| A variety of plants produce toxins as a means of self-defense. Some of these toxins are ribosome inhibiting proteins, known as RIPs. The plant Phytolacca rivinoides leaves produce three different toxin isoforms of 29 kD, 32 kD, and 34/35 W. These toxins, named rivinins, share sequence similarity with the well-characterized toxin from Phytolacca americana (PAP). The 29 kD has 95% sequence homology to PAP, while the 32 kD, and 34/35 kD have 81% homology. Interestingly, the first 19 residues of these rivinins share a 95% homology with α-PAP, a protein whose mRNA has been identified but whose expressed protein has not been found. Two genes were found to code for all three toxins, riv4 for the 29 kD rivinin protein, and riv7 for rivinin 32 and rivinin 34/35. Both of the genes code for a 29 kD protein. Conconavalin A sepharose chromatography and endoglycosidase H digestion of the rivinin 34/35 showed that this protein is glycosylated. The riv4 and riv7 genes were cloned into several vectors (pGEX, pET 23d, pET 25b(+)) and although all constructs expressed protein, none expressed an active toxin, most likely because there are 2 internal disulfide bonds that were not made correctly or at all. Refolding experiments did not produce active protein.; Ricin toxin A chain (RTA) is a potent ribosome inhibiting protein, attacking ribosomes at a rate of 1500/min, but how RTA enters the cytoplasm from an intracellular compartment after endocytosis is not known. It has been postulated that a hydrophobic stretch of amino acids at the C-terminus may be important in membrane translocation of RTA to the cytosol. Charged point mutations at isoleucine 251 suggested that this residue was important in translocation, but proteolytic instability of these proteins made analysis difficult. Isoleucine 251 was changed to alanine to further test this hypothesis. |
