Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons

Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, we performed an unbiased survey of C-degrons in budding yeast. We identified over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C-terminome. Combining machine learning, high-throughput mutagenesis and genetic screens revealed that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis. Overall design: Multiplexed protein stability (MPS) profiling using DNA amplicon-seq data. For this, a library of peptide variants (based on inserted random or pre-determined DNA sequences) fused to a tandem fluorescent protein timer (tFT) is expressed from yeast plasmids and sorted into stability bins using the tFT readout. The contained variable regions are sequenced and the occurrences of all observed DNA sequence variants are counted per stability bin. The distributions of read counts are summarized in the form of a protein stability index (PSI) for each peptide.