Budding yeast SKP1 encodes an evolutionarily conserved kinetochore protein required for cell cycle progression

Abstract

The budding yeast SKP1 gene, identified as a dosage suppressor of a known kinetochore protein mutant, encodes an intrinsic 22.3 kDa subunit of CBF3, a multiprotein complex that binds centromere DNA in vitro. Temperature-sensitive mutations in SKP1 define two distinct phenotypic classes. skp1-4 mutants arrest predominantly as large budded cells with a G2 DNA content and short mitotic spindle, consistent with a role in kinetochore function. skp1-3 mutants, however, arrest predominantly as multiply budded cells with a G1 DNA content, suggesting an additional role during the G1/S phase. Identification of Skp1p homologs from C. elegans, A. thaliana, and H. sapiens indicates that SKP1 is evolutionarily highly conserved. Skp1p therefore represents an intrinsic kinetochore protein conserved throughout eukaryotic evolution and may be directly involved in linking kinetochore function with the cell cycle-regulatory machinery.

Figure 1

SKP1 Genomic DNA, Mutant Alleles, and Epitope Tag Fusion Proteins (A) Restriction map of the cloned SKP1 gene and flanking regions. The vector multiple cloning site (mcs) represents the original left end of the library clone where a genomic Sau3A site was ligated to a BamHI site in the vector. (B) The skp1-Δ1 allele was constructed by replacement of the 0.73 kb PstI–Eco57I segment with a 1.8 kb TRP1 gene containing fragment (shown in bold). (C) Schematic structures of the E1 and HA amino-terminal epitope-tagged Skp1p fusion constructs. The E1–Skp1p fusion is under transcriptional control of the GAL1 promotor; the HA–Skp1p fusion is under the transcriptional control of the ADH2 promotor. (D) Schematic of the skp1 temperature-sensitive alleles used in phenotypic studies. (E) Schematic of skp1-Δ2. The 28 amino acid in-frame deletion corresponds to amino acid residues 37–64.

Figure 2

Mobility Shift Analysis of Skp1p in CBF3–CEN DNA Complexes DNA–protein complexes formed with ³²P-labeled CDEIII probe and whole-cell extracts were analyzed on a nondenaturing polyacrylamide gel (Doheny et al. 1993). Antibodies were added to preformed complexes, and samples were incubated for 20 min at room temperature before gel analysis. Unbound probe was run off the bottom of the gel. (A) Lanes 1, 2, and 11 are controls. Lanes 3–6, extracts from cells containing E1-tagged or untagged Ctf13p or Skp1p proteins demonstrating an intrinsic mobility shift due to the 60 amino acid epitope fusion in each case. Lanes 7–10, extracts from cells containing E1-tagged Ctf13p or Skp1p subsequently incubated with or without antibodies directed against the E1 epitope as indicated. Lanes 7 and 9 are controls for lanes 8 and 10. (B) Lanes 1–6, extracts containing either HA-tagged or untagged Ctf13p or Skp1p subsequently incubated with or without an antibody directed against the HA epitopeas indicated. Lanes 7 and 8 are from an independent transformant (repeat of lanes 5 and 6). Lane 9 is a control.

Figure 3

Chromosome Segregation Phenotypes of skp1-3 and skp1-4 Mutants Yeast strains carrying integrated skp1 temperature-sensitive alleles were tested for chromosome missegregation phenotypes using the colony color sectoring assay (Koshland and Hieter 1987). Loss of the nonessential marker chromosome gives a red sector in a white colony. skp1-3 mutants exhibit a wild-type phenotype (very rarely sectored colonies) when transformed with vector only. skp1-4 mutants exhibit dramatically increased rates of chromosome loss (highly sectored colonies) when transformed with vector only. Introduction of the CTF13 gene on a low copy (CEN) vector or high copy (2μ) vector suppresses the chromosome missegregation phenotype of the skp1-4 mutant. Introduction of the NDC10 gene on a 2μ vector has no effect.

Figure 4

Phenotypic Analysis of skp1 Temperature-Sensitive Mutants (A) Shown are G1 arrest (at nonpermissive temperature) of skp1-3 cells and G2/M accumulation (at permissive temperature) and G2/M arrest (at nonpermissive temperature) of skp1-4 cells, as analyzed by flow cytometry (Gerring et al. 1990). The number of cells is depicted on the vertical axis, with fluorescent intensity of emitted light (proportional to DNA content) on the horizontal axis. Logarithmically growing cultures at 25°C were split and incubated at either 25°C or 37°C for 3 hr prior to analysis. (B) Terminal arrest morphologies of skp1-3 or skp1-4 cells after 3 or 6 hr at 37°C. Cells were stained with 4′,6-diamidino-2-phenylindole and photographed for fluorescence staining and by phase contrast. The fields shown were chosen to emphasize specific phenotypes and are not necessarily representative of overall frequencies.

Figure 5

Quantitation of Cell and Nuclear Morphology SKP1/SKP1 wild-type, skp1-3/skp1-3 and skp1-4/skp1-4 homozygous mutants, and skp1-3/skp1-4 heteroallelic diploids were grown to logarithmic phase at 25°C and shifted to 37°C for 3 and 6 hr, and nuclear and bud morphology were scored. The criteria used for each morphologic class scored are shown schematically above the columns. The numbers shown represent percentages of the total cells scored (far right column). Asterisk, in this population of cells, 67% had the nucleus at the neck and 33% had the nucleus spanning the neck (n = 203).

Figure 6

Multiple Protein Sequence Alignments of Skp1p Homologs The seven Skp1p homologs, from the six species indicated and the chlorella virus genome (PBCV-1), are aligned and ordered by decreasing similarity to the human protein. The consensus is derived from positions that have a 5 out of 7 identical match or better. Hs, H. sapiens; Cp, C. porcellus; Dd, D. discoideum; At, A. thaliana; Sc, S. cerevisiae; CV, PBCV-1 chlorella virus; Ce, C. elegans. Asterisks mark the positions of temperature-sensitive mutations.