Systematic analyses of all possible ubiquitin mutants

Abstract

We quantified the affects of all point mutations in ubiquitin on yeast growth. These fitness analyses delineated the physical requirements at each amino acid position in ubiquitin. We identified a stability window where many ubiquitin mutants were capable of populating a natively folded state, but unable to support yeast growth. Many mutants within this stability window accumulated as high molecular‐weight species in cells indicating that they were efficiently attached to substrates but inefficiently targeted to the proteasome. In addition, we identified a founding member of a new class of ubiquitin mutants that failed to accumulate in cells unless substrate conjugation was blocked with a second mutant in the C‐terminal tail. Heteronuclear NMR indicated that this destabilizing mutation reduces the unfolding temperature while retaining a ground‐state structure similar to wild‐type that enables binding to proteasome receptors. Based on these results, we propose that dynamics within the native ubiquitin structure are critical for proteasome targeting and that ubiquitin stability to unfolding is required for efficient recycling during proteasome‐mediated substrate degradation.