Free ricin a chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts

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 ill 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.