The plant toxin ricin is one of the most potent and lethal substances known. Ricin inhibits protein synthesis by removing a specific adenine from the highly conserved ␣-sarcin/ricin loop in the large rRNA. Very little is known about how ricin interacts with ribosomes and the molecular mechanism by which it kills cells. To gain insight to the mechanism of ricin-induced cell death, we set up yeast (Saccharomyces cerevisiae) as a simple and genetically tractable system to isolate mutants defective in cytotoxicity. Ribosomes were depurinated in yeast cells expressing the precursor form of the A chain of ricin (pre-RTA), and these cells displayed apoptotic markers such as nuclear fragmentation, chromatin condensation, and accumulation of reactive oxygen species. We conducted a large-scale mutagenesis of pre-RTA and isolated a panel of nontoxic RTA mutants based on their inability to kill yeast cells. Several nontoxic RTA mutants depurinated ribosomes and inhibited translation to the same extent as wild-type RTA in vivo. The mutant proteins isolated from yeast depurinated ribosomes in vitro, indicating that they were catalytically active. However, cells expressing these mutants did not display hallmarks of apoptosis. These results provide the first evidence that the ability to depurinate ribosomes and inhibit translation does not always correlate with ricin-mediated cell death, indicating that ribosome depurination and translation inhibition do not account entirely for the cytotoxicity of ricin.

Shiga toxins (Stx1 and Stx2) are produced by E. coli O157:H7, which is a leading cause of foodborne illness. The A subunits of Stx1 (Stx1A) and Stx2 (Stx2A) are ribosome inactivating proteins (RIPs) that inhibit translation by removing an adenine from the highly conserved α-sarcin ricin loop (SRL) of the large rRNA. Here, we used mutagenesis in S. cerevisiae to identify residues critical for cytotoxicity of Stx1A and Stx2A. The A subunits depurinated the SRL, inhibited translation and caused apoptotic-like cell death in yeast. Single mutations in Asn75, Tyr77, Glu167 and Arg176 reduced the cytotoxicity of both toxins around 10-fold. However, Asn75 and Tyr77 were more critical for the depurination activity of Stx2A, while Arg176 was more critical for the depurination activity of Stx1A. The crystal structures of the two proteins lack electron density for some surface loops, including one which is adjacent to the active site in both molecules. Modeling these loops changed neither the secondary nor the tertiary structures of the rest of the protein. Analysis of solvent accessible surface areas indicated that Asn75 and Tyr77 are more exposed in Stx2A, while Arg176 is more exposed in Stx1A, indicating that residues with higher surface exposure were more critical for enzymatic activity. Double mutations at Glu167 and Arg176 eliminated the depurination activity and cytotoxicity of both toxins. C-terminal deletions of A chains eliminated cytotoxicity of both toxins, but showed functional differences. Unlike Stx1A, cytotoxicity of Stx2A was lost before its ability to depurinate ribosomes. These results identify residues that affect enzymatic activity and cytotoxicity of Stx1A and Stx2A differently and demonstrate that the function of these residues can be differentiated in yeast. The extent of ribosome depurination and translation inhibition did not correlate with the extent of cell death, indicating that depurination of the SRL and inhibition of translation are not entirely responsible for cell death.

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