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The cells of the endosperm of castor bean seeds (Ricinus communis) undergo programmed cell death during germination, after their oil and protein reserves have been mobilized. Nuclear DNA fragmentation first was observed at day 3 in the endosperm cells immediately adjacent to the cotyledons and progressed across to the outermost cell layers by day 5. We also detected the accumulation of small organelles known as ricinosomes, by using an antibody against a cysteine endoprotease. By the time the nuclear DNA was susceptible to heavy label by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, the ricinosomes had released into the cytoplasm their content of cysteine endoprotease, which became activated because of the cleavage of its propeptide. The cysteine endoprotease is distinguished by a Cterminal KDEL sequence, although it is not retained in the lumen of the endoplasmic reticulum and is a marker for ricinosomes. Homologous proteases are found in the senescing tissues of other plants, including the petals of the daylily. Ricinosomes were identified in this tissue by electron microscopy and immunocytochemistry. It seems that ricinosomes are not unique to Ricinus and play an important role in the degradation of plant cell contents during programmed cell death.Ricinus communis ͉ Hemerocallis sp. ͉ papain-type KDEL peptidase

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 (⌽-Arg/Lys2) is different from the processing substrate specificity (Cys-Xxx2/Xxx-Cys2) 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 Lonprotease. Specific antibodies against the peroxisomal Degprotease 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.Arabidopsis thaliana ͉ Ca 2ϩ signal ͉ Citrullus vulgaris ͉ monomer/dimer equilibrium N umerous matrix enzymes have to be imported from the cytosol into peroxisomes, in plants especially into glyoxysomes for seed storage oil mobilization or into leaf peroxisomes for photorespiration. The majority of these enzymes are imported in their mature form and targeted by a C-terminal SKL designated peroxisomal targeting signal 1 (PTS1) (1). In a few matrix enzymes, the peroxisomal targeting signal 2 (PTS2) with the consensus RL-X5-HL is located in the N-terminal 30 to 50 amino acids of the protein (2). In plants, these are four enzymes of the glyoxylate cycle and ␤-oxidation of fatty acids: glyoxysomal malate dehydrogenase (gMDH), glyoxysomal citrate synthase (gCS), acyl-CoA oxidase, and thiolase. In mammals, three enzymes with a PTS2 have been identified: thiolase, alkyl-DHAP synthase, and phytanoyl-CoA hydroxylase. In higher eukaryotes, such as plants and mammals, the PTS2 is removed on import; a Cys is consistently found near the cleavage site [supporting information (SI) Table 2]. In lower eukaryotes, such as yeasts, a PTS2 is present in the N terminus of the mature subunit of thiolase and amine oxidase (2) (SI Table 2). The Cys in position P2 is required for processing the presequences of gCS and gMDH in pumpkin; deletion of the...

The ricinosome (precursor protease vesicle) is an organelle found exclusively in plant cells. Ricinosomes contain a 45-kDa pro-cysteine endopeptidase (CysEP) with a C-terminal KDEL endoplasmic reticulum retention signal. CysEP is a member of a unique group of papain-type cysteine peptidases found specifically in senescing and ricinosome-containing tissues. During seed development in the castor oil plant (Ricinus communis L.), the cells of the nucellus are killed as the major seed storage organ, the cellular endosperm, expands and begins to accumulate reserves. The destruction of the maternal seed tissues is a developmentally programmed cell death. castor bean ͉ papain-type KDEL peptidase ͉ seed maturation

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 β-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.

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.Ricinus communis ͉ papain-type KDEL peptidase P rogrammed cell death (PCD) in higher plants and animals fulfills multiple functions in the differentiation or turnover of tissues and is triggered by pathogens at and around the site of infection as a mechanism to prevent proliferation of the disease (1, 2). Examples for elimination by PCD of organs that serve temporary functions are tadpole tails during metamorphosis and the endosperm of the castor bean during germination. In plants, the PCD of whole organs such as leaves, flowers, and fruits is designated as senescence (3).Biochemical pathways accompanying PCD or apoptosis in mammals have been elucidated in detail (reviewed in ref. 4). Plant cells undergoing senescence by PCD express papain-type cysteine endoproteases with a C-terminal KDEL sequence (5). They attract attention because they are present in the senescing endosperm cells of castor bean (Ricinus communis) as relatively inactive 45-kDa proenzymes until the final stages of PCD, at which time they are processed into the 35-kDa mature form with a 50-100 times higher activity (6).The cysteine endopeptidase from senescing castor bean endosperm (CysEP) is homologous to papain-type KDEL cysteine endopeptidases from senescing flower petals of daylily, the maturing French bean pods, and the cotyledons of germinating mung bean and vetch (6). In castor bean, daylily, and mung bean, the KDEL-tailed propeptidase is stored in a special organelle, called ricinosome (5, 6) or KDEL-tailed cysteine proteinaseaccumulating vesicles (KV) (7). Ricinosomes were first described and ultrastructurally characterized in the endosperm of R. communis (8, 9), whereas KV were described as cytoplasmic ''foci'' in Vigna radiata (10). The mature CysEP was originally purified from germinating castor bean endosperm (11). Sequencing of cDNA clones from the endosperm of germinating seedlings and from developing seeds established the presence of a presequence for cotranslational targeting into the lumen of the endoplasmic reticulum (ER), an N-terminal propeptide and a C-terminal KDEL motif. Immunogold l...

In yeasts and mammals, PEX10 encodes an integral membrane protein with a C3HC4 RING finger motif in its C-terminal domain and is required for peroxisome biogenesis and matrix protein import. In humans, its dysfunction in peroxisome biogenesis leads to severe Zellweger Syndrome and infantile Refsum disease. Here we show that dysfunction of a homologous gene in Arabidopsis leads to lethality at the heart stage of embryogenesis, impairing the biogenesis of peroxisomes, lipid bodies, and protein bodies. In a T-DNA insertion mutant disrupting the fourth exon of the Ath-PEX10 gene, ultrastructural analyses fail to detect peroxisomes characteristic for wild-type embryogenesis. Storage triacyl glycerides are not assembled into lipid bodies (oil bodies; oleosomes) surrounded by the phospholipid-protein monolayer membrane. Instead, the dysfunctional monolayer membranes, which derive from the bilayer membrane of the endoplasmic reticulum, accumulate in the cytosol. Concomitantly the transfer of the storage proteins from their site of synthesis at the endoplasmic reticulum to the vacuoles is disturbed. The mutant can be rescued by transformation with wild-type AthPEX10 cDNA. Transformants of wild-type Hansenula polymorpha cells with the AthPEX10 cDNA did produce the encoded protein without targeting it to peroxisomes. Additionally, the cDNA could not complement a Hansenula pex10 mutant unable to form peroxisomes. The ultrastructural knockout phenotype of AthPEX10p suggests that this protein in Arabidopsis is essential for peroxisome, oleosome, and protein transport vesicle formation.

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. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced b-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.