The Plant Chemetics Lab, Chemical Genomics Centre of the Max Planck Society, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
PLoS One. 2012;7(3):e32422. doi: 10.1371/journal.pone.0032422. Epub 2012 Mar 2.
RD21-like proteases are ubiquitous, plant-specific papain-like proteases typified by carrying a C-terminal granulin domain. RD21-like proteases are involved in immunity and associated with senescence and various types of biotic and abiotic stresses. Here, we interrogated Arabidopsis RD21 regulation and trafficking by site-directed mutagenesis, agroinfiltration, western blotting, protease activity profiling and protein degradation. Using an introduced N-glycan sensor, deglycosylation experiments and glyco-engineered N. benthamiana plants, we show that RD21 passes through the Golgi where it becomes fucosylated. Our studies demonstrate that RD21 is regulated at three post-translational levels. Prodomain removal is not blocked in the catalytic Cys mutant, indicating that RD21 is activated by a proteolytic cascade. However, RD21 activation in Arabidopsis does not require vacuolar processing enzymes (VPEs) or aleurain-like protease AALP. In contrast, granulin domain removal requires the catalytic Cys and His residues and is therefore autocatalytic. Furthermore, SDS can (re-)activate latent RD21 in Arabidopsis leaf extracts, indicating the existence of a third layer of post-translational regulation, possibly mediated by endogenous inhibitors. RD21 causes a dominant protease activity in Arabidopsis leaf extracts, responsible for SDS-induced proteome degradation.
RD21 样蛋白酶是普遍存在的、植物特异性的组织蛋白酶样蛋白酶，其特征是携带 C 末端颗粒素结构域。RD21 样蛋白酶参与免疫反应，并与衰老和各种生物及非生物胁迫有关。在这里，我们通过定点突变、农杆菌浸润、western blot、蛋白酶活性分析和蛋白质降解等方法研究了拟南芥 RD21 的调控和运输。利用引入的 N-糖传感器、去糖基化实验和糖基工程化的 N. benthamiana 植物，我们表明 RD21 通过高尔基体，在那里它被岩藻糖基化。我们的研究表明，RD21 在三个翻译后水平受到调控。催化半胱氨酸突变体中没有阻止前导肽的去除，这表明 RD21 通过蛋白水解级联被激活。然而，拟南芥中 RD21 的激活不需要液泡加工酶（VPEs）或类 Aleurain 蛋白酶 AALP。相比之下，颗粒素结构域的去除需要催化半胱氨酸和组氨酸残基，因此是自催化的。此外，SDS 可以在拟南芥叶片提取物中重新激活潜伏的 RD21，这表明存在第三层翻译后调控，可能由内源性抑制剂介导。RD21 在拟南芥叶片提取物中引起显性蛋白酶活性，负责 SDS 诱导的蛋白质组降解。
