Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Rudy Maor,Alex Jones,Thomas S Nühse,David J Studholme,Scott C Peck,Ken Shirasu

doi:10.1074/mcp.m600408-mcp200

Abstract

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

Highlights

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells
A common problem encountered when enriching ubiquitinated proteins is the presence of highly active deubiquitinating enzymes [23]
Given the high sequence conservation of Ub, we investigated whether the Ub-interacting motif (UIM) and ISO fusion proteins can be used to enrich for ubiquitinated proteins from other plant species

Summary

EXPERIMENTAL PROCEDURES

Plant Material—An Arabidopsis (ecotype Landsberg erecta) cell suspension line [28] was used to extract proteins. Proteins were extracted using 10 ml of 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5% glycerol, and 1% Triton X-100 (TBSGT) supplemented with one tablet of Complete EDTA-free protease inhibitor mixture (Roche Applied Science), 10 mM iodoacetamide, 1 ␮g/ml DNase I, and 10 ␮g/ml RNase. Samples were resolved from the peptide trap and loaded onto the SCX part of the biphasic column using a 40-min gradient of 0 – 40% buffer B (0.1% formic acid in HPLC-grade acetonitrile) followed by a 10-min gradient of 40 –98% buffer B. Cell lysates were cleared by centrifuging twice at 3,500 rpm for 10 min each, and recombinant proteins were immunoprecipitated by the Anti-HA Affinity Matrix (Roche Applied Science).

RESULTS

Ubiquitination sites identifieda

DISCUSSION

Peptide sequence

Exonuclease family protein

Lysine residue