Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots.
Quantitative proteomics analysis of the Arg/N-end rule pathway of targeted degradation in Arabidopsis roots.
复制标题
DOI：
10.1002/pmic.201400530
复制
发表时间：
2015-07
期刊：
影响因子：
3.4
通讯作者：
Theodoulou FL
中科院分区：
文献类型：
--
作者：
Zhang H;Deery MJ;Gannon L;Powers SJ;Lilley KS;Theodoulou FL
根据Arg/N端规则通路，含有碱性N末端的蛋白质是拟南芥E3连接酶PROTEOLYSIS6(PRT6)降解的靶标。精氨酸基转移酶ATe1和2翻译后精氨酸化后，蛋白质也可以成为Prt6底物。在这里，我们对Arg/N-end规则突变体ate1/2和prt6进行了定量蛋白质组学研究，以探讨这一途径对根蛋白质组的影响。串联质量标签标记在突变体中发现了少量丰度增加的蛋白质，其中一些代表转录因子的下游靶标，已知是N-末端规则底物。用样品(尾巴)的末端胺同位素标记结合三重二甲基标记分离N-末端多肽，鉴定出1465个唯一的N-末端。在游离的neo-N末端中，稳定残基过多，但不稳定残基在N-末端规则突变体中没有明显富含。大多数游离的neo-N末端是在细胞器靶向信号被切割后被发现的，因此区隔作用可能是野生型N-末端中存在不稳定残基的部分原因。我们的数据表明，PRT6对拟南芥根的整体蛋白质组没有明显的影响，可能参与了相对较少的调节蛋白的受控降解。所有MS数据都已存储在ProteomeXchange中，标识符为PXD001719(http://proteomecentral.proteomexchange.org/dataset/PXD001719).
According to the Arg/N-end rule pathway, proteins with basic N-termini are targeted for degradation by the Arabidopsis thaliana E3 ligase, PROTEOLYSIS6 (PRT6). Proteins can also become PRT6 substrates following post-translational arginylation by arginyltransferases ATE1 and 2. Here, we undertook a quantitative proteomics study of Arg/N-end rule mutants, ate1/2 and prt6, to investigate the impact of this pathway on the root proteome. Tandem mass tag labelling identified a small number of proteins with increased abundance in the mutants, some of which represent downstream targets of transcription factors known to be N-end rule substrates. Isolation of N-terminal peptides using terminal amine isotope labelling of samples (TAILS) combined with triple dimethyl labelling identified 1465 unique N-termini. Stabilising residues were over-represented among the free neo-N-termini, but destabilising residues were not markedly enriched in N-end rule mutants. The majority of free neo-N-termini were revealed following cleavage of organellar targeting signals, thus compartmentation may account in part for the presence of destabilising residues in the wild-type N-terminome. Our data suggest that PRT6 does not have a marked impact on the global proteome of Arabidopsis roots and is likely involved in the controlled degradation of relatively few regulatory proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD001719 (http://proteomecentral.proteomexchange.org/dataset/PXD001719).