FIG 4 .

ScCst6 phosphorylation at position S266 is crucial for ScNCE103 regulation. (A) ScCst6-His, overexpressed in the WT under 5% CO₂, was affinity purified and subjected to SDS-PAGE. Corresponding bands were excised and digested with trypsin-LysC, AspN, GluC, or LysargiNase. Additionally, in-solution digestion for trypsin-LysC and GluC was performed. Phosphorylated peptides were enriched and analyzed by LC-MS/MS. Nineteen different phosphorylation sites, indicated by “P,” were identified in at least 2 of 10 independent experiments. Newly identified phosphorylation sites are shown in boldface, and previously known sites are not in boldface. Underlined are the 3 serine residues that were conserved in S. cerevisiae, C. glabrata, and C. albicans. Among them, only S266 was found to be phosphorylated in the WT, noted within a box. (B) Site-specific mutation of S266 was performed to investigate direct influence on ScNCE103 expression under changing CO₂ conditions. Expression levels were normalized to WT ScNCE103 expression in 5% CO₂. Changing S266 to alanine (cst6Δ + CST6^S266A) showed a significant upregulation of ScNCE103^CO2, while ScNCE103^air levels were unaltered. This is comparable to expression changes of the sch9Δ mutant and proves the critical role of Cst6 S266 for CA regulation. The bars show means ± standard deviations from 5 independent experiments. Significance against the cst6Δ + CST6 strain, calculated by a two-sided t test for unpaired samples, was defined as P ≤ 0.01 (**).