his region by Aurora B/Ipl1 may represent a conserved feature of the CPC. Sli15 phosphorylation by Ipl1 is not required for chromosome biorientation Yeast cells relying on either non-phosphorylatable or phosphomimic alleles of SLI15 were fully viable, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19636622 in contrast to cells in which conditional mutations in either the SLI15 IN-box region or IPL1 causes lethality due to failed chromosome biorientation under restrictive conditions. This implies that both sli1520A and sli15-20D strains should be capable of promoting efficient chromosome biorientation. Consistently, we could detect no significant change in the efficiency of chromosome biorientation in either strain, in agreement with the properties of a similar nonphosphorylatable sli15 mutant. Our data therefore indicate that constitutive phosphorylation of Sli15 on the Ipl1 sites is 12 Ipl1-Dependent Phosphorylation of Sli15 unlikely to interfere with chromosome biorientation despite the benomyl hypersensitivity of the sli15-20D strain. The robust spindle 2883-98-9 site association of Sli15-20A in pre-anaphase cells could potentially reduce kinetochore-localized CPC, but efficient chromosome biorientation in the sli15-20A strain indicates that Ipl1 can still gain proper access to its substrates on incorrectly attached kinetochores. Deletion of the entire N-terminal domain of Sli15, which mediates its association with the other CPC components involved in centromere targeting, also drives Sli15 onto the preanaphase spindle but has little or no effect on the efficiency of chromosome biorientation or chromosome segregation. While this questions the importance of centromere targeting of the CPC at least in yeast, it underlines the view that an abnormal CPC association with the pre-anaphase spindle is not an obstacle to achieving efficient chromosome biorientation. Sli15 phosphorylation by Ipl1 affects its interaction with spindle microtubules Phosphorylation 
of the central domain of Sli15 on its cyclindependent kinase sites is known to regulate its interaction with spindle PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19639073 microtubules, and alanine substitution of either just ser-335 or all six Cdk phosphorylation sites drives Sli15 onto the spindle prematurely in metaphase-arrested cells. Both our nonphosphorylatable Sli15-20A protein and another similar Sli15 mutant show strong localization to the metaphase spindle that mimics the effect of inhibiting Ipl1, indicating that phosphorylation by Ipl1 is involved in restricting interaction of the CPC with the spindle in pre-anaphase cells. Consistent with its reduced spindle localization in vivo, Sli15-20D was completely defective in binding microtubules in vitro whereas Sli15-20A and wild-type Sli15 bound microtubules well. Binding of the wild-type protein was strongly reduced following phosphorylation by Ipl1, indicating that phosphorylation of Sli15 by Ipl1 directly affects its affinity for microtubules and confirming that addition of multiple negative charges to the microtubule domain is inhibitory to binding. The opposite behavior of Sli15-20A and Sli15-20D in relation to microtubule association is therefore in contrast to alanine or aspartate substitutions at the six Cdk phosphorylation sites in Sli15, both of which conferred similar behavior. This indicates that Sli15-20D is not just behaving as non-phosphorylatable form of Ipl1-Dependent Phosphorylation of Sli15 Sli15 but that it has distinct properties conferred by the negativelycharged side chains of the glutamate and aspartate residues. Thus the poor i