RESUMEN
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.
Asunto(s)
Apoptosis/fisiología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cisteína Endopeptidasas/metabolismo , Retículo Endoplásmico/enzimología , Precursores Enzimáticos/metabolismo , Oligopéptidos/metabolismo , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Pared Celular/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Activación Enzimática , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Concentración de Iones de Hidrógeno , Hipocótilo/citología , Hipocótilo/enzimología , Hipocótilo/genética , Hipocótilo/fisiología , Oligopéptidos/genética , Especificidad de Órganos , Epidermis de la Planta/citología , Epidermis de la Planta/enzimología , Epidermis de la Planta/genética , Epidermis de la Planta/fisiología , Hojas de la Planta/citología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Eliminación de Secuencia , Especificidad por SustratoRESUMEN
Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca(2+)-removal and towards the dimer upon Ca(2+)-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca(2+)/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Peroxisomas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Calmodulina/metabolismo , Cromosomas Artificiales de Levadura/genética , Dimerización , Proteínas Sensoras del Calcio Intracelular/genética , Proteínas Sensoras del Calcio Intracelular/metabolismo , Proteínas Sensoras del Calcio Intracelular/fisiología , Péptido Hidrolasas/metabolismo , Filogenia , Proteínas Recombinantes , Alineación de Secuencia , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiologíaRESUMEN
Programmed cell death (PCD) in plants is a prerequisite for development as well as seed and fruit production. It also plays a significant role in pathogen defense. A unique group of papain-type cysteine endopeptidases, characterized by a C-terminal endoplasmic reticulum (ER) retention signal (KDEL CysEP), is involved in plant PCD. Genes for these endopeptidases have been sequenced and analyzed from 25 angiosperms and gymnosperms. They have no structural relationship to caspases involved in mammalian PCD and homologs to this group of plant cysteine endopeptidases have not been found in mammals or yeast. In castor beans (Ricinus communis), the CysEP is synthesized as pre-pro-enzyme. The pro-enzyme is transported to the cytosol of cells undergoing PCD in ER-derived vesicles called ricinosomes. These vesicles release the mature CysEP in the final stages of organelle disintegration triggered by acidification of the cytoplasm resulting from the disruption of the vacuole. Mature CysEP digests the hydroxyproline (Hyp)-rich proteins (extensins) that form the basic scaffold of the plant cell wall. The KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated Hyp residues of the extensins. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2 and AtCEP3) are expressed in tissues undergoing PCD. In transgenic Arabidopsis plants expressing ß-glucuronidase under the control of the promoters for these three genes, cell- and tissue-specific activities were mapped during seedling, flower and seed development. KDEL CysEPs participate in the collapse of tissues in the final stage of PCD and in tissue re-modeling such as lateral root formation.
Asunto(s)
Arabidopsis/citología , Arabidopsis/enzimología , Muerte Celular , Cisteína Endopeptidasas/metabolismo , Ricinus/citología , Ricinus/enzimología , Ácidos/metabolismo , Arabidopsis/crecimiento & desarrollo , Pared Celular/metabolismo , Retículo Endoplásmico/enzimología , Endospermo/crecimiento & desarrollo , Endospermo/metabolismo , Glicoproteínas/metabolismo , Células Vegetales/enzimología , Proteínas de Plantas/metabolismo , Proteolisis , Ricinus/crecimiento & desarrollo , Semillas/enzimología , Semillas/crecimiento & desarrollo , Especificidad por Sustrato , Vacuolas/metabolismoRESUMEN
The integral peroxisomal membrane proteins PEX10, PEX2, and PEX12 contain a zinc RING finger close to the C terminus. Loss of function of these peroxins causes embryo lethality at the heart stage in Arabidopsis. Preventing the coordination of Zn(2+) ions by amino acid substitutions in PEX10, PEX2, and PEX12 and overexpressing the resulting conditional sublethal mutations in WT uncovered additional functions of PEX10. Plants overexpressing DeltaZn-mutant PEX10 display deformed peroxisomal shapes causing diminished contact with chloroplasts and possibly with mitochondria. These changes correlated with impaired metabolite transfer and, at high CO(2), recoverable defective photorespiration plus dwarfish phenotype. The N-terminal PEX10 domain is critical for peroxisome biogenesis and plant development. A point mutation in the highly conserved TLGEEY motif results in vermiform peroxisome shape without impairing organelle contact. Addition of an N-terminal T7 tag to WT PEX0 resulted in partially recoverable reduced growth and defective inflorescences persisting under high CO(2). In contrast, plants overexpressing PEX2-DeltaZn-T7 grow like WT in normal atmosphere, contain normal-shaped peroxisomes, but display impaired peroxisomal matrix protein import. PEX12-DeltaZn-T7 mutants exhibit unimpaired import of matrix protein and normal-shaped peroxisomes when grown in normal atmosphere. During seed germination, glyoxysomes form a reticulum around the lipid bodies for mobilization of storage oil. The formation of this glyoxysomal reticulum seemed to be impaired in PEX10-DeltaZn but not in PEX2-DeltaZn-T7 or PEX12-DeltaZn-T7 plants. Both cytosolic PEX10 domains seem essential for peroxisome structure but differ in metabolic function, suggesting a role for this plant peroxin in addition to the import of matrix protein via ubiquitination of PEX5.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Peroxisomas/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Dióxido de Carbono/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación de la Expresión Génica de las Plantas , Glioxisomas/metabolismo , Glioxisomas/ultraestructura , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Metabolómica/métodos , Microscopía Confocal , Microscopía Electrónica , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Peroxinas , Receptor de la Señal 1 de Direccionamiento al Peroxisoma , Peroxisomas/ultraestructura , Fotosíntesis , Plantas Modificadas Genéticamente , Dominios RING Finger/genética , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Dedos de Zinc/genéticaRESUMEN
KDEL-tailed cysteine endopeptidases are a group of papain-type peptidases found in senescing tissue undergoing programmed cell death (PCD). Their genes have so far been cloned and analyzed in 12 angiosperms. They are synthesized as proenzymes with a C-terminal KDEL endoplasmatic reticulum retention signal, which is removed with the prosequence to activate enzyme activity. We previously identified three genes for KDEL-tailed cysteine endopeptidases (AtCEP1, AtCEP2, AtCEP3) in Arabidopsis thaliana. Transgenic plants of A. thaliana expressing ß-glucuronidase (GUS) under the control of the promoters for the three genes were produced and analyzed histochemically. GUS activity was promoter- and tissue-specific GUS activity during seedling, flower, and root development, especially in tissues that collapse during final stages of PCD, and in the course of lateral root formation. KDEL-tailed cysteine endopeptidases are unique in being able to digest the extensins that form the basic scaffold for cell wall formation. The broad substrate specificity is due to the structure of the active site cleft of the KDEL-tailed cysteine endopeptidase that accepts a wide variety of amino acids, including proline and glycosylated hydroxyproline of the hydroxyproline rich glycoproteins of the cell wall.
RESUMEN
Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca(2+) removal toward the monomer and on Ca(2+) addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (Phi-Arg/Lys downward arrow) is different from the processing substrate specificity (Cys-Xxx downward arrow/Xxx-Cys downward arrow) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lon-protease. Specific antibodies against the peroxisomal Deg-protease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.