Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.
Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.
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DOI：
10.1007/s11103-013-0157-6
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发表时间：
2014-04
影响因子：
5.1
通讯作者：
Gietl, Christine
中科院分区：
文献类型：
--
作者：
Hierl, Georg;Hoewing, Timo;Isono, Erika;Lottspeich, Friedrich;Gietl, Christine
蓖麻蛋白体是内质网衍生的细胞器，储存与程序性细胞死亡（PCD）相关的KDEL-tailed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个KDEL-CysEP（AtCEP 1、AtCEP 2和AtCEP 3），其在经历PCD的营养组织和生殖组织中差异表达。这些拟南芥蛋白酶还没有在生化水平上的特点，也没有本地化细胞内。在这项研究中，我们表征了AtCEP 2。包括在内源启动子控制下表达的前肽和KDEL靶向序列的3xHA-mCherry-AtCEP 2基因融合体使我们能够“离体”分离AtCEP 2。纯化的蛋白质被证明是在pH依赖性的方式激活。然而，活化后，蛋白酶活性是pH无关的。底物特异性的分析表明，AtCEP 2接受脯氨酸附近的切割位点，这是一个罕见的功能，具体的KDEL-CysEPs。mCherry-AtCEP 2在叶、下胚轴和根的表皮层中检测到;在根中，主要在伸长区和根冠中发现。与ER膜标记物的共定位显示，mCherry-AtCEP 2储存在两种不同类型的ER衍生的细胞器中：10 μm长的纺锤形细胞器以及直径约1 μm的圆形囊泡。长的细胞器似乎是内质网体，这是发现专门在大肠杆菌。圆形囊泡与首次在蓖麻子中描述的蓖麻蛋白体非常相似。本研究首次证明了蓖麻蛋白体在拟南芥中的存在，并可能在PCD和发育组织重塑领域开辟新的研究途径。本文的在线版本（doi：10.1007/s11103-013-0157-6）包含补充材料，可供授权用户使用。
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. The online version of this article (doi:10.1007/s11103-013-0157-6) contains supplementary material, which is available to authorized users.
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影响因子：
7.2
作者：
Matsushima, R;Kondo, M;Hara-Nishimura, I
通讯作者：
Hara-Nishimura, I
DOI：
10.1073/pnas.0711980105
发表时间：
2008-02-12
影响因子：
11.1
作者：
Cannon, Maura C.;Terneus, Kimberly;Kieliszewski, Marcia J.
通讯作者：
Kieliszewski, Marcia J.
DOI：
10.1073/pnas.97.7.3718
发表时间：
2000-03-28
影响因子：
11.1
作者：
Cutler, SR;Ehrhardt, DW;Somerville, CR
通讯作者：
Somerville, CR
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF
影响因子：
2.7
作者：
Berger, F;Linstead, P;Haseloff, J
通讯作者：
Haseloff, J