Ricin B Chain Targeted to the Endoplasmic Reticulum of Tobacco Protoplasts Is Degraded by a CDC48- and Vacuole-independent Mechanism*

Abstract

The B chain of ricin was expressed and delivered to the endoplasmic reticulum of tobacco protoplasts where it disappeared with time in a manner consistent with degradation. This turnover did not occur in the vacuoles or upon secretion. Indeed, several lines of evidence indicate that, in contrast to the turnover of endoplasmic reticulum-targeted ricin A chain in the cytosol, the bulk of expressed ricin B chain was degraded in the secretory pathway.Ricin is a heterodimeric plant protein consisting of a catalytic ribosome-inactivating polypeptide (the A chain, or RTA) 5 disulfide-bonded to a galactose-specific lectin (the B chain, or RTB) (1). In this form, it is able to enter mammalian cells to reach the endoplasmic reticulum (ER) where, following toxin reduction, the RTA subunit is exported to the cytosol in a process that probably exploits some or all phases of the quality control pathway known as ER-associated protein degradation (ERAD) (2, 3). Although a significant proportion of RTA is eventually degraded by proteasomes, a fraction appears to uncouple from this pathway to refold and inactivate substrate ribosomes (4). This inactivation results from a specific depurination of 28 S rRNA at a site essential for the binding of elongation factors during protein synthesis (5). In mammalian cells, the fate of endocytosed RTB is not known. During the biosynthesis of ricin in the producing castor oil plant, the protein initially folds within the ER lumen. However, retro-translocation of RTA is avoided by the translation and ER segregation of an A-B precursor (proricin) that is incompetent for such a step (6). Instead, the ER-sequestered precursor is transported to vacuoles by virtue of a targeting signal that lies in a propeptide linking the two polypeptides. This internal sequence is removed by proteolysis only when proricin reaches the safe haven of storage vacuoles (7). In this way, sensitive plant ribosomes remain undamaged in the wake of large scale synthesis of a highly toxic protein.We have shown previously that in plant cells, in contrast to the fate of proricin, ER-sequestered RTA (rather like RTA reduced from ricin in the mammalian ER) was susceptible to proteasomal degradation following its retro-translocation and deglycosylation in the cytosol (8). As in mammalian cells, however, a fraction of dislocated RTA was able to refold to inhibit protein synthesis. This was the first demonstration of an operational retro-translocation pathway in plant cells (9, 10), and it highlighted the danger to the plant cell of expressing damaged transcripts or prematurely processed proricin. In contrast, when RTA was co-expressed with RTB, where both nascent proteins contained an ER signal peptide, a disulfide-bonded holotoxin was generated and subsequently secreted from the cell (8). The presence, on one or other of the subunits, of the previously characterized vacuolar targeting sequence, directed this holotoxin to vacuoles in a route akin to that of the proricin precursor (7, 11). These findings clearly showed that co-expr...

Signal peptide‐regulated toxicity of a plant ribosome‐inactivating protein during cell stress

“…Mutations (T22N and A24V) were therefore introduced into the saporin signal peptide in order to inhibit its cleavage by signal peptidase. This mutated signal peptide has previously been shown to cause the retention of a normally secreted protein, ricin B chain, in the ER (Chamberlain et al. , 2008).…”

A context-independent N-glycan signal targets the misfolded extracellular domain of Arabidopsis STRUBBELIG to endoplasmic-reticulum-associated degradation

“…BRI1–9 degradation in A. thaliana was proteasome-dependent, but BRI1–5 disposal could not be blocked in the same way by MG132 treatment [ 18 , 19 ]. Two distinct degradation pathways were also observed for ricin A and B chains when expressed in tobacco protoplasts [ 38 ]. In line with findings for SUBEX-C57Y, these data highlight that different ERAD substrates are also cleared by distinct routes in plants.…”

The Golgi Localization of GnTI Requires a Polar Amino Acid Residue within Its Transmembrane Domain

“…increasing the pH in the organelle, the catalytic activity of proteases is diminished, which may result in the accumulation of proteins targeted for vacuolar degradation (Chamberlain et al, 2008). Pharmacological inhibition with MG132 or concanamycin A did not result in substantial changes in the pattern of AtGnTI-Q23A-mRFP degradation products.…”