Plant Sciences Department, Rothamsted Research, Harpenden, AL5 2JQ, UK.
Cambridge Centre for Proteomics, Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, CB2 1QR, UK.
New Phytol. 2018 May;218(3):1106-1126. doi: 10.1111/nph.14909. Epub 2017 Nov 23.
The N-end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking. To investigate the impact of the Arg/N-end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT-TAILS) for relative quantification of N-terminal peptides in prt6, an Arabidopsis thaliana N-end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6). TMT-TAILS identified over 4000 unique N-terminal peptides representing c. 2000 protein groups. Forty-five protein groups exhibited significantly increased N-terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm α-cruciferin was delayed in prt6 seedlings. N-termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII-dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript. We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action.
靶向蛋白降解的 N 端规则途径是植物中多种过程的重要调节剂,但关于其对蛋白质组的影响的详细知识尚缺乏。为了研究 Arg/N 端规则途径对黄化幼苗蛋白质组的影响,我们使用末端胺同位素标记的底物串联质量标签(TMT-TAILS)对拟南芥 N 端规则突变体 prt6 中的 N 端肽进行相对定量,该突变体缺乏 E3 连接酶 PROTEOLYSIS6(PRT6)。TMT-TAILS 鉴定出超过 4000 个独特的 N 端肽,代表约 2000 个蛋白质组。在 prt6 幼苗中,45 个蛋白质组的 N 端肽丰度显著增加,包括 cruciferins,主要的种子贮藏蛋白,它们受 PRT6 的已知底物 Group VII Ethylene Response Factor(ERFVII)转录因子的调控。prt6 幼苗中胚乳α-cruciferin 的动员被延迟。PRT6 下调了几种蛋白酶的 N 端,包括 RD21A。RD21A 的转录物、蛋白质和活性水平在很大程度上依赖于 ERFVII 下调。相比之下,组织蛋白酶 B3 的蛋白质和活性被 ERFVII 独立于转录物上调。我们提出,该途径的 PRT6 分支通过控制 ERFVII 的作用,以复杂的方式调节蛋白酶的活性,并通过控制 ERFVII 的作用优化从种子到幼苗的储存储备动员。