Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Abstract

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways. Video LinkThe video component of this article can be found at

Mapping the Landscape of a Eukaryotic Degronome

“…Re-cloning of the degrons was necessary to eliminate the possibility of including Ura3p mutations that may arise as a result of prolonged exposure to 5FOA and might destabilize the reporter regardless of its fused peptide. Following re-insertion of the tested degron, we measured single colony minimal doubling times (MDTs) (Cohen et al, 2014) and reporter Ura3p protein levels at exponential phase ( Figures 3A-3C). The observed differences in steady-state levels were in good agreement with reporter protein half-lives ( Figures 3D and 3E), indicating that, indeed, the fused peptides, rather than mutations in the URA3 gene, correspond to the different growth kinetics.…”

Cycloheximide Chase Analysis of Protein Degradation in <em>Saccharomyces cerevisiae</em>

“…Similarly, growth-based reporter assays reflect steady state protein levels over an extended time period without discriminating between the influences of synthesis and degradation [15][16][17][18][19][20] . It is possible to infer the contribution of degradative processes to steady state protein levels by comparing abundance before and after inhibiting specific components of the degradative mechanism (e.g., by pharmacologically inactivating the proteasome 21 or knocking out a gene hypothesized to be required for degradation 13 ).…”

A reference-based protein degradation assay without global translation inhibitors

“…A variety of protein degradation assays are currently in use, including pulse-chases as well as proteomescale techniques based on mass spectrometry (19,(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48).…”