The aurora B kinase promotes inner and outer kinetochore interactions in budding yeast

Abstract

The kinetochore is the macromolecular protein complex that mediates chromosome segregation. The Dsn1 component is crucial for kinetochore assembly and is phosphorylated by the Aurora B kinase. We found that Aurora B phosphorylation of Dsn1 promotes the interaction between outer and inner kinetochore proteins in budding yeast.

Keywords: Dsn1; Ipl1/Aurora B protein kinase; chromosome segregation; kinetochore.

Figure 1

The Dsn1-S240A, S250A mutant that lacks Ipl1 phosphorylation sites is inviable and has defective interactions with inner kinetochore proteins. (A) Dsn1 contains two Ipl1/Aurora B phosphorylation motifs (R/K-R/K-X-pS/T-V/I/L/X). Alignment shows the conservation of Ser residues (shown in boldface type). (B) Dsn1-S240A, S250A cells are inviable and rescued by the additional mutation of S264A. Serial dilutions (fivefold) of dsn1Δ cells containing DSN1 on a URA3, CEN vector and the indicated integrated point mutants (SBY2318, SBY5948, SBY5949, SBY5950, SBY5952, and SBY6072) were plated on −ura and 5-FOA plates. Cells that need to maintain the URA3, DSN1 vector for viability are sensitive to 5-FOA. (C and D) Dsn1-S240A, S250A, S264A shows a weakened interaction with the inner-kinetochore components Cse4/CENP-A, Mif2/CENP-C, and Ctf19. Dsn1-Flag proteins containing the indicated mutations (SBY7902, SBY8037, SBY7904, and SBY8123) were immunoprecipitated with anti-Flag antibodies and analyzed by SDS–PAGE followed by immunoblotting against representative kinetochore proteins (C) or silver staining (D) as previously described (Akiyoshi et al. 2010). Note that Mif2/CENP-C is not detectable in lysate and the asterisk (*) indicates proteins that nonspecifically co-purify with all Flag-tagged proteins in D. (E and F). Dsn1-Flag was immunoprecipitated from lysates prepared from WT (SBY7441) or ipl1-321 (SBY8120) strains shifted to 37° for 3 hr and analyzed by SDS–PAGE followed by immunoblotting against representative kinetochore proteins (E) or silver staining (F). Strains are listed in supporting information, Table S1.

Figure 2

Outer kinetochore proteins have a weakened association with inner kinetochores in dsn1-S240A, S250A, S264A cells. (A) Centromeric minichromosomes containing kinetochores were purified from strains via a LacI-Flag protein that recognizes lacO repeats in the minichromosomes as previously described (Akiyoshi et al. 2009). (B) Minichromosomes were purified from strains containing the indicated Dsn1 mutants (SBY8338, SBY8339, SBY8340, and SBY8341) and analyzed via immunoblot with the indicated antibodies. (C) Minichromosomes were purified from WT and ipl1-321 strains (SBY7824 and SBY7823) that had been shifted to 37° for 3 hr and analyzed via immunoblot with the indicated antibodies. (D) All cells expressing GFP-tagged Dsn1 (SBY7774), Dsn1-S240A, S250A, S264A (SBY7776), and Dsn1-S240D, S250D (SBY7775) mutants show a normal bilobed kinetochore distribution in vivo. Bar, 5 µm. (E) Dsn1-S240A, S250A, S264A cells, but not dsn1-S240D, S250D cells, exhibit a genetic interaction with the spindle checkpoint mutant mad2Δ. Serial dilutions (fivefold) of dsn1-S240A, S250A, S264A (left; SBY5952, SBY11599, SBY5956, and SBY11601, respectively) or dsn1-S240D, S250D cells (right; SBY5950, SBY11599, SBY5954, and SBY11600, respectively) with or without mad2Δ were plated on YPD at 30°. (F) Model for the role of Dsn1 phosphorylation by the Aurora B kinase. Strains are listed in Table S1.