It’s Time for Some “Site”-Seeing: Novel Tools to Monitor the Ubiquitin Landscape in Arabidopsis thaliana

Unraveling K63 Polyubiquitination Networks by Sensor-Based Proteomics

“…Alternative approaches to enrich for ubiquitinated peptides, using K-«-GG antibodies recognizing ubiquitin remnants after trypsin digestion, have been used successfully in mammals (Kim et al, 2011;Udeshi et al, 2013) and may be the method of choice to improve the detection of K63 polyubiquitination sites. Recently, a COFRADIC-based ubiquitinome using protein precipitation with a methanol/chloroform mix right after extraction to kill DUB activities also allowed the identification of thousands of ubiquitination sites in Arabidopsis (Walton et al, 2016). Therefore, a lot of effort will be required to optimize linkage-specific ubiquitinomes in the future by adapting these different above-mentioned methods to sensor-based purification.…”

“…To evaluate the relevance of the identified proteins, we compared our K63 ubiquitinome with previously published Arabidopsis ubiquitinomes. The recently established TUBE/USU and COFRADIC ubiquitinomes serve as references (Kim et al, 2013;Walton et al, 2016), but these studies monitored ubiquitination with no resolution on linkage types and, therefore, provide no hint of the function of a particular ubiquitination event. Over 32% (35 out of 107 proteins) and 22% (23 out of 107 proteins) of the proteins identified as K63 polyubiquitinated in our analyses also were identified in previous TUBE/USU and COFRADIC proteomes, respectively (Fig.…”

“…These include enzymes localized in the cytosol as well as in various organelles and mostly involved in carbohydrate, amino acid, and vitamin metabolism. Several enzymes were identified (Kim et al, 2013;Walton et al, 2016). B, Functional categorization of K63 polyubiquitinated proteins using Gene Ontogeny (GO) annotations from The Arabidopsis Information Resource (TAIR) and agriGO databases.…”

“…The deubiquitination of ubiquitinated Lys residues is then used to attach a tag that allows the specific isolation of ubiquitinated peptides during subsequent COFRADIC runs. This approach led to the identification of over 3,000 ubiquitination sites in 1,600 proteins (Walton et al, 2016).…”

“…Even though our ability to detect ubiquitinated proteins has improved dramatically in recent years (Kim et al, 2013;Walton et al, 2016), a major hurdle in our understanding of ubiquitin biology remains due to the existence of several forms of ubiquitination, associated with many different cellular outputs. The lack of resolution of specific ubiquitin type detection in published ubiquitinomes has prevented us from making biological sense of these large data sets and has greatly hampered our ability to tackle with precision the roles of subtypes of ubiquitin.…”