These findings, together with the observation that de novo protein synthesis is critical for Pmk1 activation, strongly suggest that an unknown branch regulates the signaling of the absence of glucose to the cell integrity pathway. Pmk1 activity is required for fission yeast adaptation from fermentative to respiratory metabolism, as evidenced by the moderate growth defect displayed by Pmk1-less cells in respiratory media. Our results support that Pmk1 reinforces the adaptive response of fission yeast to the nutritional stress by enhancing the activity of the SAPK pathway at two different click here levels: i- by

positively targeting Atf1 transcription factor to allow timely and full expression of genes involved in growth adaptation to respiratory metabolism, and ii- by enhancing signal transmission to Sty1, the core MAPK of the SAPK pathway. Methods Strains, growth Protein Tyrosine Kinase inhibitor conditions, stress treatments and plasmids The S. pombe strains employed in this study are listed in Table  1. They were grown with shaking at 28°C in either YES or EMM2 minimal medium with 7% of glucose (repressing conditions) MI-503 order to a final OD600 of 0.5 (actual glucose concentration = 6% as determined by the glucose oxidase method) [12]. Then the cells were recovered by filtration and resuspended

in the same medium lacking glucose and osmotically equilibrated with either 3% glycerol, 3% glycerol plus 0.1% glucose, 2.8% glycerol plus 0.5% glucose, 2.5% glycerol plus 1% glucose, or 2% glycerol plus 3% ethanol. In hypertonic stress experiments cultures were supplemented with 0.6 M KCl. In some of the experiments N-acetyl cysteine (NAC; final concentration 30 mM) or cycloheximide (final G protein-coupled receptor kinase concentration 100 μg/ml) were added to the glucose-rich based cultures [12]. Plasmids pREP41-rho1(T20N) and pREP41-GST-cdc42(T17N) express dominant

negative alleles of Rho1 and Cdc42 under the control of the attenuated variant (41X) of the thiamine-repressible promoter nmt1, respectively [17]. Cells containing these plasmids were first grown in EMM2 glucose rich medium with or without 10 μM thiamine for about 18 h, and transferred to osmotically equilibrated medium without glucose. Solid media were supplemented with 2% agar (Difco). Transformation of yeast strains was performed by the lithium acetate method [35]. Culture media were supplemented with adenine, leucine, histidine or uracil (100 mg/l, all obtained from Sigma Chemical Co.) depending on the requirements for each particular strain. Table 1 S . pombe strains used in this study* Strain Genotype Source/Reference MM1 h+ Madrid et al. [17] MM2 h- Madrid et al. [17] MI200 h+ pmk1-Ha6H::ura4 + Madrid et al. [12] MI201 h- pmk1-Ha6H::ura4 + Madrid et al. [12] LS116 h+ pmk1::KanR pmk1(K52E)-GFP:: leu1 + Sánchez-Mir et al. [36] MI702 h- pyp2-13myc::ura4 + Madrid et al.