Yeast PKA holoenzyme and condensate targeted and label-free proteomics

Protein kinase A (PKA) is a conserved kinase essential for many biological processes linked to growth, development and metabolism. The PKA catalytic subunit is expressed as multiple isoforms in diverse eukaryotes; however, their contribution to ensuring specificity in response to environmental cues remains poorly defined. In this study, we aimed to dissect the role of the Saccharomyces cerevisiae PKA isoforms, Tpk1-3, in the thermal stress response. We used an absolute quantification mass spectrometry approach to investigate stress-induced changes in the stoichiometry of Tpk1-3 bound to the PKA regulatory subunit. We identified Tpk3 accumulation in cytoplasmic granules upon stress and used immunopurification combined with proteomics to characterise the constituents of these Tpk3 granules.