title: Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.
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.
authors.affiliation: Center of Life and Food Sciences Weihenstephan, Lehrstuhl fuer Botanik, Technische Universitaet Muenchen, Emil-Ramann-Str. 4, 85350, Freising, Germany.
authors.forename: Georg
authors.initials: G
authors.lastname: Hierl
authors.forename: Timo
authors.initials: T
authors.lastname: Höwing
authors.forename: Erika
authors.initials: E
authors.lastname: Isono
authors.forename: Friedrich
authors.initials: F
authors.lastname: Lottspeich
authors.forename: Christine
authors.initials: C
authors.lastname: Gietl
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D017360:Arabidopsis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018546:Hypocotyl / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D019441:Plant Epidermis / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D018515:Plant Leaves / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D018517:Plant Roots / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000502:physiology; D030821:Plants, Genetically Modified; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D004792:Enzyme Precursors; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C073762:lysyl-aspartyl-glutamyl-leucine; C000588661:CEP2 protein, Arabidopsis; D003546:Cysteine Endopeptidases