S5A), especially in the region of L3 that is critical for binding of PAP to the ribosome (Hudak indicated that this P1 andP2 proteins are more highly conserved among the eukaryotes than between the eukaryotes and (Fig. ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have comparable requirements for ribosome depurination, PAP has different requirements, providing evidence that this conversation of RIPs with different ribosomal proteins is responsible for their ribosome specificity. Introduction The herb toxin ricin produced by the AT 56 castor bean (23S rRNA) in the highly conserved -sarcin/ricin loop (SRL) of the large rRNA (Endo and Tsurugi, 1987; 1988). The depurination of the SRL has been reported to interfere with the elongation factor 1-dependent binding of aminoacyl-tRNA to the ribosome, as well as the GTP-dependent binding of elongation factor 2 and inhibit protein synthesis at the translocation step (Montanaro (Li at the corresponding position, but not the ribosomes from (Endo (Chan pull-down studies indicated that Stx1 interacts with the P proteins of the ribosomal stalk from human cells (McCluskey ribosomes by PAP AT 56 (Ayub relevance of these interactions to the enzymatic activity and the cytotoxicity of RIPs has not been exhibited. Furthermore, the role of the ribosome interactions in the kingdom specificity of the different RIPs is not well comprehended. Using the yeast P protein deletion mutants we present here the first evidence that this ribosomal stalk forms the docking site for RTA around AT 56 the ribosome, allowing RTA to depurinate the SRL and reduce the viability of yeast cells. We further show that this interaction with the P proteins is not a general feature of all RIPs. Results An intact ribosomal stalk is required for the conversation of RTA with the ribosomes The wild-type yeast strain (W303) and three different isogenic strains with deletions in the P protein genes were used in this study. In D45 (P2), the genes and and and examined depurination by dual-primer extension analysis at the different time points (Parikh depurination of yeast rRNA by RTA. A. Yeast ribosomes (20 pmol) were incubated with 10 ng of RTA at 30C for 0, Rabbit Polyclonal to IkappaB-alpha 15, 30 and 60 min. The rRNA was extracted and used in the dual-primer extension assay as described in the plasmids harbouring the wildtype preRTA (the precursor form of RTA) or the preRTA with a point mutation at the active site, E177K (Li plasmid were transformed into the P2 mutant. The isogenic wild-type yeast cells (W303) were transformed with the same plasmids as the control. Monoclonal antibody against the V5 epitope was used to detect RTA expression in the P1 mutant (Fig. 4A), and polyclonal antibody against RTA was used to detect RTA expression in the P2 mutant (Fig. 4B). Antibodies against the integral endoplasmic reticulum membraneprotein, dolichol-phosphate mannose synthase (Dpm1) were used as the loading control. Expression of the wildtype RTA was lower in the P1 and P2 mutants than the non-toxic active site mutant, E177K (Fig. 4A and B). The signal sequence targets the preRTA to the endoplasmic reticulum in yeast where it undergoes glycosylation as in the castor bean (Parikh expression of RTA and depurination of rRNA. Immunoblot analysis was carried out at 10 h post induction. Each strain was transformed with vector (VC), preRTA (RTA) or an inactive RTA mutant (E177K). Membrane fractions were isolated (Li in the yeast mutants that did not contain an intact ribosomal stalk. Todetermine if the reduced ribosome depurination affected the sensitivity of yeast cells to RTA, we examined the viability of the wild-type cells and the P1 and the P2 mutants expressing RTA by plating cells on glucose plates after galactose induction for different times in liquid media. Yeast cells harbouring the vacant vector or the non-toxic E177K were used as controls. As shown in Fig. 5, after 10 h of induction, RTA expression reduced the viability of the wild-type cells by almost three magnitudes compared with cells expressing E177K or harbouring the vector. In contrast, the P1 or the P2 mutant expressing RTA was more viable than the wildtype cells. Viability of the P2 mutant expressing RTA, E177K or harbouring the vector was comparable, while the P1 mutant expressing RTA displayed a slight reduction in viability compared with the P1 mutant expressing E177K or harbouring the vector. The.