SPECIFICITY OF THE CAMP-PKA PATHWAY IN CELLULAR MEMORY OF ACQUIRED HEAT STRESS RESISTANCE IN SACCHAROMYCES CEREVISIAE

María Clara Ortolá Martínez ¹ - Fiorella Galello ¹ - Sergio Nemirovsky ¹ - Vanina Zaremberg ² - Paula Portela ¹ - Silvia Rossi ¹

1 Departamento de Química Biológica, FCEN, UBA (IQUIBICEN-CONICET)
2 Department of Biological Sciences, University of Calgary, Calgary, Canada

In S. cerevisiae, cAMP-dependent protein kinase (PKA) consists of two regulatory (Bcy1) and three catalytic subunits (Tpk1, Tpk2, Tpk3). How signaling specificity arises from these isoforms remains a central question. The Tpk proteins display both shared and distinct functions, and their differential expression contributes to substrate specificity under stress. Yeast can acquire resistance to severe stress after a mild pretreatment, a phenomenon known as cellular memory. We previously demonstrated differential expression of Tpk isoforms under stress. Here we show that Tpk1 is strongly upregulated during thermal stress resistance, thereby promoting the formation of a holoenzyme preferentially enriched in this isoform. We analyzed transcriptomic, proteomic, and phosphoproteomic changes in a Tpk1-only strain and in the wild type (WT) during thermotolerance recovery (37°C pretreatment, 45°C severe stress, 25°C recovery). We identified 2,291 differentially expressed genes (DEGs) and 277 proteins (DEPs) in WT, and 1,701 DEGs and 237 DEPs in the mutant. Transcriptomics revealed enrichment of stress responses, protein folding, cell cycle, cell wall processes, and translation in both strains, with additional terms such as trehalose biosynthesis, RNA splicing, and proteasome activity in the mutant. Proteomics showed stress responses, protein folding, trehalose metabolism, carbohydrate metabolism, and telomere organization, along with strain-specific terms. However, transcript–protein fold-change correlations were poor. Phosphoproteomics identified 164 phosphopeptides in WT and 609 in the mutant, with limited overlap. Motif analysis revealed R[S/T][S/L]S as the most represented PKA motif, diverging from canonical RRXS. Our study reveals how coordinated changes in gene expression, protein abundance, and phosphorylation shape the cellular memory of acquired thermal stress resistance, determining the specificity of cAMP–PKA signaling.