Multidimensional protein identification technology (MudPIT) analysis of ubiquitinated proteins in plants.
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.
Maor
Rudy
R
The Sainsbury Laboratory, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom.
Jones
Alex
A
Nühse
Thomas S
TS
Studholme
David J
DJ
Peck
Scott C
SC
Shirasu
Ken
K
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D018744:DNA, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin