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

被引:210
作者
Kim, Do-Young [1 ]
Scalf, Mark [2 ]
Smith, Lloyd M. [2 ]
Vierstra, Richard D. [1 ]
机构
[1] Univ Wisconsin, Dept Genet, Madison, WI 53706 USA
[2] Univ Wisconsin, Dept Chem, Madison, WI 53706 USA
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
UBIQUITIN-PROTEASOME SYSTEM; 26S PROTEASOME; PHYTOCHROME DEGRADATION; SPOROPHYTE DEVELOPMENT; AFFINITY PURIFICATION; SUBCELLULAR LOCATION; NITRATE REDUCTASE; AUTOPHAGY PATHWAY; DIVERSE ARRAY; PROTEINS;
D O I
10.1105/tpc.112.108613
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:1523 / 1540
页数:18
相关论文
共 77 条
[51]   Protein domain-domain interactions and requirements for the negative regulation of Arabidopsis CDC48/p97 by the plant ubiquitin regulatory X (UBX) domain-containing protein, PUX1 [J].
Paro, Sookhee ;
Rancour, David M. ;
Bednarek, Sebastian Y. .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2007, 282 (08) :5217-5224
[52]   A proteomics approach to understanding protein ubiquitination [J].
Peng, JM ;
Schwartz, D ;
Elias, JE ;
Thoreen, CC ;
Cheng, DM ;
Marsischky, G ;
Roelofs, J ;
Finley, D ;
Gygi, SP .
NATURE BIOTECHNOLOGY, 2003, 21 (08) :921-926
[53]   Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A [J].
Raasi, S ;
Orlov, I ;
Fleming, KG ;
Pickart, CM .
JOURNAL OF MOLECULAR BIOLOGY, 2004, 341 (05) :1367-1379
[54]  
Rohrbough James G, 2006, J Biomol Tech, V17, P327
[55]   The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes [J].
Ruepp, A ;
Zollner, A ;
Maier, D ;
Albermann, K ;
Hani, J ;
Mokrejs, M ;
Tetko, I ;
Güldener, U ;
Mannhaupt, G ;
Münsterkötter, M ;
Mewes, HW .
NUCLEIC ACIDS RESEARCH, 2004, 32 (18) :5539-5545
[56]   The ubiquitin-proteasome system regulates plant hormone signaling [J].
Santner, Aaron ;
Estelle, Mark .
PLANT JOURNAL, 2010, 61 (06) :1029-1040
[57]   Tandem affinity purification and mass spectrometric analysis of ubiquitylated proteins in Arabidopsis [J].
Saracco, Scott A. ;
Hansson, Maria ;
Scalf, Mark ;
Walker, Joseph M. ;
Smith, Lloyd M. ;
Vierstra, Richard D. .
PLANT JOURNAL, 2009, 59 (02) :344-358
[58]   RED LIGHT-INDUCED FORMATION OF UBIQUITIN-PHYTOCHROME CONJUGATES - IDENTIFICATION OF POSSIBLE INTERMEDIATES OF PHYTOCHROME DEGRADATION [J].
SHANKLIN, J ;
JABBEN, M ;
VIERSTRA, RD .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (02) :359-363
[59]   The E3 ligase AtCHIP ubiquitylates FtsH1, a component of the chloroplast FtsH protease, and affects protein degradation in chloroplasts [J].
Shen, Guoxin ;
Adam, Zach ;
Zhang, Hong .
PLANT JOURNAL, 2007, 52 (02) :309-321
[60]   Ubiquitylation of an ERAD Substrate Occurs on Multiple Types of Amino Acids [J].
Shimizu, Yuichiro ;
Okuda-Shimizu, Yuki ;
Hendershot, Linda M. .
MOLECULAR CELL, 2010, 40 (06) :917-926