Sos7, an essential component of the conserved Schizosaccharomyces pombe Ndc80-MIND-Spc7 complex, identifies a new family of fungal kinetochore proteins

Abstract

Chromosome segregation is powered by the kinetochore, a large macromolecular structure assembled on centromeric chromatin. Attachment of sister chromatids to microtubules is mediated by the highly conserved tripartite KMN (acronym for KNL-1-Mis12-Ndc80) kinetochore network. In the fission yeast Schizosaccharomyces pombe, the equivalent complex is called NMS (Ndc80-MIND-Spc7). Here, we show that not all components of the NMS complex had been identified previously. A 10th NMS component exists, the essential Sos7 protein, which is a genetic and physical interaction partner of Spc7. The analysis of sos7 kinetochore-null mutant yeast strains demonstrated that Sos7 is central to NMS function. In particular, Sos7 is required for kinetochore targeting of Spc7 as well as components of the MIND complex. sos7 mutant strains show severe chromosome missegregation phenotypes and have compromised microtubule-kinetochore interactions. Sos7 is the founding member of a functionally conserved fungal kinetochore family not present in the point centromere carrying Saccharomycotina clusters, suggesting that the new Sos7 family might be a signature motif of fungi with regional centromeres.

Fig 1

Extra sos7⁺ suppresses the phenotypes of spc7 mutant strains. (A) Serial dilution patch tests (10⁴ to 10¹ cells) of the indicated spc7^ts transformants grown under plasmid-selective conditions at the indicated temperatures for 5 to 6 days. “Vector” indicates plasmid without an insert; sos7⁺ denotes the presence of this ORF plus its wild-type promoter on plasmid pUR19. The right panel shows amino acid changes present in the Spc7 variants. (B) Overexpression of sos7⁺ suppresses the chromosome missegregation and spindle defects of the spc7-23 strain grown at the restrictive temperature. Diagrammatic representation of the chromatin distribution (left) and spindle morphology (right) of mitotic spc7-23 cells (n = 100). wt, wild-type phenotype. (C) (Left) A protein extract prepared from a strain expressing endogenous Spc7-GFP was used for immunoprecipitation using anti-GFP (α-GFP) antibody, followed by Western blotting with the same anti-GFP (middle panel) or the polyclonal anti-Spc7 antibody (right panel). (Right) Photomicrographs of fixed wild-type cells incubated with the anti-Spc7 antibody and DAPI. Bar, 5 μm. (D) Kinetochore localization of Spc7-23 at 34°C is dependent on sos7⁺ overexpression. Photomicrographs of fixed spc7-23 cells overexpressing sos7⁺ incubated with the anti-Spc7 antibody and DAPI. Bar, 5 μm.

Fig 2

Sos7 is a new component of the kinetochore. (A) Localization of endogenous Sos7-GFP in fixed interphase, metaphase, and anaphase cells incubated with the anti-GFP antibody and DAPI. Bar, 5 μm. (B) Subcellular localization of Sos7-GFP in a Mis6-mCherry-expressing strain. Shown is a representative cell (n = 5) that goes through mitosis (time intervals from top to bottom: 13.5, 5, and 7.5 min). Bar, 2 μm. (C) Sos7 associates with the central domain of cen1. Cells expressing Sos7-GFP were fixed and processed for ChIP by using anti-GFP antibodies. Multiplex PCR analysis of the chromatin in immunoprecipitates shows an enrichment of the imr and cnt regions of cen1. (D) Live-cell images of 2 representative metaphase cells (n = 6) expressing Sad1-mCherry and Sos7-GFP. The top cell is seen in Movie S1 in the supplemental material.

Fig 3

Sos7 mutant strains have severe mitotic defects. (A) Serial dilution patch tests (10⁴ to 10¹ cells) of 3 sos7^ts mutant strains. The indicated strains were grown at the various temperatures for 3 to 4 days. (B) Amino acid changes found in the Sos7 mutant proteins. (C) Kinetochore localization of Sos7-Δ7 at 25°C and after 6 h at 32°C. Fixed cells were stained with our anti-Sos7 antibody and the TAT-1 antitubulin antibody. Bar, 5 μm. (D) Photomicrographs of sos7^ts cells incubated at 36°C for 6 h (a, sos7-+6; b and c, sos7-178; d and e, sos7-Δ7). Fixed cells were stained with antitubulin antibody and DAPI. Shown are all mutant phenotypes (unequally separated chromatin [cells a and b], segregated chromatin with lagging chromosomes [cell c], smeared chromatin [cell d], and nonseparated chromatin [cell e]) on an elongated spindle. (E) Quantification of the phenotypes described for panel D. The number of cells analyzed per strain and temperature was 100. (F) Photomicrographs of a mitotic sos7-Δ7 cell expressing endogenous Fta2-GFP. Cells were incubated at 36°C, fixed, and stained with antitubulin and anti-GFP antibodies. Bar, 2 μm. The number of mitotic Fta2-GFP signals analyzed was 327. (G) (Left) cen1-gfp sos7-178 cells incubated at 36°C for 6 h. Fixed cells were incubated with anti-GFP antibody, DAPI, and antitubulin antibody. Bar, 5 μm. (Right) Quantification of GFP signal distribution in wild-type and sos7^ts strains at 25°C and 36°C. The number of cells analyzed per strain and temperature was 100.

Fig 4

Sos7 and Spc7 localization interdependencies. (A) (Left panel) Western blot analysis of Sos7-GFP immunoprecipitates from equal amounts of protein extracts from wild-type (wt) and spc7-23 cells grown at the indicated temperature. γ-Tubulin, loading control. (Middle panel) Live-cell images of Sos7-GFP in wt and spc7-23 cells grown at 25°C or incubated at 33°C for 6 h. (Right panel) Quantification of these fluorescence signals. The number of cells analyzed per strain and temperature was 30. (B) (Top) Serial dilution patch tests (10⁴ to 10¹ cells) of the indicated strains grown at 18°C for 8 days. (Bottom) Live-cell images of Spc7-GFP in wt and sos7-Δ7 cells grown at 18°C. Bar, 5 μm. (Right panel) Quantification of these fluorescence signals. The number of cells analyzed per strain was 30. (C) Serial dilution patch tests (10⁴ to 10¹ cells) of sos7^ts cells transformed with plasmids overexpressing spc7⁺. Transformants were grown under plasmid-selective conditions at permissive (25°C or 30°C) or restrictive (28 to 34°C) temperatures for 5 to 6 days.

Fig 5

Interaction between Spc7 and Sos7 proteins. (A) Coimmunoprecipitation of tagged Spc7 and Sos7 proteins. Protein extracts of a Spc7-HA strain expressing plasmid-encoded Sos7-GFP variants or a vector control and of a Sos7-cMyc strain expressing plasmid-encoded Spc7-GFP variants or a vector control were used for immunoprecipitation (IP) using anti-HA and anti-GFP or anti-cMyc and anti-GFP antibodies, respectively. The immunoprecipitates were analyzed by Western blot analysis using the relevant antibodies. An example of such an immunoprecipitation is shown for the Spc7-HA strain transformed with a plasmid expressing full-length Sos7-GFP. γ-Tubulin, loading control. (Right panel) Summary of the interaction of various Spc7 and Sos7 variants. , coimmunoprecipitation; −, no coimmunoprecipitation. Raw data are shown in Fig. S5 in the supplemental material. (B) Spc7 and Sos7 interact via their C-terminal ends. Yeast 2-hybrid analysis using the indicated fusion constructs. Transformants were grown on synthetic defined medium plates with (control) or without (selective) histidine and adenine for 3 days at 30°C. “AD” and “BD” denote the Gal4 activating and binding domains, respectively. (C) Coimmunoprecipitation of differently tagged Sos7 proteins. Protein extracts of a Sos7-cMyc strain expressing plasmid-encoded Sos7-GFP variants were used for immunoprecipitation using anti-cMyc antibodies. The immunoprecipitates were analyzed by Western blot analysis using an anti-GFP antibody. (Right panel) Summary of the interaction of Sos7 variants. , coimmunoprecipitation. Raw data are shown in Fig. S6 in the supplemental material. (D) Yeast 2-hybrid analysis showing Sos7^201-264 self-interaction. Transformants were grown on SD plates with (control) or without (selective) histidine for 4 days. V, vector control; AD and BD, activating and binding domain plasmids, respectively.

Fig 6

Sos7 is required for kinetochore targeting of MIND components. (A) Serial dilution patch tests (10⁴ to 10¹ cells) of mis12-537 and nuf2-1 transformants grown under plasmid-selective conditions at the indicated temperatures for 5 days. sos7⁺, plasmid expressing sos7⁺. (B) Sos7-GFP localization in fixed mis12-537 and nuf2-1 cells incubated at the permissive (25°C) or restrictive (34°C) temperature. Diagrams above the photomicrographs show the positions of the GFP signal. Bar, 5 μm. (C) Kinetochore localization of Mis12-GFP (left panel) and Nuf2-GFP (right panel) in sos7^ts strains. sos7^ts cells expressing one of the above-mentioned tagged kinetochore proteins were grown at the indicated temperatures before fixation and stained with DAPI, anti-GFP, and TAT-1 anti-tubulin antibody. Bars, 5 μm. (D) Serial dilution patch tests of sos7-Δ7 cells transformed with plasmids expressing the indicated kinetochore proteins. Transformants were grown under plasmid-selective conditions at 25°C or 30°C for 5 to 6 days. (E) Summary of the interactions observed for the Ndc80-MIND-Spc7/Sos7 complex. An asterisk indicates that the sos7-Δ7 mutant phenotype is rescued only by extra Mis12 or Mis13, not by extra Nnf1 or Mis14.

Fig 7

Sos7 belongs to a conserved protein family. (A) Diagrammatic representation of the comparison of Sos7 proteins from S. pombe and S. japonicus. Black bars indicate regions of identity and similarity. Clockwise from top right: serial dilution patch tests (10⁴ to 10¹ cells) of sos7-Δ7 transformants expressing a vector control, S. pombe Sos7 (Spsos7⁺), S. japonicus Sos7 (Sjsos7⁺), or S. cerevisiae Kre28 (ScKRE28); sos7-+6 cells transformed with vector control or a plasmid with Sjsos7⁺; and sos7-178 cells transformed with vector control or a plasmid with Sjsos7⁺. (B) Amino acid sequence comparison of Sos7-like proteins from S. pombe, S. japonicus (XP_002174481), Aspergillus nidulans (CBF76747), Aspergillus flavus (XP_002383989), Neurospora crassa (CAE76380), and Sordaria macrospora (XP_003347116). Black boxes, ≥50% identity; gray boxes, ≥50% similarity (http://www.ch.embnet.org/software/BOX_form.html). A bracket indicates a highly conserved sequence motif. (C) Phylogenetic tree of Sos7 homologues shown in panel B. (D) Phylogenetic tree of Kre28 and V. polyspora homolog EDO18951, Z. rouxii CAR29059, A. gossypii AAS54100, C. glabrata XP_446867, L. thermotolerans CAR23808, and K. lactis XP_452234. The trees in panels C and D were engendered by PhyML 3.0 based on the maximum-likelihood method using the Jones Taylor Thornton substitution matrix (18). Bootstrap values (100 replicates) are shown. The scale bar indicates the length of the branch.