Article pubs.acs.org/jpr
Comprehensive Profiling of the Rice Ubiquitome Reveals the Significance of Lysine Ubiquitination in Young Leaves Xin Xie,†,‡ Houxiang Kang,† Wende Liu,*,† and Guo-Liang Wang*,†,§ †
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China ‡ Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China § Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *
ABSTRACT: Protein ubiquitination is a major post-translational modification that regulates development, apoptosis, responses to environmental cues, and other processes in eukaryotes. Although several ubiquitinated proteins have been identified in rice, large-scale profiling of the rice ubiquitome has not been reported because of limitations in the current analytical methods. Here, we report the first rice ubiquitome, determined by combining highly sensitive immune affinity purification and high resolution LC−MS/MS. We identified 861 di-Gly-Lys-containing peptides in 464 proteins in rice leaf cells. Bioinformatic analyses of the ubiquitome identified a variety of cellular functions and diverse subcellular localizations for the ubiquitinated proteins, and also revealed seven putative ubiquitination motifs in rice. Proteins related to binding and catalytic activity were predicted to be the preferential targets of lysine ubiquitination. A protein interaction network and KEGG analysis indicated that a wide range of signaling and metabolic pathways are modulated by protein ubiquitination in rice. Our results demonstrate the usefulness of the significantly improved method for assaying proteome-wide ubiquitination in plants. The identification of the 464 ubiquitinated proteins in rice leaves provides a foundation for the analysis of the physiological roles of these ubiquitination-related proteins. KEYWORDS: post-translational modification, lysine ubiquitination, proteomics, mass spectrometry, Oryza sativa
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INTRODUCTION Ubiquitin encodes a 76-amino-acid protein that is highly conserved among different eukaryotic organisms. The ubiquitin−proteasome system (UPS) is important for the selective degradation of proteins in eukaryotic cells and is composed of three main enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3).1,2 Many studies have shown that ubiquitination is also important in various other cellular processes, such as DNA damage repair, DNA replication, receptor endocytosis, and innate immune signaling.3,4 The identification of post-translational modification (PTM) sites is key to determining the functional roles of the modified proteins. Recent advances in mass spectrometry (MS)-based proteomics and in the high affinity purification of ubiquitinated peptides have enabled proteome-wide surveys of ubiquitination-mediated modifications.5 After the ubiquitinated proteins underwent proteolytic digestion with trypsin, the diglycine (diGly) remnant derived from the two C-terminal glycine residues of ubiquitin remained covalently linked to the modified lysines. Thus, the antibody recognizing the di-Gly remnant on lysine residues enables the affinity capture of ubiquitinated peptides.6 © XXXX American Chemical Society
The di-Gly modification causes a mass shift (114.0429 Da) of the parent peptide, which enables the identification and precise location of ubiquitination sites on the basis of peptide fragment masses. Large-scale ubiquitination site mapping by MS has been performed for both yeast and human cells. For instance, more than 100 ubiquitination sites were identified in yeast,7 and over 1000 ubiquitination sites were mapped in human proteins in four screens.8−11 Although many ubiquitinated proteins were identified in these studies, only a relatively small number of ubiquitination sites were mapped because the methods used require the enrichment of ubiquitinated proteins. Using newly developed, robust ubiquitination site identification methods based on di-Gly-Lys-specific antibody enrichment, researchers recently performed several proteome-wide in-depth ubiquitination analyses and identified a large number of ubiquitination sites in mammalian cells.6,9,12 However, the adaptability of this method to plants has not been tested. Received: September 13, 2014
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DOI: 10.1021/pr5009724 J. Proteome Res. XXXX, XXX, XXX−XXX
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Journal of Proteome Research Tryptic Digestion
Rice (Oryza sativa) is the model organism for monocot plant research and the staple food for about half of the world’s population. The resequencing of the many cultivated and wild rice lines and the deep transcriptome and proteomic analyses of the rice genome in recent years have made rice the most extensively annotated crop plant at both the DNA and the protein levels.13 Elucidation of the ubiquitome in rice cells is important for understanding the role of the UPS in regulating development and stress responses. Lysine ubiquitination plays an important role in plant physiology.14 Although numerous studies have implicated UPS regulation in various processes in rice,14 the proteome-wide identification of lysine-ubiquitinated proteins has not been accomplished in rice. The main limitation in defining plant ubiquitomes by MS is the lack of affinity methods that can efficiently enrich ubiquitin conjugates under stringent conditions. Although several researchers have studied the ubiquitome in Arabidopsis,15−18 the proteome-wide ubiquitination data are still lacking for other plant species. However, as increasing numbers of plant species are investigated with LC−MS-based proteomics, we expect that the catalog of ubiquitinated plant proteins will greatly expand. In this study, we performed a global profiling of the ubiquitome of young rice leaves using integrated proteomic techniques. We developed a streamlined approach in which di-Gly-Lyscontaining peptides are directly enriched from trypsin-digested whole rice cell protein mixtures with a commercially available, high affinity anti-di-Gly-Lys-specific antibody. By integrating highly sensitive MS and bioinformatics tools, we profiled lysine ubiquitination in rice in depth. Using this method, we identified a total of 861 di-Gly-Lys-containing peptides from 464 ubiquitinated proteins in rice seedlings. These ubiquitinated proteins regulate a wide range of processes, including cellular transport, signal transduction, proteolysis, metabolism, and response to stress stimuli. The described method enables the extensive quantification of endogenous ubiquitinated proteins in rice. Overall, this proteomic study illustrates the breadth of rice processes affected by ubiquitination and provides a general strategy for identifying the ubiquitome more effectively in other plant species.
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A 20 mg quantity of proteins was treated with 1.25 mM dithiothreitol (DTT) for 30 min at 55 °C to reduce disulfides. The resulting free cysteines were alkylated using 10 mM iodoacetamide for 15 min at room temperature in the dark. About 20 mg of DTT and iodoacetamide-treated proteins were digested overnight at 37 °C with TPCK trypsin (Worthington) at an enzyme-to-substrate ratio of 1:50 after a 4-fold dilution in 20 mM HEPES (pH 8.0). Trypsin digestion was stopped by the addition of trifluoroacetic acid to a final concentration of 1%. After the precipitates were removed by centrifugation for 20 min at 10000g, the supernatants were desalted using Sep-Pak Classic C18 cartridges (Waters) followed by lyophilization. Enrichment of Ubiquitin-Remnant-Containing Peptides
Lyophilized peptides were dissolved in immunoaffinity purification (IAP) buffer (50 mM MOPS−NaOH, pH 7.2, 10 mM Na2HPO4, and 50 mM NaCl) and spun at 10000g at 4 °C for 10 min. For each sample, 250 μg of di-Gly-Lys antibody cross-linked on agarose beads was used (PTMScan ubiquitin remnant motif K-ε-GG kit, Cell Signaling Technology) and diGly-Lys-containing peptides were enriched as previously described.12,20 Mass Spectrometry Data Acquisition
The treated samples were separated at a flow rate of 300 nL/ min with a gradient (0−8 min, 4−8% solvent B; 8−85 min, 8− 35% solvent B; 85−95 min, 35−50% solvent B; 95−102 min, 50−95% solvent B) on the UltiMate 3000 RSLC nano UHPLC system (Dionex Corporation Germering, Germany), which was directly interfaced with a Thermo Scientific LTQ-Orbitrap Velos mass spectrometer. The analytical column was purchased from Agilent (75 μm ID, 150 mm length). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The LTQ-Orbitrap mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur software (version 2.2). A single full-scan mass spectrum in the Orbitrap (350−2000 m/z, 60 000 resolution) was followed by 10 data-dependent MS/MS scans in the linear trap quadrupole (LTQ) mass analyzer using collision-induced dissociation (CID), in which the precursor mass selection window was set at 2 Da and the normalized collision energy for CID was 27 eV.
EXPERIMENTAL PROCEDURES
Rice Planting and Isolation of Samples
Plants of the japonica cultivar Nipponbare were used in this study.19 Rice seeds were surface-sterilized in 75% ethanol for 1 min and then surface-sterilized again in 40% sodium hypochlorite for 30 min. After they were washed with sterile water three times, the seeds were transferred into one-halfstrength Murashige and Skoog medium. One week later, the seedlings were transplanted into soil in a greenhouse at 25 °C. The leaves of three-week-old rice seedlings (three independent rice seedlings samples) were collected for the protein extraction described in the next section.
Database Search
The proteins and ubiquitination sites were identified with the Andromeda search engine in Max Quant (version 1.3.0.5). The tandem MS data obtained were searched against the MSU Rice Genome Database (version 6, released on May 6, 2009, downloaded from http://rice.plantbiology.msu.edu/index. shtml), which contains 67 393 annotated proteins, and then concatenated with a reverse decoy database and the protein sequences of common contaminants. The precursor mass tolerance was set at 6 ppm, and the MS/MS tolerance was set at 0.5 Da. Trypsin/P was specified as the enzyme, and two missed cleavages were permitted. During the database search, the modifications were set as follows: the fixed modification was carbamidomethylation for cysteines, and the variable modifications were Gly−Gly modification for lysines and oxidation for methionines. The false discovery rate (FDR) thresholds for proteins, peptides, and modification sites were set at 0.01. The minimum peptide length was set at 5, and a MaxQuant score was set at ≥20.
Protein Extraction
Rice leaves were ground in liquid nitrogen and resuspended in urea lysis buffer (20 mM HEPES, pH 8.0, 8 M urea, 1 mM each of sodium orthovanadate and sodium β-glycerophosphate, and 2.5 mM sodium pyrophosphate). Lysates were sonicated at 50 W output with two bursts of 10 s each; the lysates were cooled on ice for 1 min between bursts. Following lysis, tissues were centrifuged at 20000g for 20 min at 4 °C to remove insoluble material. The protein concentration of the cleared lysates was measured using the BCA Protein Assay Kit (CWBIO). B
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Figure 1. Schematic diagram of a strategy for the global identification of ubiquitinated peptides in rice.
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Bioinformatic Analysis
The Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/ ).21 The lysine ubiquitination (Kub) peptide ID was converted to a UniProt ID and then mapped to a GO ID. The Kub proteins were then further classified by GO annotation based on three categories: biological processes, molecular functions, and cellular components. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathways.22 The KEGG online service tool KAAS was used to annotate the proteins’ KEGG database description. The annotation results were mapped on the KEGG pathway database using the KEGG online service tool KEGG Mapper. The domain annotation was performed with InterProScan on the InterPro domain database via web-based interfaces and services. WoLF PSORT was used for predicting the subcellular localization.23 The CORUM database was used to annotate protein complexes. Motif-X software was used to analyze the model of the sequences with amino acids in specific positions of ubiquityl15-mers (seven amino acids upstream and downstream of the ubiquitination site) in all of the protein sequences. In addition, the IPI Arabidopsis proteome was used as the background database, and the other parameters were set to the default values. The setting parameters for searching motifs using MotifX software was “occurrences 20” and “the Bonferroni corrected P = 0.005”. Protein−protein interaction networks were analyzed with the IntAct database (http://www.ebi.ac.uk/intact/). The protein− protein interaction network map was generated with the Cytoscape software.24
RESULTS AND DISCUSSION
Integrated Strategy for Ubiquitin Proteomics in Rice
To identify lysine-ubiquitinated peptides in rice, we developed a new method that combines the highly specific enrichment of lysine-ubiquitinated peptides with highly sensitive Orbitrap mass spectrometry and bioinformatics tools. As shown in Figure 1, this approach has four steps: (1) the extraction and trypsin digestion of the whole protein lysate of rice seedling leaves; (2) the immune enrichment of lysine-ubiquitinated peptides using a di-Gly-Lys-specific monoclonal antibody; (3) the fractionation of immunoenriched peptides, followed by the analysis of the identified protein complex using a high resolution hybrid linear ion-trap/Orbitrap mass spectrometer (LTQ-OrbitrapVelos); and (4) the identification of lysine ubiquitination peptides by a database search and the characterization of the protein function, metabolic pathway, subcellular localization, ubiquitination motif, and protein interaction networks. We first isolated the total protein from 3-week-old Nipponbare seedlings. Using a new method described above, we identified 861 di-Gly-Lys-containing peptides in rice seedlings (Table S1 in the Supporting Information). The 861 di-Gly-Lys-containing peptides were mapped to 464 unique proteins, in which between 1 and 15 putative ubiquitination sites were identified (Figure 2 and Table S1 in the Supporting Information). About 62% of the proteins contained a single putative ubiquitination site. Because the trypsin proteolysis of proteins modified by NEDD8 and ISG15 generates a di-Gly remnant on modified lysines that is identical to that caused by ubiquitin modification, these modifications cannot be distinguished by MS.6 However, NEDD8-mediated modification primarily targets cullin subunits C
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Supporting Information).The functional characterization of the ubiquitinated proteins was further assigned according to the GO term annotation.21 Of the 413 GO-annotated ubiquitinated proteins, 295 are cellular process proteins, 290 are metabolic process proteins, 145 are proteins that respond to stimuli, and 96 proteins are involved in single-organism processes (Figure 3A). For the 413 ubiquitinated proteins grouped by their molecular functions, 229 have binding activity (Figure 3B), suggesting that proteins involved in DNA transcription or protein interaction are subject to massive ubiquitination in rice. The second largest group of GO-annotated ubiquitinated proteins consists of 196 proteins associated with catalytic activity (Figure 3B), indicating that enzymatic proteins are also subject to abundant ubiquitination in rice. The GO analysis of the rice ubiquitome demonstrates that ubiquitinated proteins are involved in a wide range of biological processes and have different molecular functions in rice. Most proteins are localized to specific subcellular compartments that are related to their functions in the cell. To obtain an overview of the distribution of ubiquitinated proteins in rice cells, we assigned the ubiquitinated proteins to six categories: (1) cell, (2) organelle, (3) membrane, (4) macromolecular complex, (5) membrane-enclosed lumen, and (6) extracellular region. GO analysis showed that 326 of the proteins are located in the cell (cell wall and cell envelope), 211 are located in the organelles, and 174 are localized at the membrane (Figure 3C).
Figure 2. Distribution of the ubiquitinated proteins based on their number of putative ubiquitination sites.
of cullin−RING E3 ubiquitin ligases in plants,25 and both NEDDylation and ISGylation are very rare compared to ubiquitination.26 Thus, most di-Gly remnants derived from cellular peptides are derived from ubiquitinated proteins. Therefore, in this study we refer to all di-Gly modified lysines as “ubiquitination sites”, even though a small portion of these sites might be derived from an NEDD8 or an ISG15 modification. Protein Ubiquitination Regulation of Diverse Cellular Processes in Rice
To investigate the cellular function of the ubiquitinated proteins in rice, we classified the identified proteins into different groups according to biological processes, molecular functions, and cellular components (Figure 3 and Table S2 in
Figure 3. Gene ontology functional characterization of the identified ubiquitinated proteins. (A) Distribution of the ubiquitinated proteins in terms of biological processes. (B) Distribution of the ubiquitinated proteins in terms of molecular functions. (C) Distribution of the ubiquitinated proteins in terms of cellular components. D
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Journal of Proteome Research Analysis of Ubiquitinated Lysine Motifs
interactions mediated by ubiquitinated proteins in rice. As shown in Figure 5, the network consists of a complex interconnected web with a number of ubiquitinated proteins present at key hubs, some of them with functions in a variety of cellular processes (Figure 5 and Table S4 in the Supporting Information). The most abundant subnetwork is involved in calcium sensing mediated by calmodulins and calcineurin B-like proteins (Figure 5A). In calcium sensing, the ubiquitinated calmodulin protein CAM2 interacts with the ubiquitinated calcineurin B protein CBL2. In addition, numerous WRKY transcription factors interact with CAM2, and many kinases interact with CBL2 (CIPKs). It will be interesting to determine whether those WRKY and CIPK proteins are subjected to ubiquitination during calcium signaling. Other dense protein interaction networks were mediated by 14-3-3 proteins (Figure 5B,C and Table S4 in the Supporting Information). The protein/protein interaction results suggested that lysine ubiquitination is relatively active in multiple protein complexes and signal transduction pathways in rice.
Unlike phosphorylation or acetylation sites, ubiquitination sites lack the preference for specific amino acid residues at particular positions surrounding the ubiquitinated lysine in human cells, indicating that conserved motifs might not exist in humans.8,12 The failure to identify a conserved definition of the ubiquitination site motifs, however, might be due to the use of an overly strict parameter setting during the analysis by the Motif-X program in the previous studies. Alternatively, different organisms, such as mammals and plants, may have different sequence preferences in the ubiquitination sites. To further determine the nature of the ubiquitinated lysines in rice, we used the Motif-X program to compare the position-specific frequencies of the amino acid residues surrounding ubiquitinated lysine residues with those of all lysine residues that occur in the rice proteome. Of the 861 di-Gly-Lys-containing peptides, 825 had seven or more amino acids N- and Cterminally surrounding the ubiquitinated lysine. We identified a total of seven conserved motifs for 543 unique sites, which accounted for about 61% of the sites identified (Figure 4 and
Metabolic Pathways of Ubiquitinated Proteins in Rice
Lysine ubiquitination is important in metabolic regulation.15 Although many ubiquitinated proteins have been identified in eukaryotes, a set of ubiquitinated metabolic enzymes have yet to be identified in rice. To gain insight into the ubiquitinationmediated metabolic processes in rice, we conducted a KEGG pathway analysis. The results showed that 234 ubiquitinated proteins are involved in 195 metabolic pathways (Table S5 in the Supporting Information). Below, we discussed in detail the involvement of ubiquitinated proteins in two of these metabolic pathways: glycolysis/gluconeogenesis and carbon fixation in photosynthetic organisms (Figure 6 and Figure S1 in the Supporting Information). We identified 10 ubiquitinated enzymes associated with glycolysis/gluconeogenesis (Figure 6). These enzymes play an important role in starch and sucrose metabolisms, which involve the pentose phosphate pathway and carbon fixation in photosynthetic organisms. Previous studies have shown that proteins involved in glycolysis are regulated by certain stressors. For instance, several reports revealed that fructokinase 2 is significantly upregulated when plants are under salt stress, indicating that this enzyme may have a role in responding to abiotic stresses in rice.27 Many studies also suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fructokinase 2 are vital for salt tolerance in tomato, soybean, and rice.28 Furthermore, fructose-bisphosphate aldolases are slightly upregulated under salt stress in wheat.29 The abundance of several proteins involved in the glycolysis pathway such as phosphoglucomutase and pyruvate dehydrogenase is also significantly changed in response to salt stress in rice.28 These studies revealed a close relationship between salt tolerance and the expression of glycolysis-related proteins in rice. The activation of glycolysis may protect rice seedlings from salt stress damage by supplying needed energy. Growing evidence suggests that ubiquitination regulates the flux of carbon through central metabolism.30 Our analysis showed that proteins participating in the carbon fixation pathway in photosynthesis are also highly enriched in the rice ubiquitome (Figure S1 in the Supporting Information). Via photosynthesis, plants use the energy from sunlight to convert carbon dioxide into organic compounds. Plants contain the pigment chlorophyll, which fixes carbon autotrophically through the Calvin cycle. To our knowledge, ubiquitination
Figure 4. Ubiquitinated lysine motifs in rice. (A) Ubiquitination motifs and the conservation of ubiquitination sites. The height of each letter corresponds to the frequency of that amino acid residue in that position. The central K refers to the ubiquitinated lysine. (B) The number of identified peptides containing ubiquitinated lysine in each motif.
Table S3 in the Supporting Information). These seven unique sites were named AAXXXXKub, AXXXXXKubXA, AXXXKub, AKub, EKub, KubXXA, and AXKub (Figure 4A), and they exhibit different abundances (Figure 4B) (Kub indicates the ubiquitinated lysine, and X indicates any amino acid). A survey of these motifs revealed that only two distinct residues are found upstream or downstream of the ubiquitinated lysine (Figure 4A), including neutral alanine (A) and acidic glutamic acid (E). Protein Interaction Networks for the Rice Ubiquitome
To better understand how ubiquitination might affect rice physiology and development, we generated a protein−protein interaction network for all of the ubiquitinated proteins and other rice proteins in the IntAct database (http://www.ebi.ac. uk/intact/) and we depicted this network with Cytoscape.24 The resulting complete network provides insight into the E
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Figure 5. Interaction networks of ubiquitinated proteins associated with calcium sensors (A) and 14-3-3 proteins (B,C). Ubiquitinated proteins are highlighted in yellow.
metabolism. A number of PEPC and PEPC isoforms have been found in the ubiquitome catalog of Arabidopsis, harsh hakea, lily, and castor oil plants.15,31−33 For instance, three paralogs of PEPC protein were identified in Arabidopsis using a stringent two-step affinity ubiquitinated-protein-conjugate purification method.15 RcPpc3, a plant-type PEPC encoded by Ricinus communis, is a monoubiquitinated protein in germinated castor oil seeds.32 Moreover, Lys-628 of RcPpc3 was proven to be the monoubiquitination site through the tandem mass spectrometry sequencing of a diglycinated tryptic peptide.32 Sequence alignment revealed that the Lys-628 of RcPpc3 is conserved in Arabidopsis and Oryza sativa PEPC paralogs (Figure S2 in the Supporting Information), indicating that this residue might be conserved in various plant PEPCs and is proximal to a PEP binding, catalytic domain. These
of the enzymes that are involved in this pathway has not been previously reported in rice. We identified nine ubiquitinated enzymes that are involved in photosynthesis: fructose-bisphosphate aldolase, glycolaldehyde transferase, fructose-1,6bisphosphatase, triose-phosphate isomerase (TPI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribulose-bisphosphate carboxylase, phosphoenolpyruvate carboxylase (PEPC), pyruvate-phosphate dikinase, and malate dehydrogenase. Some of these proteins are known to be associated with important biological processes. For instance, GAPDH has been implicated in the salt stress response in rice revealed in several rice proteomics studies.28 It will be interesting to assess the rice ubiquitome in response to a variety of abiotic stresses to gain in-depth insights into the function of ubiquitination in regulating metabolic pathways. PEPC is a tightly regulated enzyme that plays an important role in plant carbon F
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Figure 6. Ubiquitinated proteins in representative metabolic pathways in terms of glycolysis/gluconeogenesis in the rice cultivar Nipponbare. Ubiquitinated proteins are highlighted in green.
often requires selective affinity enrichment. In this study, we combined the high affinity enrichment of ubiquitinated peptides, highly sensitive mass spectrometry, and bioinformatics tools to analyze the rice ubiquitome. Using this integrated approach, we identified 861 peptides with ubiquitinated lysines in 464 proteins from rice seedling leaves, and thus we established a large data set of endogenous protein ubiquitination in rice. In addition, our characterization of ubiquitinated proteins indicates that the ubiquitome may play
results suggest that the modification of PEPC by ubiquitination might also be important for carbon metabolism in rice.
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CONCLUSIONS Although protein modifications are important in the regulation of various cellular processes, ubiquitination and most other modifications are not abundant. Therefore, the detection of protein modifications is a challenge for proteomics studies. Detection of such low levels of modified proteins or peptides G
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an important role in different biological processes, protein− protein interactions, and metabolic pathways. Our study demonstrates that the improved approach is useful for identifying ubiquitinated proteins on a large scale. Our results also reveal that many different proteins involved in diverse biological processes are ubiquitinated in rice plants. To obtain a comprehensive ubiquitination landscape in the model crop rice, we intend to study mutants of ubiquitination-related genes as well as ubiquitination in a variety of rice tissues under different biotic or abiotic stresses.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1. Ubiquitinated proteins in representative metabolic pathways in terms of carbon fixation in photosynthetic organisms in rice cultivar Nipponbare. Figure S2. Alignment of the four rice PEPCs amino acid sequences with castor oil plant RcPpc3 and Arabidopsis AtPPC1, 2, 3. Table S1. Identified ubiquitinated proteins and their functional annotation. Table S2. GO annotation of ubiquitinated proteins in rice. Table S3. Motif analysis of ubiquitinated proteins in rice. Table S4. Interaction networks of ubiquitinated proteins in rice. Table S5. KEGG metabolic pathways of ubiquitinated proteins in rice. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*W.L. E-mail: [email protected]. Tel: +86-10-62817045. Fax: +86-10-62817045. *G.-L.W. E-mail: [email protected]. Tel: +1-614-292 1375. Fax: + 1-614-292-4455. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Jan E. Leach and Lindsay Triplett at Colorado State University for their critical reading of this manuscript. We also thank Guoqiang Xu at Soochow University and Haiteng Deng at Tsinghua University for helpful discussions. This work was given support from the National Natural Science Foundation of China (31272034) to W.L., the 973 Project (2012CB114005) of the Ministry of Science and Technology China to G.-L.W, and the National Transgenic Crop Initiative (2012ZX08009001) to G.-L.W.
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REFERENCES
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Journal of Proteome Research (26) Kerscher, O.; Felberbaum, R.; Hochstrasser, M. Modification of proteins by ubiquitin and ubiquitin-like proteins. Annu. Rev. Cell Dev. Biol. 2006, 22, 159−180. (27) Sarhadi, E.; Bazargani, M. M.; Sajise, A. G.; Abdolahi, S.; Vispo, N. A.; Arceta, M.; Nejad, G. M.; Singh, R. K.; Salekdeh, G. H. Proteomic analysis of rice anthers under salt stress. Plant Physiol. Biochem. 2012, 58, 280−287. (28) Liu, C. W.; Chang, T. S.; Hsu, Y. K.; Wang, A. Z.; Yen, H. C.; Wu, Y. P.; Wang, C. S.; Lai, C. C. Comparative proteomic analysis of early salt stress-responsive proteins in roots and leaves of rice. Proteomics 2014, 14 (15), 1759−1775. (29) Kamal, A. H.; Cho, K.; Kim, D. E.; Uozumi, N.; Chung, K. Y.; Lee, S. Y.; Choi, J. S.; Cho, S. W.; Shin, C. S.; Woo, S. H. Changes in physiology and protein abundance in salt-stressed wheat chloroplasts. Mol. Biol. Rep. 2012, 39 (9), 9059−9074. (30) Sato, T.; Maekawa, S.; Yasuda, S.; Yamaguchi, J. Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system. Plant Signaling Behav. 2011, 6 (10), 1465−1468. (31) Shane, M. W.; Fedosejevs, E. T.; Plaxton, W. C. Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol. 2013, 161 (4), 1634−1644. (32) Uhrig, R. G.; She, Y. M.; Leach, C. A.; Plaxton, W. C. Regulatory monoubiquitination of phosphoenolpyruvate carboxylase in germinating castor oil seeds. J. Biol. Chem. 2008, 283 (44), 29650−29657. (33) Igawa, T.; Fujiwara, M.; Tanaka, I.; Fukao, Y.; Yanagawa, Y. Characterization of bacterial-type phosphoenolpyruvate carboxylase expressed in male gametophyte of higher plants. BMC Plant Biol. 2010, 10, 200.
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Comprehensive Profiling of the Rice Ubiquitome Reveals the Significance of Lysine Ubiquitination in Young Leaves Xin Xie,†,‡ Houxiang Kang,† Wende Liu,*,† and Guo-Liang Wang*,†,§ †
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China ‡ Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China § Department of Plant Pathology, The Ohio State University, Columbus, Ohio 43210, United States S Supporting Information *
ABSTRACT: Protein ubiquitination is a major post-translational modification that regulates development, apoptosis, responses to environmental cues, and other processes in eukaryotes. Although several ubiquitinated proteins have been identified in rice, large-scale profiling of the rice ubiquitome has not been reported because of limitations in the current analytical methods. Here, we report the first rice ubiquitome, determined by combining highly sensitive immune affinity purification and high resolution LC−MS/MS. We identified 861 di-Gly-Lys-containing peptides in 464 proteins in rice leaf cells. Bioinformatic analyses of the ubiquitome identified a variety of cellular functions and diverse subcellular localizations for the ubiquitinated proteins, and also revealed seven putative ubiquitination motifs in rice. Proteins related to binding and catalytic activity were predicted to be the preferential targets of lysine ubiquitination. A protein interaction network and KEGG analysis indicated that a wide range of signaling and metabolic pathways are modulated by protein ubiquitination in rice. Our results demonstrate the usefulness of the significantly improved method for assaying proteome-wide ubiquitination in plants. The identification of the 464 ubiquitinated proteins in rice leaves provides a foundation for the analysis of the physiological roles of these ubiquitination-related proteins. KEYWORDS: post-translational modification, lysine ubiquitination, proteomics, mass spectrometry, Oryza sativa
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INTRODUCTION Ubiquitin encodes a 76-amino-acid protein that is highly conserved among different eukaryotic organisms. The ubiquitin−proteasome system (UPS) is important for the selective degradation of proteins in eukaryotic cells and is composed of three main enzymes: ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3).1,2 Many studies have shown that ubiquitination is also important in various other cellular processes, such as DNA damage repair, DNA replication, receptor endocytosis, and innate immune signaling.3,4 The identification of post-translational modification (PTM) sites is key to determining the functional roles of the modified proteins. Recent advances in mass spectrometry (MS)-based proteomics and in the high affinity purification of ubiquitinated peptides have enabled proteome-wide surveys of ubiquitination-mediated modifications.5 After the ubiquitinated proteins underwent proteolytic digestion with trypsin, the diglycine (diGly) remnant derived from the two C-terminal glycine residues of ubiquitin remained covalently linked to the modified lysines. Thus, the antibody recognizing the di-Gly remnant on lysine residues enables the affinity capture of ubiquitinated peptides.6 © XXXX American Chemical Society
The di-Gly modification causes a mass shift (114.0429 Da) of the parent peptide, which enables the identification and precise location of ubiquitination sites on the basis of peptide fragment masses. Large-scale ubiquitination site mapping by MS has been performed for both yeast and human cells. For instance, more than 100 ubiquitination sites were identified in yeast,7 and over 1000 ubiquitination sites were mapped in human proteins in four screens.8−11 Although many ubiquitinated proteins were identified in these studies, only a relatively small number of ubiquitination sites were mapped because the methods used require the enrichment of ubiquitinated proteins. Using newly developed, robust ubiquitination site identification methods based on di-Gly-Lys-specific antibody enrichment, researchers recently performed several proteome-wide in-depth ubiquitination analyses and identified a large number of ubiquitination sites in mammalian cells.6,9,12 However, the adaptability of this method to plants has not been tested. Received: September 13, 2014
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Journal of Proteome Research Tryptic Digestion
Rice (Oryza sativa) is the model organism for monocot plant research and the staple food for about half of the world’s population. The resequencing of the many cultivated and wild rice lines and the deep transcriptome and proteomic analyses of the rice genome in recent years have made rice the most extensively annotated crop plant at both the DNA and the protein levels.13 Elucidation of the ubiquitome in rice cells is important for understanding the role of the UPS in regulating development and stress responses. Lysine ubiquitination plays an important role in plant physiology.14 Although numerous studies have implicated UPS regulation in various processes in rice,14 the proteome-wide identification of lysine-ubiquitinated proteins has not been accomplished in rice. The main limitation in defining plant ubiquitomes by MS is the lack of affinity methods that can efficiently enrich ubiquitin conjugates under stringent conditions. Although several researchers have studied the ubiquitome in Arabidopsis,15−18 the proteome-wide ubiquitination data are still lacking for other plant species. However, as increasing numbers of plant species are investigated with LC−MS-based proteomics, we expect that the catalog of ubiquitinated plant proteins will greatly expand. In this study, we performed a global profiling of the ubiquitome of young rice leaves using integrated proteomic techniques. We developed a streamlined approach in which di-Gly-Lyscontaining peptides are directly enriched from trypsin-digested whole rice cell protein mixtures with a commercially available, high affinity anti-di-Gly-Lys-specific antibody. By integrating highly sensitive MS and bioinformatics tools, we profiled lysine ubiquitination in rice in depth. Using this method, we identified a total of 861 di-Gly-Lys-containing peptides from 464 ubiquitinated proteins in rice seedlings. These ubiquitinated proteins regulate a wide range of processes, including cellular transport, signal transduction, proteolysis, metabolism, and response to stress stimuli. The described method enables the extensive quantification of endogenous ubiquitinated proteins in rice. Overall, this proteomic study illustrates the breadth of rice processes affected by ubiquitination and provides a general strategy for identifying the ubiquitome more effectively in other plant species.
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A 20 mg quantity of proteins was treated with 1.25 mM dithiothreitol (DTT) for 30 min at 55 °C to reduce disulfides. The resulting free cysteines were alkylated using 10 mM iodoacetamide for 15 min at room temperature in the dark. About 20 mg of DTT and iodoacetamide-treated proteins were digested overnight at 37 °C with TPCK trypsin (Worthington) at an enzyme-to-substrate ratio of 1:50 after a 4-fold dilution in 20 mM HEPES (pH 8.0). Trypsin digestion was stopped by the addition of trifluoroacetic acid to a final concentration of 1%. After the precipitates were removed by centrifugation for 20 min at 10000g, the supernatants were desalted using Sep-Pak Classic C18 cartridges (Waters) followed by lyophilization. Enrichment of Ubiquitin-Remnant-Containing Peptides
Lyophilized peptides were dissolved in immunoaffinity purification (IAP) buffer (50 mM MOPS−NaOH, pH 7.2, 10 mM Na2HPO4, and 50 mM NaCl) and spun at 10000g at 4 °C for 10 min. For each sample, 250 μg of di-Gly-Lys antibody cross-linked on agarose beads was used (PTMScan ubiquitin remnant motif K-ε-GG kit, Cell Signaling Technology) and diGly-Lys-containing peptides were enriched as previously described.12,20 Mass Spectrometry Data Acquisition
The treated samples were separated at a flow rate of 300 nL/ min with a gradient (0−8 min, 4−8% solvent B; 8−85 min, 8− 35% solvent B; 85−95 min, 35−50% solvent B; 95−102 min, 50−95% solvent B) on the UltiMate 3000 RSLC nano UHPLC system (Dionex Corporation Germering, Germany), which was directly interfaced with a Thermo Scientific LTQ-Orbitrap Velos mass spectrometer. The analytical column was purchased from Agilent (75 μm ID, 150 mm length). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The LTQ-Orbitrap mass spectrometer was operated in the data-dependent acquisition mode using the Xcalibur software (version 2.2). A single full-scan mass spectrum in the Orbitrap (350−2000 m/z, 60 000 resolution) was followed by 10 data-dependent MS/MS scans in the linear trap quadrupole (LTQ) mass analyzer using collision-induced dissociation (CID), in which the precursor mass selection window was set at 2 Da and the normalized collision energy for CID was 27 eV.
EXPERIMENTAL PROCEDURES
Rice Planting and Isolation of Samples
Plants of the japonica cultivar Nipponbare were used in this study.19 Rice seeds were surface-sterilized in 75% ethanol for 1 min and then surface-sterilized again in 40% sodium hypochlorite for 30 min. After they were washed with sterile water three times, the seeds were transferred into one-halfstrength Murashige and Skoog medium. One week later, the seedlings were transplanted into soil in a greenhouse at 25 °C. The leaves of three-week-old rice seedlings (three independent rice seedlings samples) were collected for the protein extraction described in the next section.
Database Search
The proteins and ubiquitination sites were identified with the Andromeda search engine in Max Quant (version 1.3.0.5). The tandem MS data obtained were searched against the MSU Rice Genome Database (version 6, released on May 6, 2009, downloaded from http://rice.plantbiology.msu.edu/index. shtml), which contains 67 393 annotated proteins, and then concatenated with a reverse decoy database and the protein sequences of common contaminants. The precursor mass tolerance was set at 6 ppm, and the MS/MS tolerance was set at 0.5 Da. Trypsin/P was specified as the enzyme, and two missed cleavages were permitted. During the database search, the modifications were set as follows: the fixed modification was carbamidomethylation for cysteines, and the variable modifications were Gly−Gly modification for lysines and oxidation for methionines. The false discovery rate (FDR) thresholds for proteins, peptides, and modification sites were set at 0.01. The minimum peptide length was set at 5, and a MaxQuant score was set at ≥20.
Protein Extraction
Rice leaves were ground in liquid nitrogen and resuspended in urea lysis buffer (20 mM HEPES, pH 8.0, 8 M urea, 1 mM each of sodium orthovanadate and sodium β-glycerophosphate, and 2.5 mM sodium pyrophosphate). Lysates were sonicated at 50 W output with two bursts of 10 s each; the lysates were cooled on ice for 1 min between bursts. Following lysis, tissues were centrifuged at 20000g for 20 min at 4 °C to remove insoluble material. The protein concentration of the cleared lysates was measured using the BCA Protein Assay Kit (CWBIO). B
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Figure 1. Schematic diagram of a strategy for the global identification of ubiquitinated peptides in rice.
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Bioinformatic Analysis
The Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/ ).21 The lysine ubiquitination (Kub) peptide ID was converted to a UniProt ID and then mapped to a GO ID. The Kub proteins were then further classified by GO annotation based on three categories: biological processes, molecular functions, and cellular components. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathways.22 The KEGG online service tool KAAS was used to annotate the proteins’ KEGG database description. The annotation results were mapped on the KEGG pathway database using the KEGG online service tool KEGG Mapper. The domain annotation was performed with InterProScan on the InterPro domain database via web-based interfaces and services. WoLF PSORT was used for predicting the subcellular localization.23 The CORUM database was used to annotate protein complexes. Motif-X software was used to analyze the model of the sequences with amino acids in specific positions of ubiquityl15-mers (seven amino acids upstream and downstream of the ubiquitination site) in all of the protein sequences. In addition, the IPI Arabidopsis proteome was used as the background database, and the other parameters were set to the default values. The setting parameters for searching motifs using MotifX software was “occurrences 20” and “the Bonferroni corrected P = 0.005”. Protein−protein interaction networks were analyzed with the IntAct database (http://www.ebi.ac.uk/intact/). The protein− protein interaction network map was generated with the Cytoscape software.24
RESULTS AND DISCUSSION
Integrated Strategy for Ubiquitin Proteomics in Rice
To identify lysine-ubiquitinated peptides in rice, we developed a new method that combines the highly specific enrichment of lysine-ubiquitinated peptides with highly sensitive Orbitrap mass spectrometry and bioinformatics tools. As shown in Figure 1, this approach has four steps: (1) the extraction and trypsin digestion of the whole protein lysate of rice seedling leaves; (2) the immune enrichment of lysine-ubiquitinated peptides using a di-Gly-Lys-specific monoclonal antibody; (3) the fractionation of immunoenriched peptides, followed by the analysis of the identified protein complex using a high resolution hybrid linear ion-trap/Orbitrap mass spectrometer (LTQ-OrbitrapVelos); and (4) the identification of lysine ubiquitination peptides by a database search and the characterization of the protein function, metabolic pathway, subcellular localization, ubiquitination motif, and protein interaction networks. We first isolated the total protein from 3-week-old Nipponbare seedlings. Using a new method described above, we identified 861 di-Gly-Lys-containing peptides in rice seedlings (Table S1 in the Supporting Information). The 861 di-Gly-Lys-containing peptides were mapped to 464 unique proteins, in which between 1 and 15 putative ubiquitination sites were identified (Figure 2 and Table S1 in the Supporting Information). About 62% of the proteins contained a single putative ubiquitination site. Because the trypsin proteolysis of proteins modified by NEDD8 and ISG15 generates a di-Gly remnant on modified lysines that is identical to that caused by ubiquitin modification, these modifications cannot be distinguished by MS.6 However, NEDD8-mediated modification primarily targets cullin subunits C
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Supporting Information).The functional characterization of the ubiquitinated proteins was further assigned according to the GO term annotation.21 Of the 413 GO-annotated ubiquitinated proteins, 295 are cellular process proteins, 290 are metabolic process proteins, 145 are proteins that respond to stimuli, and 96 proteins are involved in single-organism processes (Figure 3A). For the 413 ubiquitinated proteins grouped by their molecular functions, 229 have binding activity (Figure 3B), suggesting that proteins involved in DNA transcription or protein interaction are subject to massive ubiquitination in rice. The second largest group of GO-annotated ubiquitinated proteins consists of 196 proteins associated with catalytic activity (Figure 3B), indicating that enzymatic proteins are also subject to abundant ubiquitination in rice. The GO analysis of the rice ubiquitome demonstrates that ubiquitinated proteins are involved in a wide range of biological processes and have different molecular functions in rice. Most proteins are localized to specific subcellular compartments that are related to their functions in the cell. To obtain an overview of the distribution of ubiquitinated proteins in rice cells, we assigned the ubiquitinated proteins to six categories: (1) cell, (2) organelle, (3) membrane, (4) macromolecular complex, (5) membrane-enclosed lumen, and (6) extracellular region. GO analysis showed that 326 of the proteins are located in the cell (cell wall and cell envelope), 211 are located in the organelles, and 174 are localized at the membrane (Figure 3C).
Figure 2. Distribution of the ubiquitinated proteins based on their number of putative ubiquitination sites.
of cullin−RING E3 ubiquitin ligases in plants,25 and both NEDDylation and ISGylation are very rare compared to ubiquitination.26 Thus, most di-Gly remnants derived from cellular peptides are derived from ubiquitinated proteins. Therefore, in this study we refer to all di-Gly modified lysines as “ubiquitination sites”, even though a small portion of these sites might be derived from an NEDD8 or an ISG15 modification. Protein Ubiquitination Regulation of Diverse Cellular Processes in Rice
To investigate the cellular function of the ubiquitinated proteins in rice, we classified the identified proteins into different groups according to biological processes, molecular functions, and cellular components (Figure 3 and Table S2 in
Figure 3. Gene ontology functional characterization of the identified ubiquitinated proteins. (A) Distribution of the ubiquitinated proteins in terms of biological processes. (B) Distribution of the ubiquitinated proteins in terms of molecular functions. (C) Distribution of the ubiquitinated proteins in terms of cellular components. D
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Journal of Proteome Research Analysis of Ubiquitinated Lysine Motifs
interactions mediated by ubiquitinated proteins in rice. As shown in Figure 5, the network consists of a complex interconnected web with a number of ubiquitinated proteins present at key hubs, some of them with functions in a variety of cellular processes (Figure 5 and Table S4 in the Supporting Information). The most abundant subnetwork is involved in calcium sensing mediated by calmodulins and calcineurin B-like proteins (Figure 5A). In calcium sensing, the ubiquitinated calmodulin protein CAM2 interacts with the ubiquitinated calcineurin B protein CBL2. In addition, numerous WRKY transcription factors interact with CAM2, and many kinases interact with CBL2 (CIPKs). It will be interesting to determine whether those WRKY and CIPK proteins are subjected to ubiquitination during calcium signaling. Other dense protein interaction networks were mediated by 14-3-3 proteins (Figure 5B,C and Table S4 in the Supporting Information). The protein/protein interaction results suggested that lysine ubiquitination is relatively active in multiple protein complexes and signal transduction pathways in rice.
Unlike phosphorylation or acetylation sites, ubiquitination sites lack the preference for specific amino acid residues at particular positions surrounding the ubiquitinated lysine in human cells, indicating that conserved motifs might not exist in humans.8,12 The failure to identify a conserved definition of the ubiquitination site motifs, however, might be due to the use of an overly strict parameter setting during the analysis by the Motif-X program in the previous studies. Alternatively, different organisms, such as mammals and plants, may have different sequence preferences in the ubiquitination sites. To further determine the nature of the ubiquitinated lysines in rice, we used the Motif-X program to compare the position-specific frequencies of the amino acid residues surrounding ubiquitinated lysine residues with those of all lysine residues that occur in the rice proteome. Of the 861 di-Gly-Lys-containing peptides, 825 had seven or more amino acids N- and Cterminally surrounding the ubiquitinated lysine. We identified a total of seven conserved motifs for 543 unique sites, which accounted for about 61% of the sites identified (Figure 4 and
Metabolic Pathways of Ubiquitinated Proteins in Rice
Lysine ubiquitination is important in metabolic regulation.15 Although many ubiquitinated proteins have been identified in eukaryotes, a set of ubiquitinated metabolic enzymes have yet to be identified in rice. To gain insight into the ubiquitinationmediated metabolic processes in rice, we conducted a KEGG pathway analysis. The results showed that 234 ubiquitinated proteins are involved in 195 metabolic pathways (Table S5 in the Supporting Information). Below, we discussed in detail the involvement of ubiquitinated proteins in two of these metabolic pathways: glycolysis/gluconeogenesis and carbon fixation in photosynthetic organisms (Figure 6 and Figure S1 in the Supporting Information). We identified 10 ubiquitinated enzymes associated with glycolysis/gluconeogenesis (Figure 6). These enzymes play an important role in starch and sucrose metabolisms, which involve the pentose phosphate pathway and carbon fixation in photosynthetic organisms. Previous studies have shown that proteins involved in glycolysis are regulated by certain stressors. For instance, several reports revealed that fructokinase 2 is significantly upregulated when plants are under salt stress, indicating that this enzyme may have a role in responding to abiotic stresses in rice.27 Many studies also suggested that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fructokinase 2 are vital for salt tolerance in tomato, soybean, and rice.28 Furthermore, fructose-bisphosphate aldolases are slightly upregulated under salt stress in wheat.29 The abundance of several proteins involved in the glycolysis pathway such as phosphoglucomutase and pyruvate dehydrogenase is also significantly changed in response to salt stress in rice.28 These studies revealed a close relationship between salt tolerance and the expression of glycolysis-related proteins in rice. The activation of glycolysis may protect rice seedlings from salt stress damage by supplying needed energy. Growing evidence suggests that ubiquitination regulates the flux of carbon through central metabolism.30 Our analysis showed that proteins participating in the carbon fixation pathway in photosynthesis are also highly enriched in the rice ubiquitome (Figure S1 in the Supporting Information). Via photosynthesis, plants use the energy from sunlight to convert carbon dioxide into organic compounds. Plants contain the pigment chlorophyll, which fixes carbon autotrophically through the Calvin cycle. To our knowledge, ubiquitination
Figure 4. Ubiquitinated lysine motifs in rice. (A) Ubiquitination motifs and the conservation of ubiquitination sites. The height of each letter corresponds to the frequency of that amino acid residue in that position. The central K refers to the ubiquitinated lysine. (B) The number of identified peptides containing ubiquitinated lysine in each motif.
Table S3 in the Supporting Information). These seven unique sites were named AAXXXXKub, AXXXXXKubXA, AXXXKub, AKub, EKub, KubXXA, and AXKub (Figure 4A), and they exhibit different abundances (Figure 4B) (Kub indicates the ubiquitinated lysine, and X indicates any amino acid). A survey of these motifs revealed that only two distinct residues are found upstream or downstream of the ubiquitinated lysine (Figure 4A), including neutral alanine (A) and acidic glutamic acid (E). Protein Interaction Networks for the Rice Ubiquitome
To better understand how ubiquitination might affect rice physiology and development, we generated a protein−protein interaction network for all of the ubiquitinated proteins and other rice proteins in the IntAct database (http://www.ebi.ac. uk/intact/) and we depicted this network with Cytoscape.24 The resulting complete network provides insight into the E
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Figure 5. Interaction networks of ubiquitinated proteins associated with calcium sensors (A) and 14-3-3 proteins (B,C). Ubiquitinated proteins are highlighted in yellow.
metabolism. A number of PEPC and PEPC isoforms have been found in the ubiquitome catalog of Arabidopsis, harsh hakea, lily, and castor oil plants.15,31−33 For instance, three paralogs of PEPC protein were identified in Arabidopsis using a stringent two-step affinity ubiquitinated-protein-conjugate purification method.15 RcPpc3, a plant-type PEPC encoded by Ricinus communis, is a monoubiquitinated protein in germinated castor oil seeds.32 Moreover, Lys-628 of RcPpc3 was proven to be the monoubiquitination site through the tandem mass spectrometry sequencing of a diglycinated tryptic peptide.32 Sequence alignment revealed that the Lys-628 of RcPpc3 is conserved in Arabidopsis and Oryza sativa PEPC paralogs (Figure S2 in the Supporting Information), indicating that this residue might be conserved in various plant PEPCs and is proximal to a PEP binding, catalytic domain. These
of the enzymes that are involved in this pathway has not been previously reported in rice. We identified nine ubiquitinated enzymes that are involved in photosynthesis: fructose-bisphosphate aldolase, glycolaldehyde transferase, fructose-1,6bisphosphatase, triose-phosphate isomerase (TPI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribulose-bisphosphate carboxylase, phosphoenolpyruvate carboxylase (PEPC), pyruvate-phosphate dikinase, and malate dehydrogenase. Some of these proteins are known to be associated with important biological processes. For instance, GAPDH has been implicated in the salt stress response in rice revealed in several rice proteomics studies.28 It will be interesting to assess the rice ubiquitome in response to a variety of abiotic stresses to gain in-depth insights into the function of ubiquitination in regulating metabolic pathways. PEPC is a tightly regulated enzyme that plays an important role in plant carbon F
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Figure 6. Ubiquitinated proteins in representative metabolic pathways in terms of glycolysis/gluconeogenesis in the rice cultivar Nipponbare. Ubiquitinated proteins are highlighted in green.
often requires selective affinity enrichment. In this study, we combined the high affinity enrichment of ubiquitinated peptides, highly sensitive mass spectrometry, and bioinformatics tools to analyze the rice ubiquitome. Using this integrated approach, we identified 861 peptides with ubiquitinated lysines in 464 proteins from rice seedling leaves, and thus we established a large data set of endogenous protein ubiquitination in rice. In addition, our characterization of ubiquitinated proteins indicates that the ubiquitome may play
results suggest that the modification of PEPC by ubiquitination might also be important for carbon metabolism in rice.
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CONCLUSIONS Although protein modifications are important in the regulation of various cellular processes, ubiquitination and most other modifications are not abundant. Therefore, the detection of protein modifications is a challenge for proteomics studies. Detection of such low levels of modified proteins or peptides G
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an important role in different biological processes, protein− protein interactions, and metabolic pathways. Our study demonstrates that the improved approach is useful for identifying ubiquitinated proteins on a large scale. Our results also reveal that many different proteins involved in diverse biological processes are ubiquitinated in rice plants. To obtain a comprehensive ubiquitination landscape in the model crop rice, we intend to study mutants of ubiquitination-related genes as well as ubiquitination in a variety of rice tissues under different biotic or abiotic stresses.
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1. Ubiquitinated proteins in representative metabolic pathways in terms of carbon fixation in photosynthetic organisms in rice cultivar Nipponbare. Figure S2. Alignment of the four rice PEPCs amino acid sequences with castor oil plant RcPpc3 and Arabidopsis AtPPC1, 2, 3. Table S1. Identified ubiquitinated proteins and their functional annotation. Table S2. GO annotation of ubiquitinated proteins in rice. Table S3. Motif analysis of ubiquitinated proteins in rice. Table S4. Interaction networks of ubiquitinated proteins in rice. Table S5. KEGG metabolic pathways of ubiquitinated proteins in rice. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*W.L. E-mail: [email protected]. Tel: +86-10-62817045. Fax: +86-10-62817045. *G.-L.W. E-mail: [email protected]. Tel: +1-614-292 1375. Fax: + 1-614-292-4455. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Jan E. Leach and Lindsay Triplett at Colorado State University for their critical reading of this manuscript. We also thank Guoqiang Xu at Soochow University and Haiteng Deng at Tsinghua University for helpful discussions. This work was given support from the National Natural Science Foundation of China (31272034) to W.L., the 973 Project (2012CB114005) of the Ministry of Science and Technology China to G.-L.W, and the National Transgenic Crop Initiative (2012ZX08009001) to G.-L.W.
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