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: Oryza sativa; lysine ubiquitination; mass spectrometry; post-translational modification; proteomics.