Free Ricin A Chain, Proricin, and Native Toxin Have Different Cellular Fates When Expressed in Tobacco Protoplasts

Abstract

The catalytic A subunit of ricin can inactivate eukaryotic ribosomes, including those of Ricinus communis where the toxin is naturally produced. How such plant cells avoid intoxication has remained an open question. Here we report the transient expression of a number of ricin A chain-encoding cDNA constructs in tobacco protoplasts. Ricin A chain entered the endoplasmic reticulum lumen, where it was efficiently glycosylated, but it was toxic to the cells and disappeared with time in a brefeldin A-insensitive manner, suggesting reverse translocation to the cytosol and eventual degradation. Proricin (the natural precursor form containing A and B chains joined together by a linker sequence) was glycosylated, transported to the vacuole, and processed to its mature form, but was not toxic. Free ricin A chain and proricin were not secreted, whereas free ricin B chain was found entirely in the extracellular medium. The coexpression of ricin A and B chains resulted in the formation of disulfide-linked, transport-competent heterodimers, which were secreted, with a concomitant reduction in the observed cytotoxicity. These results suggest that the production of ricin as a precursor is essential for its routing to the vacuole and for protection of ricin-producing cells.Ricin is a cytotoxin present in the endosperm of Ricinus communis seeds, where it accumulates in protein bodies (storage vacuoles) to 5% of the total particulate protein (1). Structurally, ricin is a heterodimeric glycoprotein comprising a ribosome-inactivating A chain (RTA) 1 and a galactose-binding B chain (RTB) covalently linked by a single disulfide bond. RTA (glycosylated molecular mass ϳof 32 kDa) catalyzes the removal of a single adenine from a highly conserved loop of 28 S/26 S/25 S rRNA within the context of a eukaryotic ribosome (2). Ribosomes depurinated in this manner are unable to bind the elongation factor-2⅐GTP complex, and protein synthesis is blocked at the translocation step of the elongation cycle (3). The precise activity of RTA varies depending on the source of ribosomes. Thus, a single A chain molecule can depurinate 1000 -2000 mammalian cell ribosomes/min under physiological conditions (2). This can be measured in vitro as a DC 50 (the concentration causing 50% depurination) of ϳ5 ng/ml under standard conditions. Although ricin A chain is certainly active against tobacco ribosomes, the DC 50 value is 650 ng/ml, showing that tobacco ribosomes are ϳ130-fold less sensitive than mammalian or salt-washed yeast ribosomes (4). Nevertheless, such is the potency of RTA that should it begin to accumulate within the cytosol of tobacco leaf protoplasts, the protein biosynthetic capacity of the expressing cells would be severely compromised.When heterodimeric ricin is presented to the surface of mammalian cells, RTB opportunistically binds to membrane components with exposed galactose residues. Toxin molecules are then endocytosed to a specific internal compartment from which RTA translocates to reach the cytosol, where the ribosomes are located. The...

Ricin A chain without its partner B chain is degraded after retrotranslocation from the endoplasmic reticulum to the cytosol in plant cells

“…In this heterologous system the proricin precursor was quantitatively targeted to vacuoles, as determined by the appearance of processed RTA and RTB with time (10). However, during this earlier investigation, we observed that the RTA precursor (comprising the 35-residue presequence in front of mature RTA), when synthesized without the intervening linker or RTB, did not travel along the secretory pathway but disappeared in a time-dependent, brefeldin A (BFA)-insensitive fashion.…”

“…However, during this earlier investigation, we observed that the RTA precursor (comprising the 35-residue presequence in front of mature RTA), when synthesized without the intervening linker or RTB, did not travel along the secretory pathway but disappeared in a time-dependent, brefeldin A (BFA)-insensitive fashion. We also showed that RTA disappearance was tightly coupled to a reduction in protein synthesis, suggesting that RTA might be able to access the cytosol at some stage during its degradation (10), possibly by exploiting an ER quality-control mechanism that normally deals with aberrant proteins.…”

“…Preproricin, RTA, and phaseolin DNA constructs in the expression vector pDHA have been described (10). For constitutive expression in transgenic tobacco, pDHA-containing RTA E177D (27) was inserted into the HindIII site of the binary vector pGA470 (28), which was then used to transform Agrobacterium tumefaciens EHA105 (29) by electroporation.…”

“…We previously have demonstrated that these biosynthetic events can be reproduced in tobacco protoplasts (10). In this heterologous system the proricin precursor was quantitatively targeted to vacuoles, as determined by the appearance of processed RTA and RTB with time (10).…”

The Thylakoid ΔpH/ΔΨ Are Not Required for the Initial Stages of Tat-dependent Protein Transport in Tobacco Protoplasts

“…Homogenization of protoplasts and incubation media was performed by adding to the frozen samples 2 volumes of ice-cold homogenization buffer (150 mM TrisCl, 150 mM NaCl, 1.5 mM EDTA, and 1.5% (w/v) Triton X-100, pH 7.5) supplemented with complete protease inhibitor mixture (Roche Applied Science). Immunoprecipitation of expressed polypeptides was performed as described (26), using rabbit polyclonal antisera raised against pea 23K or GFP (Molecular Probes, Eugene, OR). Immunoselected proteins were analyzed by 15% (w/v) reducing SDS-PAGE and fluorography.…”