Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association

Abstract

DASH is a microtubule- and kinetochore-associated complex required for proper chromosome segregation and bipolar attachment of sister chromatids on the mitotic spindle. We have undertaken a genetic and biochemical analysis of the DASH complex and uncovered a strong genetic interaction of DASH with the Ras/protein kinase A (PKA) pathway. Overexpression of PDE2 or deletion of RAS2 rescued the temperature sensitivity of ask1-3 mutants. Ras2 negatively regulates DASH through the PKA pathway. Constitutive PKA activity caused by mutation of the negative regulator BCY1 is toxic to DASH mutants such as ask1 and dam1. In addition, we have discovered two novel subunits of DASH, Hsk2 and Hsk3 (helper of Ask1), which are microproteins of fewer than 75 amino acids, as dosage suppressors of ask1 mutants. These are essential genes that colocalize with DASH components on spindles and kinetochores and are present in the DASH complex. Mutants in hsk3 arrest cells in mitosis with short spindles and broken spindle structures characteristic of other DASH mutants. Hsk3 is critical for the integrity of the DASH complex because in hsk3 mutants the association of Dam1, Duo1, Spc34, and Spc19 with Ask1 is greatly diminished. We propose that Hsk3 acts to incorporate Ask1 into the DASH complex.

FIG. 1.

The Ras-cAMP pathway genetically interacts with ask1-3 mutants. (A) Genetic diagram of the Ras/PKA pathway. Regulatory interactions are represented by arrows for positive interactions and by bars for negative regulatory interactions. Question marks represent hypothetical interactions not previously documented in the literature that could explain the genetic interactions observed in our suppressor analysis. (B) ras2 deletion but not ras1 deletion rescues the temperature sensitivity of ask1-3 mutants. Cells of the indicated genotypes were serially 10-fold diluted, and YPD plates were spotted with them at either room temperature (RT), 30°C, or 35°C (restrictive temperature) to assess their growth phenotypes. (C) bcy1 deletion blocks the suppression by ras2 deletion and exacerbates the growth defect of ask1-3 mutants. Cells of the indicated genotypes were serially 10-fold diluted, and YPD plates were spotted with them at room temperature, 30°C, or 35°C. (D) ras2 deletion cannot suppress the temperature lethality of ask1-2 mutants. YPD plates were streaked with ask1-2, ask1-2 ras2Δ, and ras2Δ strains at room temperature or 37°C to assess their growth phenotypes. (E) ras2 deletion cannot bypass the lethality of ask1 deletion mutants. YPD or SC-5-FOA plates were streaked with ask1Δ, ask1Δ ras2Δ and ras2Δ containing pJBN81 (pRS426-ASK1).

FIG. 2.

The Ras-cAMP pathway genetically interacts with dam1-1 and duo1-2 mutants. (A) Overexpression of PDE2 rescued the temperature sensitivity of dam1-1 and duo1-2 mutants. Plates were streaked with dam1-1 mutant cells with vector alone or p2μ-PDE2 at 35°C. Plates were streaked with duo1-2 mutant cells with vector alone or p2μ-PDE2 at 37°C. (B) ras2 but not ras1 deletion suppresses the temperature lethality of dam1-1. dam1-1, dam1-1 ras2Δ, dam1-1 ras1Δ, ras1Δ, and ras2Δ cells were serially 10-fold diluted, and YPD plates were spotted with them at either room temperature (RT), 30°C, or 35°C (restrictive temperature) to assess their growth phenotypes. (C) bcy1 is required for the suppression of dam1-1 by ras2. Plates were spotted with 10-fold serial dilutions of strains of the indicated genotypes and incubated at room temperature, 30°C, or 35°C.

FIG. 3.

HSK2 and HSK3 are suppressors of ask1-2 mutants. (A) ask1-2 mutants containing either the complete ORF or an N-terminal truncation of HSK2 or HSK3 under Gal control were streaked onto YP galactose plates and incubated at room temperature (RT) or at 37°C, the nonpermissive temperature for ask1-2 mutants. The growth phenotype was examined after 3 days. The nucleotide (nt) positions of the deletions of the ORFs are diagrammed below. Nucleotide 1 refers to the A of the ATG start codon. (B) Hsk2 and Hsk3 are essential for cell growth. Tetrad dissection of the HSK2/Δ hsk2 and HSK3/Δhsk3 heterozygous diploid gave two viable and two dead spores. (C) Alignments of S. cerevisiae (Sc) Hsk2 and Hsk3 with their orthologues in Schizosaccharomyces pombe (SP) and Kluyveromyces lactis (Kl).

FIG. 4.

Hsk2 and Hsk3 are spindle and centromere-binding proteins. (A) Hsk2 and Hsk3 localize along the mitotic spindle and SPB. Cells carrying the integrated HSK2-HA or HSK3-HA genes were fixed for indirect immunostaining with anti-HA and antitubulin antibodies. (B) ChIP analysis of HSK2-HA and HSK3-HA. Binding of Hsk2 and Hsk3 to CEN3, CEN16, and a noncentromeric region, PGK1, was analyzed by ChIP with anti-Myc or anti-HA antibodies. (C) Hsk3-HA centromere binding is dependent on intact spindle structures. cdc13-1 HSK3-HA cells were α-factor arrested for 3 h at 25°C and released into YPD prewarmed to 32°C with or without nocodazole (Noc). Seventy minutes later, cells were collected for ChIP analysis. An untagged cdc13-1 strain was included as a control.

FIG. 5.

Hsk2 and Hsk3 are components of the DASH complex. (A) Hsk2 associates with Ask1. (Top) Extracts from ASK1-MYC, HSK2-HA, and ASK1-MYC HSK2-HA strains were immunoprecipitated (IP) with anti-HA or anti-Myc antibodies and probed with either anti-HA or anti-Myc as indicated. Input represents 2% of the total. (B) Hsk3 associates with Ask1. (Top) Extracts from ASK1-MYC, HSK3-HA, and ASK1-MYC HSK3-HA strains were immunoprecipitated with anti-HA or anti-Myc antibodies and probed with either anti-HA or anti-Myc as indicated. (C) Hsk3 associates with Dam1. (Top) Extracts from DAM1-MYC HSK3-HA strains were immunoprecipitated and probed with either anti-Myc or anti-HA antibodies. (Bottom) A longer exposure of the anti-HA Western blot. (D) Hsk3 is a component of the DASH complex. TAP-tagged purification of the DASH complex from ASK1-TAP and ASK1-TAP HSK3-HA strains was resolved by 4 to 20% polyacrylamide SDS-PAGE and stained with Coomassie blue or Western blotted with anti-HA antibodies.

FIG. 6.

HSK3 is required for mitotic progression and spindle integrity. hsk3-16, hsk3-41, and hsk3-44 mutants were α-factor arrested for 3 h at 25°C and released into YPD at 37°C. Cells were collected for DNA and spindle staining at 60, 90, and 180 min after release from the α-factor block. The white arrows show examples of broken spindles. WT, wild type.

FIG. 7.

The integrity of the DASH complex depends upon Hsk3. TAP tag purification of the DASH complex from ASK1-TAP and ASK1-TAP hsk3-16, ASK1-TAP hsk3-41, and ASK1-TAP hsk3-44 mutant strains was resolved by 4 to 20% polyacrylamide SDS-PAGE, and the strain was stained with Coomassie blue. WT, wild type.