Identification of ubiquitinated proteins in Arabidopsis

Abstract

Ubiquitin (Ub) is a small peptide that is covalently attached to proteins in a posttranslational reaction. Ubiquitination is a precise regulatory system that is present in all eukaryotic organisms and regulates the stability, the activity, the localization and the transport of proteins. Ubiquitination involves different enzymatic activities, in which the E3 ligases catalyze the last step recruiting of the target for labelling with ubiquitin. Genomic analyses have shown that the ubiquitin-proteasome system involves a large number of proteins in plants, as approximately 5% of the total protein belongs to this pathway. In contrast to the high number of E3 ligases of ubiquitin identified, very few proteins regulated by ubiquitination have been described. To solve this, we have undertaken a new proteomic approach aimed to identify proteins modified with ubiquitin. This is based on affinity purification and identification for ubiquitinated proteins using the ubiquitin binding domain (UBA) polypeptide of the P62 protein attached to agarose beads. This P62-agarose matrix is capable of specifically binding ubiquitinated proteins. These bound proteins were digested with trypsin and the peptides separated by HPLC chromatography, spotted directly onto a MALDI target and analyzed by MALDI-TOF/TOF off-line coupled LC/MALDI-MS/MS. A total of 200 putative ubiquitinated proteins were identified. From these we found that several of the putative targets were already described in plants, as well as in other organisms, as ubiquitinated proteins. In addition, we have found that some of these proteins were indeed modified with ubiquitin in vivo. Taken together, we have shown that this approach is useful for identifying ubiquitinated protein in plants.

COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis

“…One possible RUB function might thus be the protection of CUL1 from erroneous ubiquitination and degradation by blocking a potential ubiquitination site, although this is unlikely because neddylation of human CUL1 is required for CUL1 in vitro autoubiquitination (66). In Arabidopsis, CUL1 monoubiquitination was reported only recently (67). However, this modification did not induce CUL1 degradation under the tested conditions.…”

Unraveling the Ubiquitome of the Human Malaria Parasite

“…Using this filter, we find 73 Plasmodium proteins, ϳ1.3% of the translated genome, that were specifically detected in the ubiquitin immunoprecipitations, 24 of them with low spectral counts. For comparison, previous works using similar technique (same antibody and same denaturing conditions) identified 345 ubiquitin conjugates in human cells (21), ϳ1.7% of the translated genome, and 200 ubiquitin conjugates in A. thaliana (16), ϳ0.8% of the translated genome).…”

“…In human cells, proteins involved in translation/protein synthesis represent 18% of the immunoprecipitated ubiquitin conjugates (21). This value drops down to 4.5% in A. thaliana (16). Proteins involved in transcription represent 4% of the data set (i.e.…”

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

“…In order to find the direct targets of the proteasome we therefore aimed at identifying ubiquitylated proteins directly by affinity chromatography using the p62 ubiquitin-binding domain (UBA) (23,60). Using UBAs from various proteins, either alone or in combination, together with non-denaturing buffer conditions have already identified 1556 different Arabidopsis proteins (22)(23)(24)(25)(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.…”