Identification of novel degradation signals in yeast
The coupling of yeast growth and stability of a selection marker allows isolation of surviving population whose plasmid DNA is then subjected to deep sequencing. In this way we are able to identify simultaneously the degradation rates of thousands of peptides. The method is currently employed to identify degradation signals involved in protein quality control but can also be applied to investigating proteome stability in multiple organisms, from yeast to human.
Degron search: Method overview. A. Method premise - The presence of a degron will lead to lower levels of Ura3p, and therefore for faster growth in the presence of 5FOA. B. Mid-log yeast mRNA are purified, reverse transcribed to cDNA and cleaved using a 4-cutter. The resulting fragments are cloned into the c-terminus of the reporter. C. Plasmids from are transformed into yeast and kept in plasmid-selective conditions. Yeast samples are drawn at indicated time points and plasmid DNA was amplified using specific primers followed by high-throughput sequencing. D. An illustration of the expected competition and how it shapes the abundance profiles of the different clones. These profiles can be used to estimate the growth rate of each individual clone.