C-terminal sequence stability profiling in Saccharomyces cerevisiae reveals protective protein quality control pathways.
Abstract:
Protein quality control (PQC) mechanisms are essential for degradation of misfolded or dysfunctional proteins. An essential part of protein homeostasis is recognition of defective proteins by PQC components and their elimination by the ubiquitin-proteasome system, often concentrating on protein termini as indicators of protein integrity. Changes in amino acid composition of C-terminal ends arise through protein disintegration, alternative splicing or during the translation step of protein synthesis from premature termination or translational stop-codon read-through. We characterized reporter protein stability using light-controlled exposure of random C-terminal peptides (CtPC) in budding yeast revealing stabilizing and destabilizing features of amino acids at positions -5 to -1 of the C-terminus. The (de)stabilization properties of CtPC-degrons depend on amino acid identity, position as well as composition of the C-terminal sequence and are transferable. Evolutionary pressure towards stable proteins in yeast is evidenced by amino acid residues under-represented in cytosolic and nuclear proteins at corresponding C-terminal positions, but over-represented in unstable CtPC-degrons, and vice versa. Furthermore, analysis of translational stop-codon read-through peptides suggested that such extended proteins have destabilizing C-termini. PQC pathways targeting CtPC-degrons involved the ubiquitin-protein ligase Doa10 and the cullin-RING E3 ligase (CRL) SCFDas1. Overall, our data suggest a proteome protection mechanism that targets proteins with unnatural C-termini by recognizing a surprisingly large number of C-terminal sequence variants.
