The Greatwall-Endosulfine-PP2A/B55 pathway controls entry into quiescence by promoting translation of Elongator-tuneable transcripts.

Quiescent cells require a continuous supply of proteins to maintain protein homeostasis. In fission yeast, entry into quiescence is triggered by nitrogen stress, leading to the inactivation of TORC1 and the activation of TORC2. Here, we report that the Greatwall-Endosulfine-PPA/B55 pathway connects the downregulation of TORC1 with the upregulation of TORC2, resulting in the activation of Elongator-dependent tRNA modifications essential for sustaining the translation programme during entry into quiescence. This process promotes U34 and A37 tRNA modifications at the anticodon stem loop, enhancing translation efficiency and fidelity of mRNAs enriched for AAA versus AAG lysine codons. Notably, some of these mRNAs encode inhibitors of TORC1, activators of TORC2, tRNA modifiers, and proteins necessary for telomeric and subtelomeric functions. Therefore, we propose a novel mechanism by which cells respond to nitrogen stress at the level of translation, involving a coordinated interplay between the tRNA epitranscriptome and biased codon usage.

Distinct biological activity of threonine monophosphorylated MAPK isoforms during the stress response in fission yeast.

Mitogen-activated protein kinases (MAPKs) define a specific group of eukaryotic protein kinases which regulate a number of cellular functions by transducing extracellular signals to intracellular responses. Unlike other protein kinases, catalytic activation of MAPKs by MAPKKs depends on dual phosphorylation at two tyrosine and threonine residues within the conserved TXY motif, and this has been proposed to occur in an ordered fashion, where the initial phosphorylation on tyrosine is followed by phosphorylation at the threonine residue. However, monophosphorylated MAPKs also exist in vivo, and although threonine phosphorylated isoforms retain some catalytic activity, their functional significance remains to be further elucidated. In the fission yeast Schizosaccharomyces pombe MAPKs Sty1 and Pmk1 control multiple aspects of fission yeast life cycle, including morphogenesis, cell cycle, and cellular response to a variety of stressful situations. In this work we show that a trapping mechanism increases MAPKK binding and tyrosine phosphorylation of both Sty1 and Pmk1 when subsequent phosphorylation at threonine is hampered, indicating that a sequential and likely processive mechanism might be responsible for MAPK activation in this simple organism. Whereas threonine-monophosphorylated Sty1 showed a limited biological activity particularly at the transcriptional level, threonine-monophosphorylated Pmk1 was able to execute most of the biological functions of the dually phosphorylated kinase. Thus, threonine monophosphorylated MAPKs might display distinct functional relevance among eukaryotes.

Characterization of the endo-beta-1,3-glucanase activity of S. cerevisiae Eng2 and other members of the GH81 family.

The GH81 family includes proteins with endo-beta-1,3-glucanase widely distributed in yeast and fungi, which are also present in plants and bacteria. We have studied the activity of the Saccharomyces cerevisiae ScEng2 and the Schizosaccharomyces pombe SpEng1 and SpEng2 proteins. All three proteins exclusively hydrolyzed linear beta-1,3-glucan chains. Laminari-oligosaccharide degradation revealed that the minimum substrate length that the three endoglucanases were able to efficiently degrade was a molecule with at least 5 glucose residues, suggesting that the active site of the enzymes recognized five glucose units. Prediction of the secondary structure of ScEng2 and comparison with proteins of known structure allowed the identification of a 404-amino acid region with a structure similar to the Clostridium thermocellum endoglucanase CelA. This fragment showed similar enzymatic characteristics to those of the complete protein, suggesting that it contains the catalytic domain of this family of proteins. Within this domain, four conserved Asp and Glu residues (D518, D588, E609, and E613) are necessary for enzymatic activity.