FIGURE 4:

The localization and activity of CPC proteins are not affected by the absence of Bub1 or Sgo1. (A) The localization of Sli15-GFP, Bir1-GFP, and Ipl1-GFP to preanaphase spindles is not diminished in the diploid sgo1Δ cells. SPBs are marked with Spc29-RFP. Bar = 1 μm. (B) Sli15-directed ChIP revealed a sevenfold enrichment of centromeric DNA relative to telomeric DNA in wild-type cells. This enrichment is not affected by absence of Bub1, similar to results obtained with Ndc10-directed ChIP. (C) Phosphorylation of Sli15-TAP is not affected by the presence or absence of BUB1 and SGO1. Cells were grown at 35°C, synchronized in α-factor (G1 phase) or hydroxyurea (S phase), harvested, and analyzed by PAGE and Western blotting. Slower migrating forms of Sli15-TAP were eliminated by mutation of the Ipl1 kinase domain but not by deletion of SGO1 or BUB1. (D) The phosphorylation of Dam1, the crucial substrate of Ip1l in the release of microtubule attachments, is unaltered by the deletion of BUB1 (top, 30°C) but is abolished in the ipl1-321 mutants at 37°C (bottom). Two independent clones of both the bub1Δ and ipl1-321 mutants were tested, and the separation between phosphoforms of Dam1-myc9 was enhanced by adding 10 μM Phos-Tag AAL-107 (Kinoshita et al., 2006) to the polyacrylamide gel mixture. The slower migrating forms of Dam1-myc9 are sensitive to alkaline phosphatase treatment (top).