Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis  

Abstract

The posttranslational addition of ubiquitin (Ub) profoundly controls the half-life, interactions, and/or trafficking of numerous intracellular proteins. Using stringent two-step affinity methods to purify Ub-protein conjugates followed by high-sensitivity mass spectrometry, we identified almost 950 ubiquitylation substrates in whole Arabidopsis thaliana seedlings. The list includes key factors regulating a wide range of biological processes, including metabolism, cellular transport, signal transduction, transcription, RNA biology, translation, and proteolysis. The ubiquitylation state of more than half of the targets increased after treating seedlings with the proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-Leu-al), strongly suggesting that Ub addition commits many to degradation by the 26S proteasome. Ub-attachment sites were resolved for a number of targets, including six of the seven Lys residues on Ub itself with a Lys-48>Lys-63>Lys-11>>>Lys-33/Lys-29/Lys-6 preference. However, little sequence consensus was detected among conjugation sites, indicating that the local environment has little influence on global ubiquitylation. Intriguingly, the level of Lys-11-linked Ub polymers increased substantially upon MG132 treatment, revealing that they might be important signals for proteasomal breakdown. Taken together, this proteomic analysis illustrates the breadth of plant processes affected by ubiquitylation and provides a deep data set of individual targets from which to explore the roles of Ub in various physiological and developmental pathways.

Disrupting Autophagy Restores Peroxisome Function to anArabidopsis lon2Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

“…It will be interesting to learn whether Arabidopsis NBR1 (Svenning et al, 2011;ZientaraRytter et al, 2011), which functions in autophagy of insoluble ubiquitinated aggregates (Zhou et al, 2013), similarly targets peroxisomes for pexophagy in Arabidopsis and whether any ubiquitinated proteins in the peroxisome membrane are required for pexophagy. It is noteworthy that several peroxisomal proteins are found in a recent proteomic analysis of ubiquitinated Arabidopsis proteins (Kim et al, 2013).…”

Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A

“…For those proteins that were identified in the single and the tandem affinity enrichment experiment and also fulfilled the above mentioned cut-off criteria, we correlated the number of spectra for each protein in the individual experiments. (25), and Kim et al, (26).…”

“…The identification of ubiquitylated proteins was mainly achieved using ubiquitin binding domain (UBAs) affinity chromatography and mass spectrometry (22)(23)(24)(25)(26). Here we report a similar approach for the identification of ubiquitylated Arabidopsis proteins, but using a different experimental method, which allowed us to detect proteins that until now have been refractory to identification.…”

Autophagy-Related Proteins Are Required for Degradation of Peroxisomes inArabidopsisHypocotyls during Seedling Growth

“…An excess of peroxisomal proteins such as PEROXIN5 (PEX5), the receptor of peroxisomal targeting signal type 1, could be polyubiquitylated and degraded by proteasomes, as in budding yeast (Saccharomyces cerevisiae) (Platta et al, 2004). The degradation of peroxisomal proteins by proteasomes is supported by the recent identification of Arabidopsis ICL as a ubiquitylation target (Kim et al, 2013). A third possible pathway for peroxisomal degradation is pexophagy, autophagy that is selective for the degradation of peroxisomes (reviewed by Till et al, 2012).…”