Arabidopsis Bax Inhibitor-1 inhibits cell death induced by pokeweed antiviral protein in Saccharomyces cerevisiae.

Birsen Cakir,Nilgun Tumer

doi:10.15698/mic2015.02.190

Birsen Cakir, Nilgun Tumer

Open Access

https://doi.org/10.15698/mic2015.02.190

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Abstract

Apoptosis is an active form of programmed cell death (PCD) that plays critical roles in the development, differentiation and resistance to pathogens in multicellular organisms. Ribosome inactivating proteins (RIPs) are able to induce apoptotic cell death in mammalian cells. In this study, using yeast as a model system, we showed that yeast cells expressing pokeweed antiviral protein (PAP), a single-chain ribosome-inactivating protein, exhibit apoptotic-like features, such as nuclear fragmentation and ROS production. We studied the interaction between PAP and AtBI-1 (Arabidopsis thaliana Bax Inhibitor-1), a plant anti-apoptotic protein, which inhibits Bax induced cell death. Cells expressing PAP and AtBI-1 were able to survive on galactose media compared to PAP alone, indicating a reduction in the cytotoxicity of PAP in yeast. However, PAP was able to depurinate the ribosomes and to inhibit total translation in the presence of AtBI-1. A C-terminally deleted AtBI-1 was able to reduce the cytotoxicity of PAP. Since anti-apoptotic proteins form heterodimers to inhibit the biological activity of their partners, we used a co-immunoprecipitation assay to examine the binding of AtBI-1 to PAP. Both full length and C-terminal deleted AtBI-1 were capable of binding to PAP. These findings indicate that PAP induces cell death in yeast and AtBI-1 inhibits cell death induced by PAP without affecting ribosome depurination and translation inhibition.

Highlights

Ribosome inactivating proteins (RIPs) that are toxins isolated from plants, fungus, or bacteria catalytically inactivate eukaryotic as well as prokaryotic ribosomes by removing single adenine residues from the universally conserved sarcin/ricin loop (SRL) of the large rRNA [1,2,3,4]
We investigated the ability of pokeweed antiviral protein (PAP) to induce cell death in yeast
We present evidence here that cells expressing PAP, a type I ribosome inactivating protein, exhibit nuclear fragmentation characterized by DAPI stained multiple regions in the nucleus with extensive vacuolization

Summary

Introduction

Ribosome inactivating proteins (RIPs) that are toxins isolated from plants, fungus, or bacteria catalytically inactivate eukaryotic as well as prokaryotic ribosomes by removing single adenine residues from the universally conserved sarcin/ricin loop (SRL) of the large rRNA [1,2,3,4]. Pokeweed antiviral protein (PAP), a single chain type I RIP, isolated from leaves of pokeweed plants (Phytolacca americana), removes specific adenine and guanine residues from the SRL [1, 6, 7]. This enzymatic activity interferes with the binding of eEF-2 (elongation factor 2) thereby inhibiting protein synthesis at the translocation step [8, 9]. It has been reported that the antiviral activity of PAP can be separated from rRNA depurination [16] These results suggested that PAP might interfere with virus replication by a mechanism other than host ribosome inactivation. Similar activity of other RIPs on supercoiled double-stranded DNA templates was observed with dianthin, gelonin, cinnamomin and saporin [18,19,20]

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