RRC ID 70153
著者 Hierl G, Höwing T, Isono E, Lottspeich F, Gietl C.
タイトル Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.
ジャーナル Plant Mol Biol
Abstract 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.
巻・号 84(6)
ページ 605-20
公開日 2014-4-1
DOI 10.1007/s11103-013-0157-6
PMID 24287716
PMC PMC3950626
MeSH Apoptosis / physiology* Arabidopsis / cytology Arabidopsis / enzymology* Arabidopsis / genetics Arabidopsis / physiology Arabidopsis Proteins / genetics Arabidopsis Proteins / metabolism* Cell Wall / metabolism Cysteine Endopeptidases / chemistry Cysteine Endopeptidases / genetics Cysteine Endopeptidases / metabolism* Endoplasmic Reticulum / enzymology* Enzyme Activation Enzyme Precursors / chemistry Enzyme Precursors / genetics Enzyme Precursors / metabolism* Hydrogen-Ion Concentration Hypocotyl / cytology Hypocotyl / enzymology Hypocotyl / genetics Hypocotyl / physiology Oligopeptides / genetics Oligopeptides / metabolism* Organ Specificity Plant Epidermis / cytology Plant Epidermis / enzymology Plant Epidermis / genetics Plant Epidermis / physiology Plant Leaves / cytology Plant Leaves / enzymology Plant Leaves / genetics Plant Leaves / physiology Plant Roots / cytology Plant Roots / enzymology Plant Roots / genetics Plant Roots / physiology Plants, Genetically Modified Protein Sorting Signals / genetics Protein Transport Recombinant Fusion Proteins Sequence Deletion Substrate Specificity
IF 3.302
リソース情報
シロイヌナズナ / 植物培養細胞・遺伝子 pst08738 pst12055