Ubiquitin S65 proteomics: Association of ubiquitin mutants with ubiqiutin binding domains

Ubiquitylation is an essential post-translational modification that regulates numerous cellular processes, most notably protein degradation. Ubiquitin itself can be post-translationally modified by phosphorylation, with nearly every serine, threonine, and tyrosine residue having the potential to be phosphorylated. However, the effect of this modification on ubiquitin function is largely unknown. Here, we performed in vivo and in vitro characterization of the effects of phosphorylation of yeast ubiquitin at position serine 65. We find ubiquitin S65 phosphorylation to be regulated under oxidative stress, occurring in tandem with the restructuring of the ubiquitin landscape into a highly polymeric state. Phosphomimetic mutation of S65 recapitulates the oxidative stress phenotype, causing a dramatic accumulation of ubiquitylated proteins and a proteome-wide reduction of protein turnover rates. Importantly, this mutation impacts ubiquitin chain disassembly, chain linkage distribution, and substrate targeting. These results demonstrate that phosphorylation represents an additional mode of ubiquitin regulation with broad implications in cellular physiology.