ABSTRACT In Saccharomyces cerevisiae, cAMP regulates a number of different cellular processes, such as cell growth, metabolism, stress resistance and gene transcription. The intracellular target for this second messenger in yeast cells is the cAMP-dependent protein kinase (PKA). The way in which a broad specificity protein kinase mediates one right physiological response after cAMP increase indicates that specificity is highly regulated in the cAMP / PKA system. Here we address the mechanism through which cAMP-PKA signalling mediates its response to heat shock thermotolerance in Saccharomyces cerevisiae . Yeast PKA is a tetrameric holoenzyme composed of a regulatory (Bcy1) subunit dimer and two catalytic subunits (Tpk1, Tpk2 and Tpk3). PKA subunits are differentially expressed under certain stress conditions. In the present study we show that, although the mRNA levels of TPK1 are upregulated upon heat shock at 37°C, no change is detected in Tpk1 protein levels. The half-life of TPK1 mRNA increases and this mRNA condensates in cytoplasmic foci upon thermal stress. The resistance of TPK1 mRNA foci to cycloheximide-induced disassembly, together with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 mRNA foci and TPK1 expression were also evaluated during thermotolerance. The crosstalk of cAMP-PKA pathway and cell wall integrity (CWI) signalling was also studied. Wsc3 sensor and other components of the CWI pathway are necessary for the upregulation of TPK1 mRNA upon heat shock conditions. The assembly in cytoplasmic foci upon thermal stress shows to be dependent of Wsc3. Finally, evidence of an increase in the abundance of Tpk1 in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favorable conditions The results indicate the existence of a mechanism that exclusively regulates Tpk1 subunit expression and therefore contributing to the specificity of cAMP-PKA. SUMMARY STATEMENT PKA subunits are differentially expressed under heat-shock conditions. The mRNA of the TPK1 subunit is upregulated upon heat-shock at 37°C and thermotolerance, the half-life increases upon heat-stress and also this transcript condensates in cytoplasmic foci upon thermal stress and thermotolerance. The resistance to cycloheximide treatment of TPK1 mRNA foci together with the analysis by polysome profiling suggest that TPK1 mRNA is impaired for entry into translation upon thermal stress. An increase in Tpk1 protein and PKA activity was detected after the heat stress treatments. Cell Wall Integrity pathway, through Wsc3 sensor, is involved in TPK1 expression. Heat-stress regulates TPK1 expression through this pathway from an intermediate step of the cascade and independently of the upstream elements of the CWI pathway activation. These results demonstrate a new crosstalk between the two signalling pathways. The increment in Tpk1-dependent PKA activity during cell adaptation to heat stress might contribute to the overall cellular fitness when more favorable environmental conditions are restored. The results indicate the existence of a mechanism that exclusively regulates TPK1 subunit expression and therefore contributing to the specificity of cAMP-PKA.

Heat Stress Regulates the Expression ofTPK1Gene at Transcriptional and Post-Transcriptional Levels inSaccharomyces cerevisiae