| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
Schizosaccharomyces pombe expresses two homologs of mammalian 14-3-3 genes : rad24, which is involved in checkpoint control, and rad25, the function of which is considered to be supplementary to rad24 activity. In this study, we present evidence that both Rad24 and Rad25 act as negative regulators of Byr2 (MAPKKK). Overexpression of either rad24 or rad25 reduced sporulation efficiency in homothallic wild-type cells. In contrast, the sporulation efficiency of rad24 and rad25 null cell was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1 null cells, but not in byr2 or ste4 null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1S214DT218D. Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 in the yeast two-hybrid system. The formation of complexes in vivo was also confirmed by immuno-coprecipitation. Furthermore, we showed that an antibody against phospho-p44/42 MAP kinase recognized elevated phosphorylation levels of Spk1 protein (MAPK) in rad24 and rad25 null cells, and that the mRNA level of mam2, which is regulated by Ras1, was clearly elevated in ras1 rad24 and ras1 rad25 null cells relative to ras1 null cells. The cellular localization of Rad24, Rad25 and Byr2 was examined using fusion to green fluorescent protein (GFP). While Byr2-GFP translocated from the cytoplasm to the plasma membrane in response to nutrient starvation, Rad24-GFP and Rad25-GFP remained in the cytoplasm. Our of all results consistently indicate that Rad24 and Rad25 bind to Byr2 in the cytoplasm and negatively regulate Byr2 signaling in the sexual development in S.pombe.
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