Analysis of Ipl1-mediated phosphorylation of the Ndc80 kinetochore protein in Saccharomyces cerevisiae

Abstract

Phosphorylation of the Ndc80 kinetochore protein by the Ipl1/Aurora B kinase reduces its microtubule binding activity in vitro. We found that kinetochore-bound Ndc80 is phosphorylated on Ipl1 sites in vivo, but this phosphorylation is not essential. Instead, we show that additional Ipl1 targets contribute to segregation and the spindle checkpoint.

Figure 1.—

An N-terminal Ndc80 mutant lacking Ipl1 phosphorylation sites is benomyl sensitive but spindle checkpoint proficient. (A) A schematic of Ndc80 shows that it contains seven putative Ipl1 phosphorylation Ser/Thr sites (shown in boldface type) in the unstructured N terminus. Phosphorylation sites detected by MS are underlined. Minichromosomes were purified from cells expressing Gip4 from the galactose promoter (SBY6247) for 3 hr and subjected to MS analysis (Kim et al. 2007 and unpublished data). Yeast strains used in this study are listed in supporting information, Table S1. (B) Phosphorylation of the Ndc80-7A phospho-deficient mutant by Ipl1 is reduced in vitro. Ndc80-WT-Myc (WT, SBY7258) and Ndc80-7A-Myc (7A, SBY7259) proteins were immunoprecipitated with anti-Myc antibodies and used as substrates in vitro for an Ipl1 kinase assay as previously described (Buvelot et al. 2003). M, molecular weight markers. (C) Ndc80-7A mutant cells are viable, but show hypersensitivity to benomyl that is rescued by endogenous NDC80. Serial dilutions (fivefold) of NDC80-WT and ndc80-7A cells in the presence or absence of an endogenous NDC80 were plated on YPD plates with or without 7.5 μg/ml benomyl (SBY7258, SBY7259, SBY6859, and SBY6860). (D) Ndc80-7A cells do not exhibit defects in cell-cycle progression. Cells containing Pds1-Myc and either NDC80-WT (SBY7120) or ndc80-7A (SBY7123) were released from G1 and lysates were prepared at the indicated time points and monitored for Pds1-Myc by immunoblot as previously described (Biggins et al. 1999). (E) Ndc80-7A cells activate the spindle checkpoint in response to microtubule depolymerization. The experiment in D was repeated by releasing cells into 10 μg/ml nocodazole.

Figure 2.—

Multiple phosphorylations on Ndc80 are important for proper microtubule dynamics. (A) Ndc80-6A mutants exhibit varying degrees of sensitivity to benomyl. Cells containing six mutations were assayed for benomyl sensitivity as in Figure 1C (SBY7257, SBY7255, SBY7252, SBY7328, SBY7329, SBY7326, SBY7327, SBY7258, and SBY7259). (B) Ndc80-4A, ndc80-2A, and ndc80-3A mutants do not exhibit benomyl hypersensitivity, indicating that there is no specific site required for the phenotype (SBY7258, SBY7259, SBY7296, SBY7325, and SBY8597).

Figure 3.—

The Ndc80 N-terminal domain is not the sole Ipl1 downstream target for segregation and spindle checkpoint activation. (A and B) Ndc80-7A mutant cells do not bypass the checkpoint in response to a defect in cohesion (A; mcd1-1) or bipolar spindle assembly (B; mps2-1). Cells containing either NDC80-WT (SBY7131, SBY7118) or ndc80-7A (SBY7133, SBY7121) were arrested in G1, released to 37°, and analyzed as in Figure 1D. (C) Ndc80-7A is synthetically lethal with deg-ipl1. Serial dilutions (fivefold) of deg-ipl1 NDC80-WT (SBY8325), deg-ipl1 ndc80-7A (SBY8241), and WT (SBY818) cells were plated onto YPD with or without 25 μg/ml Dox. (D and E) The ndc80-7A deg-ipl1 mutant exhibits chromosome missegregation but does not activate the spindle checkpoint. Cells containing deg-ipl1 NDC80-WT or deg-ipl1 ndc80-7A were arrested in G1 for 2 hr and then 25 μg/ml Dox was added for an additional 1.5 hr. Cells were released into media containing Dox and either fixed at 140 min for microscopy (D), as previously described (Biggins et al. 1999) [examples of proper segregation (left), missegregation to the mother cell (middle), or daughter cell (right) are shown] or monitored for Pds1 levels at the indicated time points (E).