Deep quantitative phosphoproteomics identifies non-canonical pH-sensitive yeast phosphorylation networks

Changes in cellular pH act as potent upstream signals that rewire phosphorylation networks controlling cellular function and disease. In yeast, this response is thought to arise mainly from pH-dependent perturbations to membrane integrity and cell wall stress, which are relayed through TORC2- and PKC-dependent signaling pathways. Yet several proteins exhibit acid-dependent phosphorylation independently of TORC2 and PKC, pointing to additional, uncharacterized acid stress signaling mechanisms. To investigate this possibility, we performed SILAC-based deep phosphoproteomics to distinguish phosphoproteome changes that are dependent on plasma membrane and cell wall stress from those that are independent. Across more than 19,000 unique phosphosites, we identified over 1,000 sites whose acid stress-dependent regulation occurs outside the canonical acid stress pathways. These noncanonical targets were significantly enriched in proteins associated with the plasma membrane, GTPase-mediated signaling, and endocytosis. Motif analysis revealed enrichment of acidophilic substrates implicating the membrane-tethered casein kinase Yck1 as a major mediator of this response. In contrast, canonical acid stress signaling preferentially involved basophilic kinase substrates, while acid-repressed responses were enriched for proline-containing phosphosites involved in cell cycle progression. Collectively, these results uncover a distinct acid-responsive phosphorylation network that operates independently of plasma membrane and cell wall integrity signaling.