Enrichment and analysis of rice seedling ubiquitin-related proteins using four UBA domains (GST-qUBAs)

• Published in: Plant science (Limerick) Vol. 229; pp. 172 - 180
• Main Authors: Meng, Qingshi, Rao, Liqun, Pan, Yinghong
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier B.V 01.12.2014
• Subjects: Affinity enrichment; Amino Acid Sequence; Chromatography, Affinity; Glutathione Transferase - metabolism; Mass spectrometry; Molecular Sequence Data; Oryza - metabolism; Oryza sativa; Plant Proteins - chemistry; Plant Proteins - metabolism; Polyubiquitin - metabolism; Protein Processing, Post-Translational; Protein Structure, Tertiary; Recombinant Fusion Proteins - metabolism; Seedlings - metabolism; Ubiquitin; Ubiquitin - chemistry; Ubiquitin - metabolism; Ubiquitin ligases; Ubiquitin-associated domain; Ubiquitin-Protein Ligases - metabolism; Ubiquitinated Proteins - isolation & purification; Ubiquitin; DUBs; CAA; E3s; E1s; E2s; IAA; HCD; Ubiquitin ligases; Oryza sativa; Ubiquitin-associated domain; UBA; PMSF; FDR; UBD; MIU; UIM; IPTG; Affinity enrichment; GST-qUBAs; Mass spectrometry
• ISSN: 0168-9452; 1873-2259
• DOI: 10.1016/j.plantsci.2014.09.002

•Found a UBA domain to high-efficiently interacting with polyubiquitin chain.•Developed two-step method for enriching ubiquitinated proteins.•The enrichment efficiency of ubiquitinated protein increased more than three times.•170 ubiquitin-related proteins were identified from rice seedling. 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.