The ribosomal stalk is required for ribosome binding, depurination of the rRNA and cytotoxicity of ricin A chain in Saccharomyces cerevisiae

Abstract

SummaryRibosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the a-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic ribosomes. Ricin depurinates the eukaryotic ribosomes more efficiently than the prokaryotic ribosomes, while PAP can depurinate both types of ribosomes. Accumulating evidence suggests that different docking sites on the ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the a-sarcin/ ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DP1 and DP2 mutants in vivo 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 similar requirements for ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their ribosome specificity.

Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

“…It was observed that although the K m values of RTA for the rat ribosome and naked 28S rRNA were similar, RTA exhibited 10 5 -fold higher catalytic activity (K cat ) on the rat ribosome than on naked 28S rRNA (31). A subsequent study revealed that RTA could interact with the C-terminal region of the ribosomal P stalk proteins (P0, P1, and P2), and yeast ribosome with the P stalk protein mutants were depurinated less than the wild-type ribosomes (32,33). To examine whether 6C2 interfered with the interaction between RTA and ribosome, and thus inhibited the activities of RTA, we performed an SPR experiment.…”

Expression of Pokeweed Antiviral Protein Isoform S1 (PAP-S1) and of Ricin-A-Chain/PAP-S1 Novel Fusion Protein (RTA/PAP-S1) in <em>Escherichia coli </em>and Their Comparative Inhibition of Protein Synthesis <em>in Vitro</em>

“…The increased activity of RTA/PAP-S1 compared to PAP-S1 alone can probably be explained by the fact that PAP-S1 and RTA do not dock onto the ribosome at the same site. Indeed, it was found that after PAP-S1 partially depurinates the E. coli ribosome, RTA is able to depurinate the same ribosome while RTA cannot depurinate an intact E. coli ribosome on its own [19][20][21][22][23][24][25][26]. …”

Development of a quantitative RT-PCR assay to examine the kinetics of ribosome depurination by ribosome inactivating proteins using Saccharomyces cerevisiae as a model

“…Monomeric ribosomes were isolated from yeast cells as previously described (Chiou et al 2008). Ribosome depurination was performed as previously described (Chiou et al 2008) using 10 mM Tris pH 7.4, 10 mM MgCl 2 , 60 mM KCl, 2 mM DTT, ricin A chain (Vector Laboratories), and 10 pmol purified yeast ribosomes at 30°C in 100 mL.…”

“…Ribosome depurination was performed as previously described (Chiou et al 2008) using 10 mM Tris pH 7.4, 10 mM MgCl 2 , 60 mM KCl, 2 mM DTT, ricin A chain (Vector Laboratories), and 10 pmol purified yeast ribosomes at 30°C in 100 mL. The reaction was stopped by the addition of 100 mL 23 extraction buffer (240 mM NaCl, 50 mM Tris-HCl pH 8.8, 20 mM EDTA, 2% SDS) and RNA was isolated by phenol extraction as described for the primer extension assay (Parikh et al 2002).…”

“…The ratio of the depurination fragment compared with the control fragment allowed for quantification of the extent of depurination (Parikh et al 2002(Parikh et al , 2005Baykal and Tumer 2007;Li et al 2007;Chiou et al 2008). Other methods to measure depurination include quantification of the adenine released from RIP depurination by HPLC, a calorimetric assay (Brigotti et al 1998;Heisler et al 2002) or an enzymatically coupled assay to continuously measure the release of adenine by RIPs (Sturm and Schramm 2009).…”