The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum

Abstract

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...

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…The molecular mass of the deglycosylated form of pro-dionain-1 derived from SDS-PAGE (ϳ50 kDa) differed from the theoretical value (ϳ35 kDa) by ϳ15 kDa. This has previously been observed for other plant cysteine proteases (42). The aberrant migration during reducing SDS-PAGE was most likely caused by acidic motifs in the active enzyme (residues 1-223) because the activated form similarly migrated abnormally at ϳ45 kDa (Fig.…”

“…Note that pro-domain glycosylation is found in the homologous human pro-cathepsin K (48) and is considered a prerequisite for functional expression of pro-papain in insect cells (49). However, because pro-domain glycosylation motifs are not always used (42) or may be absent in certain cysteine proteases, it is not a per se requirement for proper folding and processing of C1 proteases.…”

An Endoplasmic Reticulum-Derived Structure That Is Induced under Stress Conditions in Arabidopsis

“…Ricinosome accumulates the same type of proteinase, Cys-EP, which is activated during senescence of castor bean (Ricinus communis) endosperm (Schmid et al, 2001). Ricinosome was suggested to be involved in programmed cell death in plant cells (Schmid et al, 2001). These compartments found in storage organs have diameters of 0.2 to 0.5 m. The ER bodies with a characteristic shape and size (about 5 m long) are completely distinct from the other ERderived compartments.…”

“…KV was proposed to mediate the protein mobilization in the cotyledon cells of germinated seeds (Toyooka et al, 2000). Ricinosome accumulates the same type of proteinase, Cys-EP, which is activated during senescence of castor bean (Ricinus communis) endosperm (Schmid et al, 2001). Ricinosome was suggested to be involved in programmed cell death in plant cells (Schmid et al, 2001).…”

A Protein Disulfide Isomerase Plays a Role in Programmed Cell Death

“…Prompted by the observation that protein disulfide isomerase (PDI) is present in castor bean ricinosomes, ER-derived organelles that proliferate in cells undergoing PCD (Schmid et al, 2001), Ondzighi et al (pages 2205Ondzighi et al (pages -2220 investigated the role of PDI in PCD. Of the 12 members of the Arabidopsis PDI family, the authors focused on PDI5, which is ;25% smaller than the other members and yet contains both of the characteristic thioredoxin catalytic domains.…”