Center of Life and Food Sciences Weihenstephan, Lehrstuhl fuer Botanik, Technische Universitaet Muenchen, Emil-Ramann-Str. 4, 85350, Freising, Germany.
Plant Mol Biol. 2014 Apr;84(6):605-20. doi: 10.1007/s11103-013-0157-6. Epub 2013 Nov 28.
Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 "ex vivo". The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.
瑞西诺体是一种特殊的内质网衍生细胞器,它储存与程序性细胞死亡(PCD)相关的 KDEL 尾部半胱氨酸内肽酶(KDEL-CysEP)的无活性前体形式。拟南芥基因组编码三种 KDEL-CysEP(AtCEP1、AtCEP2 和 AtCEP3),它们在经历 PCD 的营养组织和生殖组织中差异表达。这些拟南芥蛋白酶尚未在生化水平上进行表征,也没有在细胞内进行定位。在这项研究中,我们对 AtCEP2 进行了表征。一个包含前肽和 KDEL 靶向序列的 3xHA-mCherry-AtCEP2 基因融合体,在内源启动子的控制下表达,使我们能够“离体”分离出 AtCEP2。纯化的蛋白被证明以 pH 依赖性方式激活。然而,在激活后,蛋白酶活性与 pH 无关。底物特异性分析表明,AtCEP2 接受靠近切割位点的脯氨酸,这是 KDEL-CysEPs 的一个罕见特征。mCherry-AtCEP2 被检测到在叶片、下胚轴和根的表皮层中;在根中,它主要存在于伸长区和根冠中。与内质网膜标记物的共定位表明,mCherry-AtCEP2 储存在两种不同类型的内质网衍生细胞器中:10 μm 长的纺锤形细胞器以及直径约为 1 μm 的圆形小泡。长细胞器似乎是内质体,它仅存在于十字花科植物中。圆形小泡非常类似于首次在蓖麻中描述的瑞西诺体。这项研究首次提供了瑞西诺体在拟南芥中存在的证据,并可能为 PCD 和发育组织重塑领域的研究开辟新的途径。