The N-terminal Ricin Propeptide Influences the Fate of Ricin A-chain in Tobacco Protoplasts

Abstract

The plant toxin ricin is synthesized in castor bean seeds as an endoplasmic reticulum (ER)-targeted precursor. Removal of the signal peptide generates proricin in which the mature A-and B-chains are joined by an intervening propeptide and a 9-residue propeptide persists at the N terminus. The two propeptides are ultimately removed in protein storage vacuoles, where ricin accumulates. Here we have demonstrated that the N-terminal propeptide of proricin acts as a nonspecific spacer to ensure efficient ER import and glycosylation. Indeed, when absent from the N terminus of ricin A-chain, the non-imported material remained tethered to the cytosolic face of the ER membrane, presumably by the signal peptide. This species appeared toxic to ribosomes. The propeptide does not, however, influence catalytic activity per se or the vacuolar targeting of proricin or the rate of retrotranslocation/degradation of A-chain in the cytosol. The likely implications of these findings to the survival of the toxin-producing tissue are discussed.Ricin is a heterodimeric protein produced in the seeds of the castor oil plant Ricinus communis where it accumulates in the protein storage vacuoles (PSV) 3 of endosperm cells. Mature ricin consists of a ribosome-inactivating A-chain (RTA) linked by a disulfide bond and non-covalent interactions to a galactose binding B-chain (RTB). This heterodimer is toxic to mammalian cells because it can bind via RTB to a variety of galactosylated cell surface molecules and, following retrograde transport to the endoplasmic reticulum (ER) and delivery of RTA to the cytosol, irreversibly inactivate ribosomes. RTA is a potent N-glycosidase that depurinates 28 S/25 S/26 S ribosomal RNA (1, 2) at a site in the ribosome that is critical for binding elongation factor-2 ternary complexes (3, 4). This leads to a halt in protein synthesis and, ultimately, cell death.Although the ribosomes of Ricinus endosperm cells are susceptible to RTA-mediated depurination (5), intoxication of the producing tissue is avoided. Co-translational ER import is accompanied by N-glycosylation (6), disulfide bond formation (7), and proteolytic cleavage of the signal peptide (8), the first 26 residues of a 35-residue presequence at the N terminus of preproricin (9) (Fig. 1). Imported proricin consists of a 9-residue N-terminal propeptide, the mature RTA sequence, a 12-residue linker propeptide, and RTB. This precursor is catalytically inactive (10) because the RTB moiety sterically obstructs the substrate binding site of RTA, as it does in mature ricin heterodimers. In this form, proricin is delivered to PSV, and the mature RTA-RTB heterodimer is generated by the endoproteolytic removal of both the N-terminal and internal propeptides (7,(11)(12)(13)(14). Ricin holotoxin accumulates within the confines of the endosperm vacuoles to 5% of the total particulate protein (15, 16).The ricin precursor and its constituent subunits have been well studied in the heterologous system of tobacco protoplasts (17)(18)(19)(20). Although it is clear that th...

The Role of CDC48 in the Retro-translocation of Non-ubiquitinated Toxin Substrates in Plant Cells

“…To investigate whether RTA was retained inside the membranes we carried out a protease protection experiment using cell extracts containing ER microsomes (25). Accordingly, at the 3-h chase point, when RTA was largely sensitive to proteinase K under normal conditions (Fig.…”

“…The data presented clearly demonstrate that the retro-translocation of these proteins requires the participation of CDC48, and is irrespective of their glycosylation or ubiquitination status. tion E177D, lysine substitutions K4R and K239R, and the removal of the proricin signal peptide and N-terminal propeptide have also been previously documented (18,24,25). All derivative constructs used in this work were generated using the QuikChange TM in vitro mutagenesis system (Stratagene, La Jolla, CA) using the following mutagenic primers (and their reverse complements, not shown): a stop codon was introduced into the prepro-RCA sequence (26) immediately after the open reading frame of the A chain to generate pRCA A using 5Ј-CCT-CCACCGTCGTCAGAGTTTTAGTTGCTTATAAGGCCA-GTGGTGCC-3Ј, and the equivalent active site substitution E176D was introduced into pRCA A using 5Ј-GGTTTGCAT-CCAAATGATTTCAGACGCAGCAAGATTCCAGTACA-TTG-3Ј.…”

Ricin Trafficking in Plant and Mammalian Cells

“…The 9 residue propeptide at the N -terminus of RTA after cleavage of the signal peptide acts as a spacer that influences the efficiencies of both co-translational transport across the ER membrane and core glycosylation [22]. Newly synthesized proricin in the ER lumen lacks the 26 residue signal peptide, which was cleaved co-translationally, but still includes the N -terminal propeptide and the 12 residue linker peptide.…”

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

“…To prepare the construct encoding RTB with its native signal peptide and propeptide, the 35-amino acid ricin presequence (containing the ricin ER signal peptide (17) and N-terminal propeptide (18)) was fused to the 5Ј end of the mature RTB coding sequence via overlapping mutagenic PCR, using the primers 5Ј-TCTAGAATGAAACCGGGAGG-3Ј and 5Ј-ACA-AACATCAGCGTTGTTATCCTC-3Ј to amplify the ricin presequence, 5Ј-GAGGATAACAACGCTGATGTTTGTATG-3Ј and 5Ј-CTGCAGTCAAAATAATGGTAACCA-3Ј to amplify RTB, and the first and fourth of these primers to fuse the two overlapping segments together. Restriction enzyme sites are underlined.…”

“…During ricin biosynthesis, proricin reaches the protein storage vacuoles of the Ricinus communis endosperm with a 9-residue propeptide at the N terminus of RTA and a 12-residue linker peptide joining RTA to RTB (7,18). In the vacuole, both of these are cleaved to generate mature ricin holotoxin (43).…”