Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation

Abstract

Crosstalk between different types of post-translational modifications (PTMs) on the same protein molecule adds specificity and combinatorial logic to signal processing, but has not been characterized on a large-scale basis. Here, we developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation, and these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

“…Ubiquitylation plays a prominent role in hexose transporters degradation (Horak and Wolf, 1997; Nijland et al, 2016; Roy et al, 2014), whereas phosphorylation is the primary mechanism for regulating cellular signaling (Humphrey et al, 2015). However, distinct phosphorylation sites are often used in conjunction with ubiquitylation in degradation, and these distinct sites are more highly conserved than the entire set of phosphorylation sites (Swaney et al, 2013). Hxt2 can be ubiquitinated at residue K26 and this triggers its inactivation.…”

“…Importantly, transcription of these genes is repressed at high glucose concentration, while under those conditions, Hxt2 and Hxt7 proteins at the membrane are targeted to the vacuole for degradation (Kruckeberg et al, 1999; Ye et al, 2001). Hxt7 and Hxt2 can be ubiquitinated and phosphorylated at N‐terminal residues (Swaney et al, 2013) and this likely contributes to their degradation.…”

“…It was previously reported that high glucose induced degradation of the Hxt7 transporter is blocked after truncation of its N‐terminal tail, suggesting that this region bear signaling information (Krampe and Boles, 2002). Because of the analogy between Hxt2 and Hxt7, both being subjected to ubiquitination and phosphorylation of N‐terminal residues (Swaney et al, 2013), we hypothesize that replacing the N‐terminal tail of Hxt2 by the corresponding region of Hxt11 will allow stable expression of Hxt2 at the membrane at high glucose concentration. Our data show that the chimeric Hxt11/2 transporter indeed is stably expressed at the plasma membrane under those conditions.…”

Systematic Characterization and Prediction of Post-Translational Modification Cross-Talk *

“…For example, the interplay between phosphorylation and lysine acetylation was systematically investigated by the deletion perturbations in the Mycoplasma pneumonia genome (21). The relationship between phosphorylation and ubiquitylation was studied after proteasome inhibition on Saccharomyces cerevisiae (22). These studies suggested that crosstalks between different PTMs were extensive and remained to be discovered.…”

Proteomic analysis of post translational modifications in cyanobacteria

“…The interplay between two PTMs was studied in both prokaryote and eukaryote [127,128], which suggested that cross-talk between different PTMs was extensive and remained to be discovered. The large-scale PTM proteomics data revealed that PTM cross-talk may also exist in cyanobacteria since many proteins were discovered to have more than one PTM [23,25,27].…”