Comprehensive Profiling of the Rice Ubiquitome Reveals the Significance of Lysine Ubiquitination in Young Leaves

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.

蛋白质泛素化（protein ubiquitination）是真核生物（eukaryotes）中调控发育、细胞凋亡、环境应答及其他生理过程的关键翻译后修饰（post-translational modification）。尽管目前已在水稻中鉴定出多种泛素化蛋白（ubiquitinated proteins），但受限于当前分析方法的不足，水稻泛素化组（ubiquitome）的大规模全景分析尚未见报道。本研究结合高灵敏度免疫亲和纯化（immune affinity purification）与高分辨液相色谱-串联质谱（LC–MS/MS）技术，首次完成了水稻泛素化组的全面鉴定。我们在水稻叶片细胞的464个蛋白中，共识别出861条含二甘氨酸-赖氨酸（di-Gly-Lys）的肽段。对该泛素化组的生物信息学分析显示，这些泛素化蛋白具备多样的细胞功能与亚细胞定位（subcellular localizations），并在水稻中发现了7种潜在的泛素化基序（ubiquitination motifs）。具备结合活性与催化活性的蛋白被预测为赖氨酸泛素化的优先作用靶标。蛋白质相互作用网络与京都基因与基因组百科全书（KEGG）分析表明，蛋白质泛素化可调控水稻内广泛的信号传导与代谢通路。本研究结果证实了经显著优化的方法可有效用于植物全蛋白质组泛素化修饰的检测。水稻叶片中464个泛素化蛋白的鉴定，为后续解析这类泛素化相关蛋白的生理功能奠定了坚实基础。