Wat1/pop3, a conserved WD repeat containing protein acts synergistically with checkpoint kinase Chk1 to maintain genome ploidy in fission yeast S. pombe

Abstract

Aberrant chromosome segregation defects can lead to aneuploidy, a common characteristic of human solid tumors. Aneuploidy is generated due to defects in the mitotic spindle or due to inefficient mitotic checkpoint response. We have isolated a novel mutant allele of wat1, a WD repeat containing protein that exhibits conditional synthetic lethality with chk1 knock out. We observed only a marginal decrease in the level of α tubulin protein level in wat1-17 mutants after prolong exposure at semi permissive temperature. Interestingly the protein level of α-tubulin was reduced in the chk1Δ wat1-17 double mutant at 18°C with defective microtubule structure. Consistent with loss of microtubule structure in the chk1 deletion background, the double mutant of wat1-17 chk1Δ was hypersensitive to the microtubule destabilizing agent TBZ suggesting severe defects in microtubule integrity in wat1-17 mutant in the absence of Chk1. Combination of wat1-17 with the chk1 deletion also aggravates the defects in the maintenance of genome ploidy. The mutation in wat1-17 was mapped to Cys 233 that was changed to tyrosine. Based on the molecular modeling studies, we hypothesize that the substitution of the bulky Tyr residue at Cys233 position in wat1-17 mutant results in conformational changes. This in turn can affect its intercations with other interacting partners and perturb the overall functions of the Wat1 protein.

Figure 1. The ts17/wat1-17 mutant allele exhibit conditional lethality with chk1 knockout.

Indicated strains were grown at 25°C, serially diluted and spotted on YEA plates. Plates were incubated at indicated temperature for 3 days except 18°C plate that was incubated for 7 days before taking photographs.

Figure 2. The wat1-17 chk1 delete cells are hypersensitive to microtubule destabilizing agent.

A. Indicated strains were grown at 25°C, serially diluted and spotted on YEA plate or plate containing 10 ug/ml thiabendazole. Plates were incubated at 25°C for 3-4 days before taking photographs. B. Indicated strains were grown till mid log phase at 25°C and then shifted at 18°C for 36 hr, fixed with 70% ethanol and stained with DAPI. About 250 cells were counted for the presence of aberrant nuclei and percentage was calculated. Scale bar: 10 µm.

Figure 3. The wat1-17 chk1 delete cells shows reduced α tubulin levels and defects in mictrotubule structure.

A. The wild type, wat1-17 and wat1-17 chk1Δ cells were grown at permissive temperature till mid log phase then shifted at 18°C for indicated time. Protein lysate was prepared as described in material and methods, samples were run on 10% SDS PAGE, transferred on nitrocellulose membrane and probed with anti α-tubulin antibody. Anti-cdc2 antibody was used as loading control. Signals were quantitated on Gel Doc system (Life Technologies) and protein ratio was calculated. The asterisk indicates a non specific band. B. Indicated strains were grown at 25°C and shifted at 18°C for 48 hr. Cells were processed for immunoflourescence microscopy using anti α- tubulin antibody. Scale bar: 10 µm.

Figure 4. The diploidisation of wat1-17 and wat1-17 chk1Δ strain.

A. Wild type, wat1-17, chk1Δ and wat1-17chk1Δ double mutant were grown up to mid log phase, about 1000 cells were spread on YEA plates containing 1.5 µg/ml Phloxine B. All the plates were incubated at 25°C for 3–4 days before taking photographs. B. FACS analysis of wild type, chk1Δ, wat1-17, wat1-17chk1Δ mutants. The asynchronous cultures were grown at 25°C and shifted to 18°C, samples were taken at 12 h interval, fixed and stained with the propidium iodide. Samples were analyzed for BD FACS caliber for DNA content analysis.

Figure 5. Mapping of wat1-17 mutation and its conservation with human Lst8.

A. Location of mutation in wat1-17 gene. B. ESPript generated sequence alignment of Wat1 and human Lst8. Secondary structure assignment was according to crystal structure Lst8 (PDB-ID, 4JSP).

Figure 6. Molecular Modeling analysis of Wat1 and its interaction with Prp2.

A. 3D model of S. pombe Wat1 showing heptad WD repeats. Close view of region of interest where C233Y mutation lies. Upper panel shows wild type Wat1 having Cys 233 (colored in red). Lower panel shows model of mutant Wat1 having Tyr at 233 position(colored in red). Images were generated with the help of Chimera1.6. B. The Wat1 mutant protein fails to interact with Prp2 in a yeast two hybrid interaction assay. Prp2 Protein was used as prey, fused with activation domain (pACT2) and the Wat1 or Wat1 mutant protein was fused to the DNA-binding domain (pAS2) as bait. Interaction was analyzed using LacZ as reporter gene on SD-trp-leu plates containing X-gal and HIS marker as a reporter gene on SD-trp-leu plate lacking histidine. 3AT was used to prevent any leaky expression of HIS marker gene.