Abstract
We have previously shown that ribosomal protein L3 is required for pokeweed antiviral protein (PAP), a type I ribosome inactivating protein, to bind to ribosomes and depurinate the α-sarcin/ricin loop (SRL) in yeast. Co-expression of the N-terminal 99 amino acids of yeast L3 (L3Δ99) with PAP in transgenic tobacco plants completely abolished the toxicity of PAP. In this study, we investigated the interaction between PAP and L3Δ99 in Saccharomyces cerevisiae. Yeast cells co-transformed with PAP and L3Δ99 showed markedly reduced growth inhibition and reduced rRNA depurination by PAP, compared to cells transformed with PAP alone. Co-transformation of yeast with PAP and L3Δ21 corresponding to the highly conserved N-terminal 21 amino acids of L3Δ99, reduced the cytotoxicity of PAP. PAP mRNA and protein levels were elevated and L3Δ99 or L3Δ21 mRNA and protein levels were reduced in yeast co-transformed with PAP and L3Δ99 or with PAP and L3Δ21, respectively. PAP interacted with L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that the interaction between L3Δ21 and PAP may inhibit PAP-mediated depurination of the SRL, leading to a reduction in the cytotoxicity of PAP.
Highlights
Pokeweed antiviral protein (PAP), isolated from the leaves of pokeweed plants (Phytolacca americana), is a type I ribosome inactivating protein (RIP)
RIPs have the same specificity for the adenine in the sarcin/ricin loop (SRL) of the large rRNA subunit, they show different activity against ribosomes of different species
The interaction between RIPs and ribosomal substrates has been stipulated to be the cause of these differences, the molecular recognition mechanism of RIPs for ribosomes was not well-understood until we showed that PAP gains access to ribosomes by binding to the ribosomal protein
Summary
Pokeweed antiviral protein (PAP), isolated from the leaves of pokeweed plants (Phytolacca americana), is a type I ribosome inactivating protein (RIP). PAP removes a specific adenine from the highly conserved, α-sarcin/ricin loop (SRL) in the large rRNA [1,2], a process termed depurination. We have previously shown that PAP binds to ribosomal protein L3 (RPL3) in order to gain access to the SRL to depurinate the 25S rRNA in yeast [5]. We further demonstrate that the highly conserved N-terminal 21 amino acids of L3Δ99 (L3Δ21) are able to reduce the cytotoxicity of PAP. PAP binds to L3Δ21 in yeast cells in vivo and by Biacore analysis in vitro, suggesting that this interaction may block ribosome depurination by PAP in the cytosol, leading to a reduction in the cytotoxicity of PAP
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
