Selection and characterization of ricin toxin A-chain mutations in Saccharomyces cerevisiae.

Abstract

A DNA sequence encoding the A chain of ricin toxin (RTA) from the castor bean plant, Ricinus communis, was placed under GAL] promoter control and transformed into Saccharomyces cerevisiae. Induction of expression of RTA was lethal. This lethality was the basis for a selection of mutations in RTA which inactivated the toxin. A number of mutant alleles which encoded cross-reactive material were sequenced. Eight of the first nine mutant RTAs studied showed single-amino-acid changes involving residues located in the proposed active-site cleft.Ricin is the toxic lectin from Ricinus communis (castor bean) seeds. The ricin molecule is a 65,000-dalton (Da) heterodimer consisting of an A chain and a B chain linked by a disulfide bond (16). The B chain is a galatose-binding lectin which causes ricin to bind to mammalian cells. Once bound to cells, the ricin is internalized and the A chain translocates across endocytic vesicle or Golgi membranes to the cytosol. In the cytosol the ricin toxin A chain (RTA) catalytically inactivates protein synthesis by hydrolysis of the N-glycosidic bond of adenosine residue 4324 in the 28S rRNA of the eucaryotic 60S ribosomal subunit (5). This modification irreversibly inactivates the ribosome, possible by altering the binding site for elongation factors (D. Moazed, J. Robertson, and H. Noller, Nature [London], in press). A single molecule of RTA in the cytosol is sufficient to cause cell death (3).The detailed mechanism for the RNA N-glycosidase activity of RTA is unknown. X-ray crystallography shows a cleft in the second domain of RTA which resembles the active sites of other proteins that bind large polynucleotide substrates (14). Furthermore, a number of amino acid residues located in the second domain of RTA are conserved among a group of several additional toxins and RNA-binding proteins (19), suggesting that this region may be critical for catalytic activity.The genomic ricin gene encodes both the A and B proteins. The gene lacks introns and encodes a 24-amino-acid signal peptide, the A chain, a 12-amino-acid linker peptide, and a B chain (6). Cloned RTA prepared from both the genomic ricin DNA and a cDNA clone of ricin has been expressed in Escherichia coli (15,17). In each case a soluble biologically functional molecule was produced. However, procaryotic ribosomes are not a substrate for RTA (15), and no mutants with defects in enzymatic activity or other RTA functions have been described.We have been studying the expression of cloned RTA in the yeast Saccharomyces cerevisiae, whose ribosomes are sensitive to RTA. When RTA was expressed in yeast cells, growth was arrested. Plasmids encoding mutant RTA were readily identified by their failure to kill S. cerevisiae. These observations were the basis for a positive selection for mutations that inactivated RTA. By extracting both protein * Corresponding author. and nucleic acid from the yeast cells, we determined the DNA sequence of mutant RTA genes and the enzymatic properties of the proteins they encode. Our results indicate that in...

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

“…Unlike ricin A-chain mutations selected in yeast, which are predominantly at the active site (27), we have isolated mutations outside the active site that eliminate the toxicity of PAP. Several mutants that had alterations outside the active site were enzymatically active in vitro, suggesting that cytotoxicity of PAP is not due solely to its enzymatic activity.…”

Fluorescence studies on the interaction of adenine with ricin A‐chain

“…Comparison of amino acid sequences of eleven ribosome-inactivating proteins has shown that this cleft contains several perfectly conserved amino acids [71. Mutational analyses have suggested a role for RTA cleft amino acids in enzymic action [8][9][10][11]. The unique tryptophan at position 211 is one of such amino acid in the cleft: the substitution of phenylalanine for Trp-211 resulted in a nine-fold decrease in activity, suggesting an additive contribution to the RNA-binding [1 (3].…”

Inducible cell ablation in Drosophila by cold-sensitive ricin A chain

“…This residue is in the active site cleft of RTA (Frankel et al, 1989;Montfort et al, 1987) and substitutions have been shown to inactivate ricin toxicity in yeast (Frankel et al, 1989). We are not aware that the Glu-212 or Trp-212 substitutions have been tested for cold-or temperature-sensitivity.…”

“…The parental strain, JRY188, grows on medium containing either glucose or galactose as sole carbon source, but J20 fails to grow on galactose medium due to induction of RTA. Hence, any mutant J20 cell that survives on galactose medium probably carries an RTA mutation; RTA-resistant mutations are extremely rare (Frankel et al, 1989;Gould et al, 1991), and cells that fail to induce RTA because of a mutation that inactivates a GAL regulatory gene will not grow on galactose as sole carbon source. J20 cells were therefore mutagenized and plated on galactose medium at either 29°C or 18°C.…”

“…We chose RTA for temperature-inducible ablation because it has been used previously for cell ablation in mice (Landel et al, 1988), its mechanism of action is well understood (Endo and Tsurugi, 1988) and it is active in all eukaryotes tested, including yeast (Olsnes and Pihl, 1982;Frankel et al, 1989). RTA catalytically inactivates eukaryotic ribosomes by a specific depurination event in 28S rRNA, thus inhibiting protein synthesis (Endo and Tsurugi, 1988).…”