Fig. 2: QCAP degrons functionality is largely defined by their amino acid composition.

a Fluorescence-activated cell sorting of yGPS-P_lib into four gates (G1-4), each composed of 2.5 million cells, was followed by NGS and protein stability index (PSI) scoring of each peptide, leading to the formation of a peptide density map. Shown is a FACS illustration and gating of 10,000 cells. Degron cutoff at PSI < 1.7 is marked by a dashed line. a.u.: arbitrary units. b Validation of QCDPred-based degron predictions. Ten DNA fragments from the library were re-cloned into yGPS-P, followed by flow cytometry analysis. The corresponding peptides were divided into three different groups: Group I- PSI ≤ 1.62, P ≥ 0.85; Group II- PSI > 1.62, P < 0.85; Group III- PSI ≤ 1.62, P < 0.85. Stability scale: Median value of the yeG/yeC ratio in empty vector (EV) control was set as one. All other histograms were distributed accordingly. c Scatter plot comparing Kyte-Doolittle hydrophobicity amino acid scores and QCDPred probabilities. d Flow cytometry histogram of intact and mutant P3 peptide in which three amino acids were replaced with either glutamate (E) or arginine (R) residues. Stability scale was set as shown in Fig. 2B. e 3D structure of QCDPred-calculated cytosolic Pca1 degron (amino acids 289-305), based on AlphaFold Pca1 structural model #AF-p38360-F1 (Conf. (90 > pLDDT > 70). Marked by a blue color are the cysteine residues. Marked by a red color are the three amino acids that have been replaced with aspartate or arginine. pLDDT: per-residue confidence score on a scale of 0-100. f Immunoblot analysis of Pca1 protein levels. Wild type or doa10∆ (d∆) cells, expressing the indicated HA-tagged Pca1 proteins were left intact or treated with 50 μM CdCl₂ for 1 h. To determine degradation, where indicated, the translation inhibitor cycloheximide (CHX) was added to cells for 15 min before cell harvesting. Pca1 levels were measured by immunoblotting using anti-HA Abs while Pgk1 levels served as a loading control. This analysis was repeated two times. D: replacement of residues marked in 2E by red color with aspartate; R: replacement of residues marked in 2E by red color with Arginine. Source data for panels a-d and f are provided as a Source Data file.