A dominant negative 14-3-3 mutant in Schizosaccharomyces pombe distinguishes the binding proteins involved in sexual differentiation and check point.
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.
Ohshima T, Jiajun Z, Fukamachi T, Ohno Y, Senoo H, Matsuo Y, Kawamukai M