title: Enrichment and analysis of rice seedling ubiquitin-related proteins using four UBA domains (GST-qUBAs).
abstract: Protein ubiquitination is a common posttranslational modification that often occurs on lysine residues. It controls the half-life, interaction and trafficking of intracellular proteins and is involved in different plant development stages and responses to environment stresses. Four Ubiquitin-Associated (UBA) domains were sequentially fused with Glutathione S-transferase (GST) tag (GST-qUBA) as bait protein in this study. A two-step affinity protocol was successfully developed and the identification of ubiquitinated proteins and their interaction proteins increased almost threefold compared to methods that directly identify ubiquitinated proteins from crude samples. A total of 170 ubiquitin-related proteins were identified in GST-qUBAs enriched samples taken from rice seedlings. There were 134 ubiquitinated proteins, 5 ubiquitin-activating enzymes (E1s), 5 ubiquitin-conjugating enzymes (E2s), 19 ubiquitin ligases (E3s) and 7 deubiquitinating enzymes (DUBs), which all contained various key factors that regulated a wide range of biological processes. Moreover, a series of novel ubiquitinated proteins and E3s were identified that had not been previously reported. This study investigated a high-efficiency method for identifying novel ubiquitinated proteins involved in biological processes and a primary mapping of the ubiquitylome during rice seedling development, which could extend our understanding of how ubiquitin modification regulates plant proteins, pathways and cellular processes.
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China; The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Science, Beijing 100081, China; Institute of Crop Science, Chinese Academy of Agricultural Science, Beijing 100081, China.
authors.forename: Qingshi
authors.initials: Q
authors.lastname: Meng
authors.affiliation: College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China. Electronic address: raoliqun@163.com.
authors.forename: Liqun
authors.initials: L
authors.lastname: Rao
authors.affiliation: The National Key Facility for Crop Gene Resources and Genetic Improvement, Chinese Academy of Agricultural Science, Beijing 100081, China; Institute of Crop Science, Chinese Academy of Agricultural Science, Beijing 100081, China. Electronic address: panyinghong@caas.cn.
authors.forename: Yinghong
authors.initials: Y
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D005982:Glutathione Transferase / Q000378:metabolism*; D008969:Molecular Sequence Data; D012275:Oryza / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D025821:Polyubiquitin / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D036226:Seedlings / Q000378:metabolism*; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D057149:Ubiquitinated Proteins / Q000302:isolation & purification
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D057149:Ubiquitinated Proteins; D025821:Polyubiquitin; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase
keywords: Affinity enrichment; Mass spectrometry; Oryza sativa; Ubiquitin; Ubiquitin ligases; Ubiquitin-associated domain