The 14-3-3 Proteins Rad24 and Rad25 Negatively Regulate Byr2 by Affecting Its Localization in Schizosaccharomyces pombe

Abstract

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24-or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1 S214DT218D . Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.

Compartmentalized signaling of Ras in fission yeast

“…Because pheromone receptors relay mating signals across the PM, which activate Byr2, it seems appropriate that PMassociated Ras1 regulates this pathway. In support of this idea, Byr2 has been shown to associate with the PM upon sexual differentiation (21). We and others (12,22) have shown that the Ras1-Scd1 pathway is involved in protein trafficking.…”

Involvement of Moc1 in Sexual Development and Survival ofSchizosaccharomyces pombe

“…The S. pombe strains used in this study are listed in Table 1. S. pombe was grown in YESrich medium (0.5% yeast extract, 3% glucose, 225 mg/ liter adenine, histidine, leucine, uracil, and/or lysine hydrochloride) or Pombe Minimum (PM) synthetic medium, 23,24) supplemented with 75 mg/liter adenine, leucine, and/or uracil when necessary. Nitrogen-free PM medium (1% glucose without ammonium chloride) was used to culture S. pombe when the mating efficiency had to be measured.…”

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell–cell fusion

“…The Byr1 DD mutant has the two following point mutations, S214D and T218D, as described previously (Ozoe et al 2002). First, a fragment including the byr1-coding region and 5 ′ and 3 ′ extensions was amplified from genomic DNA with primers osm1837 (5 ′ -tcccccgggGATCTAATAATGCTTTG TATTAAG-3 ′ ) and osm1838 (5 ′ -aaaactgcagTTGGCTTATACG TAATTGCCAAG-3 ′ ), digested with PstI and XmaI, and ligated to similarly treated pSM1232 to generate plasmid pSM1668 (pSP72-byr1).…”