Fig. 1: Search for C-degrons using random peptide libraries.
From: Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons
a Workflow of the systematic search for C-degrons. Degenerate oligonucleotides encoding random peptides of defined length (n) are cloned into a C-degron reporter plasmid by homologous recombination in yeast. The stability of each sequence variant Xn in the pool, fused to the tandem fluorescent protein timer (tFT), is then determined using multiplexed protein stability (MPS) profiling, whereby the pool is sorted into stability bins using the tFT readout, followed by deep sequencing to determine the frequency of each variant across bins. The distribution of sequencing reads for each variant is summarized by a weighted average of reads across bins in the form of a protein stability index (PSI, Methods) scaled between 0 (unstable) and 1 (stable). b Distributions of mCherry/sfGFP ratios, determined by flow cytometry, in pooled yeast libraries expressing random peptides of different lengths (n = 4, 8 or 12 amino acids) fused to the sfGFP-mCherry timer (tFT-Xn libraries). A strain expressing the tFT alone is shown for comparison (grey). Percentage of cells expressing unstable variants (blue region, mCherry/sfGFP <0.5) is shown in each plot. N = 35000 cells per sample. c Distribution of PSIs in the tFT-X12 library determined by MPS profiling. Peptides were grouped by the C-terminal amino acid. Blue region marks putative degrons (PSI < 0.5). Centerlines mark the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend to the minimum and maximum value in each group. d Relative amino acid frequency (log2 transformed) per position in the 4726 putative degrons from the tFT-X12 library, normalized to the relative frequency in the whole library. Blue—amino acid enrichment, orange—depletion in the putative degrons. e Correlation between peptide hydrophobicity (Kyte-Doolittle hydropathy scale) and PSI in the tFT-X12 library. Blue region marks putative degrons. ρ, spearman correlation coefficient. Hydrophobicity distributions for the putative degrons and stable peptides (right). Source data are provided as a Source Data file.
