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Research ArticleLARGE-SCALE BIOLOGY ARTICLES
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Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis

Do-Young Kim, Mark Scalf, Lloyd M. Smith, Richard D. Vierstra
Do-Young Kim
Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706
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Mark Scalf
Department of Chemistry,University of Wisconsin, Madison, Wisconsin 53706
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Lloyd M. Smith
Department of Chemistry,University of Wisconsin, Madison, Wisconsin 53706
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Richard D. Vierstra
Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706
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  • For correspondence: vierstra@wisc.edu

Published May 2013. DOI: https://doi.org/10.1105/tpc.112.108613

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  • © 2013 American Society of Plant Biologists. All rights reserved.

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.

  • Glossary

    Ub
    ubiquitin
    DUB
    deubiquitylating enzyme
    MS
    mass spectrometry
    TUBE
    tandem repeated Ub binding entity
    GST
    glutathione S-transferase
    Ni-NTA
    nickel nitrilotriacetic acid
    MS/MS
    tandem MS
    LC
    liquid chromatography
    HCD
    high energy collision
    Col-0
    Columbia-0
    FDR
    false discovery rate
    MAP
    mitogen-activated protein
    PSM
    peptide spectral match
    GO
    gene ontogeny
    PEP
    phosphoenolpyruvate
    BR
    brassinosteroid
    ERAD
    endoplasmic reticulum-associated degradation
    EB
    extraction buffer
    BB
    binding buffer
    UB
    urea buffer
    • Received December 18, 2012.
    • Revised April 11, 2013.
    • Accepted April 18, 2013.
    • Published May 10, 2013.
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    Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis
    Do-Young Kim, Mark Scalf, Lloyd M. Smith, Richard D. Vierstra
    The Plant Cell May 2013, 25 (5) 1523-1540; DOI: 10.1105/tpc.112.108613

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    Advanced Proteomic Analyses Yield a Deep Catalog of Ubiquitylation Targets in Arabidopsis
    Do-Young Kim, Mark Scalf, Lloyd M. Smith, Richard D. Vierstra
    The Plant Cell May 2013, 25 (5) 1523-1540; DOI: 10.1105/tpc.112.108613
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    The Plant Cell Online: 25 (5)
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    Vol. 25, Issue 5
    May 2013
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