in our study. It therefore seemed likely that these different mechanisms would cooperate to regulate spindle association of the CPC, since blocking any of these sets of regulatory phosphorylations can drive premature spindle association of the CPC. It is therefore remarkable that interfering 
with all three pathways concurrently failed to confer any obvious additive defect. Perhaps additional redundant pathways await discovery or else the functions conferred by modulating spindle localization represent fine tuning mechanisms, improving the efficiency of chromosome segregation but not an essential component of it. Given the conservation of CPC relocalization at the metaphase to anaphase transition it is surprising that neither the conserved mechanisms governing its interaction with centromeres nor the multiple regulatory pathways that control its spindle localization appear to be essential. Sli15 phosphorylation by Ipl1 and the tension checkpoint Our work has shown that in contrast to the sli15-20A mutant, strains dependent on Sli15-20D fail to delay entry into anaphase in response to reduced tension on kinetochore-microtubule attachments