Correlating yeast growth under selective conditions to protein degradation rates  

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). In a uracil-deficient medium growth is proportional to the relative levels of Ura3. Since the reporter protein is designed so that its synthesis rate is constant, its cellular level is solely determined by its degradation rate. Thus, this method accurately recapitulates reporter protein intracellular degradation kinetics. The method has so far been 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 degradation signals. Application of the degron-URA3-based system constitutes a powerful method that can be generally adapted in any investigation aimed at monitoring changes of protein levels associated with functions of other cellular pathways. (Link to the video describing this method)

 

Measuring the degradation of a reporter substrate of the Doa10 pathway in various yeast strains. The indicated strains, expressing the reporter are incubated in SD-complete or SD-URA media and OD₆₀₀ is measured every 15 min. The Minimal Doubling Time (MDT) for each strain is calculated using MDTcalc (Cohen et al., 2014).