Mis17 is a regulatory module of the Mis6-Mal2-Sim4 centromere complex that is required for the recruitment of CenH3/CENP-A in fission yeast

Abstract

Background: The centromere is the chromosome domain on which the mitotic kinetochore forms for proper segregation. Deposition of the centromeric histone H3 (CenH3, CENP-A) is vital for the formation of centromere-specific chromatin. The Mis6-Mal2-Sim4 complex of the fission yeast S. pombe is required for the recruitment of CenH3 (Cnp1), but its function remains obscure.

Methodology/principal findings: Mass spectrometry was performed on the proteins precipitated with Mis6- and Mis17-FLAG. The results together with the previously identified Sim4- and Mal2-TAP precipitated proteins indicated that the complex contains 12 subunits, Mis6, Sim4, Mal2, Mis15, Mis17, Cnl2, Fta1-4, Fta6-7, nine of which have human centromeric protein (CENP) counterparts. Domain dissection indicated that the carboxy-half of Mis17 is functional, while its amino-half is regulatory. Overproduction of the amino-half caused strong negative dominance, which led to massive chromosome missegregation and hypersensitivity to the histone deacetylase inhibitor TSA. Mis17 was hyperphosphorylated and overproduction-induced negative dominance was abolished in six kinase-deletion mutants, ssp2 (AMPK), ppk9 (AMPK), ppk15 (Yak1), ppk30 (Ark1), wis4 (Ssk2), and lsk1 (P-TEFb).

Conclusions: Mis17 may be a regulatory module of the Mis6 complex. Negative dominance of the Mis17 fragment is exerted while the complex and CenH3 remain at the centromere, a result that differs from the mislocalization seen in the mis17-362 mutant. The known functions of the kinases suggest an unexpected link between Mis17 and control of the cortex actin, nutrition, and signal/transcription. Possible interpretations are discussed.

Figure 1. Mis17 is a hyperphosphoprotein.

A–B. An SDS-PAGE of S. pombe cell extracts was performed to detect Mis17 and Mis17-FLAG by immunoblot, using antibodies against bacterial-made Mis17 (α-Mis17 in A) and FLAG (α-FLAG in B). The wild-type strains with no tag and the chromosomally-integrated Mis17-FLAG gene expressed under the native promoter were used. Cell extracts of the Mis17-FLAG strain were treated with the calf intestine alkaline phosphatase (CIAP) in the presence (+) or absence (−) of phosphatase inhibitors (β-glycerophosphate and p-nitrophenyl phosphate). Arrowheads indicate the cross-reacting antigens. The no-tag band migrated faster than the Mis17-FLAG band with the expected MW difference. C. Mis17-FLAG chromosomally integrated in the cdc25-22 mutant and expressed under the native promoter was detected by immunoblot at 15 min intervals from 0–135 min (top). Arrowhead indicates the cross-reacting antigens. A shorter exposed image of the cross-reacting antigens is shown as the loading control (middle). The mutant cells were arrested in the G2 phase at 36°C, and released to mitosis by shifting the temperature to 26°C. Cells synchronously progressed with the timing of chromosome segregation, septation, and cytokinesis at 45, 75, and 105 min, respectively (bottom). Frequencies of cells showing two nuclei without (filled squares) or with (open squares) a septum were measured. See text for changes of the Mis17-FLAG band in the synchronous culture.

Figure 2. Overproduction of N-Mis17 is toxic.

A–C. Plasmid REP41 carrying the wild-type C-Mis17 gene (the carboxy-terminal half of Mis17) under the nmt1 promoter was made and introduced into the mis17-362 mutant. When the C-Mis17 was moderately expressed in the presence of thiamine, the plasmid rescued the ts phenotype (A). Plasmid carrying Mis17 with the substitution mutation (Mis17 S353P) failed to rescue mis17-362 (B). The mutant was not rescued by the control vector, but was rescued by plasmid carrying the wild-type full-length Mis17 gene. To visualize the localization of C-Mis17, plasmid carrying the C-Mis17-YFP was introduced into a strain that was also chromosomally integrated with Mis12, an authentic centromere/kinetochore gene that had been tagged with CFP and expressed under the native promoter. C-Mis17-YFP was overexpressed at 26°C for 16 h in the absence of thiamine (the mildest level of overexpression by the promoter nmt1 REP81). The signals of C-Mis17-YFP were localized at the centromere/kinetochore dots (C, Bar, 10 µm). D. N-Mis17 plasmid (REP41) inhibited colony formation of the wild-type strain in the absence of thiamine. E. N-Mis17-YFP in the plasmid REP81 was expressed in the strain that also carried Mis12-CFP, and observed 16 h after overexpression in the absence of thiamine at 26°C. Bar, 10 µm. F-G. The cell number of the strain carrying plasmid N-Mis17 (REP41 promoter) was counted after overexpression in the absence of thiamine (−thi) at 33°C (F). The cell viability was measured by plating the wild-type carrying vector or REP41 plasmid carrying N-Mis17 (G). After 16 h in the absence of thiamine, the majority of wild-type cells carrying N-Mis17 had lost viability. H–I. DAPI-stained S. pombe wild-type cells carrying the plasmid N-Mis17 fragment (REP41 promoter) after 16 h in the absence of thiamine at 33°C (H, Bar, 10 µm). Arrows indicate cells showing the large and small daughter nuclei typical for defects in centromere-binding proteins. Quantitative data for the frequency of chromosome missegregation were obtained by counting the number of unequal-mitotic cells in binuclear ones (I).

Figure 3. Rapid occurrence of missegregation by N-Mis17 under the TET promoter.

A. Chromosome missegregation was frequently observed after TET-induced overproduction of N-Mis17 in wild-type cells at 33°C as shown in the DAPI-stained micrograph (arrows). Bar, 10 µm. B. N-Mis17 was expressed under the TET promoter. The frequencies of chromosome segregation (%) were measured at 0 and 3 h after induction by the anhydrotetracycline (ahTET) at 33°C. C–E. The cdc25-22 mutant was transformed by plasmid carrying N-Mis17-Myc expressed under the TET promoter. Endogenous Mis17-FLAG was chromosomally integrated and expressed under the native promoter. Mis17-FLAG and N-Mis17-Myc were assayed by immunoblotting using antibodies against FLAG and Myc. Arrowhead indicates the cross-reacting antigens. The cdc25-22 mutant cells that expressed endogenous Mis17-FLAG and ectopically produced N-Mis17-Myc were arrested after 4.25 h at 36°C. See text for explanation of (C). After the release of mutant cells to 26°C, the frequencies of chromosome missegregation and the septation index were measured (D). Micrograph of missegregation (arrows) is shown in (E, Bar, 10 µm). F. Immunoblotting was done to detect Mis17 protein in the extracts of the wild-type and ts mutant mis17-362 cells at 36°C for 0–8 h. The mis17-362 mutant protein was unstable. Arrowhead indicates the cross-reacting antigens.

Figure 4. N-Mis17 overproduction does not affect endogenous centromere proteins.

A. Vector plasmid or the inducible REP41 plasmid carrying the N-Mis17-Myc gene was introduced into a strain in which endogenous Mis17-FLAG was chromosomally integrated and expressed under the native promoter. N-Mis17-Myc and Mis17-FLAG were assayed by immunoblot. The phosphorylation bands of endogenous Mis17 is shown in the upper panel (α-FLAG). Those of overproduced N-Mis17-Myc is in the lower panel (α-Myc). Arrowhead indicates the cross-reacting antigens. B. The frequency of missegregation was measured after shifting to the absence of thiamine. C. Endogenous Mis17-GFP was observed in cells that overproduced the N-Mis17 fragment in the absence of thiamine (16 h at 33°C). Bar, 10 µm. D. Mis15-GFP was observed in cells that overproduced N-Mis17 in the absence of thiamine (16 h at 33°C). Bar, 10 µm. E. Chromatin immunoprecipitation (ChIP) was done to examine whether Mis6 was bound to the central centromere in cells that overproduced N-Mis17 at 33°C. The central centromere DNAs, such as cnt1 and imr1, were precipitated with Mis6 (α-HA) in the wild-type cells that overproduced N-Mis17 (left panel). Chromosome missegregation was most frequent while Mis6 remained at the central centromere (right panel). F. Cnp1/CenH3 was observed in cells that overproduced N-Mis17 at 33°C. Cnp1-GFP chromosomally integrated and expressed under the native promoter was observed in the absence (top, overproduction for 16 h) and presence (bottom, overproduction is off) of thiamine. Bar, 10 µm. G. The frequencies of Cnp1-GFP signals at the centromere/kinetochore and the frequencies of chromosome missegregation were quantified. H. ChIP was performed for the strain that was integrated with Cnp1/CenH3-FLAG under the native promoter and transformed with plasmid carrying N-Mis17. When N-Mis17 was overproduced (16 h at 33°C), Cnp1/CenH3 was bound to the central centromeric cnt1 and imr1.

Figure 5. N-Mis17 overproduction causes sensitivity to TSA.

A. Wild type, mis6-302, mis15-68, and mis17-362 were spotted on a plate in the absence or presence of 25 µg/ml TSA at 26–36°C. See text. B. Wild-type cells that overproduced N-Mis17 by REP41 or REP81 were plated in the absence or presence of TSA (0–25 µg/ml) at 30°C. C. The N-Mis17 fragment contains eight K residues that were substituted to R. The resulting N-Mis17-8R gene was expressed under the REP41 promoter in the wild-type strain. D. The level of overproduced Mis17-N 8R was assayed compared with N-Mis17. PSTAIRE was the level of Cdc2 protein kinase assayed by the antibody PSTAIRE.

Figure 6. Negative-dominance is supported by certain protein kinases.

A. The negative dominance effect of N-Mis17 overexpression was greatly diminished in six protein kinase deletion mutants, Δssp2, Δppk9, Δppk15, Δppk30, Δlsk1, and Δwis4. Promoter is off or on in the presence or absence of thiamine, respectively. B. A cartoon for the implication of Mis17 in the Mis6-Mal2-Sim4 complex and negative dominance effect of the N-Mis17 fragment. See text.