A dominant negative 14-3-3 mutant in Schizosaccharomyces pombe distinguishes the binding proteins involved in sexual differentiation and check point

Abstract

The homothallic fission yeast Schizosaccharomyces pombe undergoes sexual differentiation when starved, but sam (skips the requirement of starvation for mating) mutants such as those carrying mutations in adenylate cyclase (cyr1) or protein kinase A (pka1) mate without starvation. Here, we identified sam3, a dominant negative allele of rad24, encoding one of two 14-3-3 proteins. Genetic mapping and whole-genome sequencing showed that the sam3 mutation comprises a change in nucleotide at position 959 from guanine to adenine, which switches the amino acid at position 185 from glutamic acid to lysine (E185K). We generated the rad24-E185K integrated mutant and its phenotype was similar to that of the sam3 mutant, including calcium sensitivity and UV non-sensitivity, but the phenotype is different from that of the Δrad24 strain. While the UV-sensitive phenotype was observed in the Δrad24 mutant, it was not observed in the sam3 and rad24-E185K mutants. The expression of the rad24-E185K gene in wild type cells induced spore formation in the nutrient rich medium, confirming rad24-E185K is dominant. This dominant effect of rad24-E185K was also observed in Δras1 and Δbyr2 diploid mutants, indicating that rad24-E185K generate stronger phenotype than rad24 null mutants. Ste11, the key transcription factor for sexual differentiation was expressed in sam3 mutants without starvation and it predominantly localized to the nucleus. The Rad24-E185K mutant protein retained its interaction with Check point kinase1 (Chk1), whereas it reduced interaction with Ste11, an RNA binding protein Mei2, and a MAPKKK Byr2, freeing these proteins from negative regulation by Rad24, that account for the sam phenotype and UV non-sensitive phenotype. Glucose depletion in rad24-E185K or Δpka1 Δrad24 double mutation induced haploid meiosis, leading to the formation of spores in haploid. The position of glutamic acid 185 is conserved in all major 14-3-3s; hence, our finding of a dominant negative allele of 14-3-3 is useful for understanding 14-3-3s in other organisms.

Fig 1. Mapping and sequencing of the mutation sites of sam3 and sam9.

(A) Chromosomal mapping of the mutation site of sam3. Mapping was conducted by crossing YUK13 (h^- ade6-216 leu1 ura4::hphMX6 rec12::kanMX6 sam3) and YUK6 (h⁹⁰ ade6-210 his7 lys1 rec12::kanMX6) strains, yielding random spores. Spores were first grown on YES, then on YES containing CaCl₂. The CaCl₂-sensitive phenotype of sam3 was co-segregated with lysine auxotrophy, caused by lys1 mutation on Chr I, at a very low ratio (4.17%), while it was co-segregated normally with histidine auxotrophy (caused by his7 mutation on Chr II) and leucine auxotrophy (caused by leu1 mutation on Chr II), and uracil auxotrophy (caused by ura4 mutation on Chr III). (B) Whole-genome sequencing of SP416 (sam3) and SP430 (sam9) mutants reveals a common mutation in rad24 in sam3 and sam9 mutants. Nucleotide G959 of the rad24 gene was changed to A, which changed glutamic acid residue 185 of Rad24 to lysine. (C) Sanger sequencing verification of the mutation sites of the rad24 genes of SP66 (WT), SP416 (sam3), and SP430 (sam9) strains. (D) Alignment of various 14-3-3s orthologues. Glu185 is conserved in all typical 14-3-3s including S. pombe Rad25, S. cerevisiae Bmh1 and Bmh2, Arabidopsis GFR12, and Homo sapiens 14-3-3 epsilon, sigma, and gamma.

Fig 2. Dominant negative effect of sam3.

(A) Mating efficiency in SP66 (WT), SP416 (sam3), FK1 (rad24-E185K), and FK3 (sam3- rad24⁺) strains. Mating efficiency was measured by counting 300 cells growing on YES-rich medium for 3 days. FK3 was derived by replacing the sam3 mutant with WT rad24. (B) Cell morphology and mating efficiency of WT and sam3 strains harboring the vector and a plasmid expressing rad24 or rad25. Mating efficiency of WT and sam3 stains harboring plasmids was measured by counting 300 cells growing on EMML medium for 4 days. No clear reversion of the mating and sporulation efficiency phenotype was observed following expression of rad24 or rad25. (C) Effects of deleting ras1, byr2, and byr1 on the rad24-E185K mutant. Cells of SPRUD (Δras1/Δras1), SPSUD (Δbyr2/Δbyr2), SPBUD (Δbyr1/Δbyr1), TOP16 (Δras1 rad24-E185K/Δras1 rad24-E185K), TOP15(Δbyr2 rad24-E185K /Δbyr2 rad24-E185K), and TOP12 (Δbyr1 rad24-E185K /Δbyr1 rad24-E185K) strains were grown on EMMAL plates for 2 days. Sporulation rates of diploid strains were measured by counting 300 cells. Averages (%) of three experiments are shown.

Fig 3. KCl, calcium, and UV sensitivity of sam3 and rad24-E185K strains.

(A) SP870 (WT), SP416 (sam3), FK1 (rad24-E185K), and FK3 (sam3-rad24⁺) cells were cultured at 30°C in liquid medium until log phase, concentrated to 1×10⁷ cells/mL, and then used to generate a 10-fold dilution series. Diluted cells were spotted onto YES plates and incubated at 30°C for 3 days in the presence of 1 M KCl or 0.2 M CaCl₂. (B) SP870 (WT), TMS1 (Δrad24), SP416 (sam3), SP418 (sam4), and FK1 (rad24-E185K) strains were grown and used to generate a 10-fold dilution series. Cells were exposed to 0, 200, or 250 J/m² UV, spotted onto YES plates, and incubated at 30°C for 3 days.

Fig 4. Expression and localization of Ste11 in the sam3 mutant.

(A) Localization of Ste11 in homothallic YUK20 (WT ste11-GFP) and YUK22 (sam3 ste11-GFP) strains before and after nitrogen starvation for 10hr. (B) YUK20 (WT) and YUK22 (sam3) strains were grown under nitrogen-starved conditions for 10 h and Ste11 expression was monitored by western blotting using anti-GFP antibody. The relative intensity of Ste11 bands was adjusted against the Cdc2 signal quantified by ImageJ (shown at the bottom). (C) Localization of Ste11 was monitored in the YUK20 (WT) strain harboring vector or a plasmid pREP42-rad24 expressing rad24 and pREP42-rad24-E185K expressing rad24-E185K, and in the YUK22 (sam3) mutant harboring vector or pREP42-rad24. Cells were grown in EMMAL medium for 24 h and Ste11 levels in the nucleus were determined upon expression of rad24-E185K.

Fig 5. Immunoprecipitation of Chk1 with Rad24 and Rad24-E185K.

(A) Cells of HRS12 (Rad24-5FLAG Chk1-13Myc) and HRS17 (Rad24-E185K-5FLAG Chk1-13Myc) were grown on YES medium to log phase, harvested, and disrupted. Rad24-5FLAG or Rad24 (E185K) protein was pulled down by FLAG-conjugated beads, and Chk1-13Myc was detected using anti-Myc antibody. The left panel indicates the input. (B) Reciprocally, using HRS12 and HRS17 strains, Chk1-13Myc was immunoprecipitated with anti-Myc antibody and detected using anti-FLAG antibody. The strengths of the Chk1, Rad24, and Rad24E185K bands were quantified by ImageJ, and the relative intensity of Chk1 vs. Rad24E185K compared with Chk1 vs. Rad24 was calculated (shown at the bottom). *Numbers indicate the relative fold change of Input / IP samples.

Fig 6. Immunoprecipitation of Ste11, Mei2, and Byr2 with Rad24 and Rad24-E185K.

(A) HRS7 (Rad24-5FLAG Ste11-GFP) and HRS27 (Rad24-FLAG Ste11-GFP) cells were grown on YES medium to log phase. Rad24-5FLAG and Rad24-E185K-5FLAG protein was pulled down by FLAG-conjugated beads, and Ste11-GFP was detected using anti-GFP antibody. The strengths of the Ste11, Rad24, and Rad24E185K bands were quantified by Image J, and the relative intensity of Ste11 vs. Rad24E185K against Chk1 vs. Rad24 was calculated (shown at the bottom). *Number indicates Input / IP samples. (B) Reciprocally, using HRS7 and HRS27 strains, Ste11-GFP immunoprecipitated with anti-GFP antibody and Rad24-5FLAG or Rad24-E185K-5FLAG protein was detected using anti-FLAG antibody. Numbers were calculated as in (A). (C) Cells of HRS8 (Rad24-5FLAG Mei2-3HA) and HRS30 (Rad24-E185K-5FLAG Mei2-3HA) were grown on YES medium to log phase. Rad24-5FLAG or Rad24-E185K-5FLAG protein was pulled down by FLAG-conjugated beads and Mei2 was detected using anti-HA antibody. Numbers were calculated as in (A). (D) Cells of the TOP2 (Rad24-5FLAG) strain harboring plasmid pSLF272U-B2 and the TOP4 (Rad24-E185K-5FLAG) strain harboring plasmid pSLF272U-B2 were grown on EMMAL. Rad24-5FLAG or Rad24-E185K-5FLAG protein was pulled down by FLAG-conjugated beads and Byr2 was detected using anti-HA antibody. Numbers were calculated as in (A).　*Numbers indicate the relative fold change of Input / IP samples after subtracting the background band intensity.

Fig 7. KCl and calcium sensitivity of rad24 pka1 mutants.

Cells of TP4-5A (h^- WT), TOP13 (h^- Δrad24), TOP4 (h^- rad24-E185K), YMP178 (h^- Δpka1), and TOP14 (h^- Δrad24 Δpka1) were spotted onto YES plates following serial dilution and incubated at 30°C for 3 days on YES medium in the presence of 0.5 M KCl, 1 M KCl, 0.1 M CaCl₂, or 0.2 M CaCl₂. YES medium containing 0.1% glucose was used as a control (lower panel).

Fig 8. Phenotype of the rad24 pka1 double deletion mutant.

SP870 (h^9liWT), TMS1 (h⁹⁰ Δrad24), TOP3 (h⁹⁰ rad24-E185K), JZ633 (h⁹⁰ Δpka1), and TOP5 (h⁹⁰ Δrad24 Δpka1) cells were grown in YES medium containing 3% or 0.1% glucose and their normal mating rate and aberrant spores were measured by counting 500 cells. Arrows indicate haploid-derived aberrant 2–4 spores and diploid-derived aberrant 8 spores. Quantification of mating ratio was performed in triplicate and p <0.05 (*) was considered statistically significant. The error bars means Standard Deviation (SD) in triplicate samples.

Fig 9. Localization of Pka1-GFP in the rad24 mutant.

YMP27 (Pka1-GFP), TOP10 (Pka1-GFP Δrad24) and TOP11 (Pka1-GFP rad24-E185K) cells were grown in EMM5S-N for 24 h. GFP was observed at 0, 6, 12, and 24 h. (B) Quantification of cytoplasmic localization of the GFP signal based on counting 500 cells at 12 h and 24 h in triplicate. Th error bars indicate SD in triplicate samples.

Fig 10. Three-dimensional structure of 14-3-3 proteins.

(A) Three-dimensional structure of S. pombe Rad24 predicted by AlphaFold. (B) Three-dimensional structure of human 14-3-3 sigma co-crystalized with 10 peptides derived from Raf1 (PDB:3IQJ). The position of E185 and the equivalent position of E182 in human 14-3-3 sigma is highlighted.