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

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.