The C‐terminus of pokeweed antiviral protein has distinct roles in transport to the cytosol, ribosome depurination and cytotoxicity

Abstract

SummaryPokeweed antiviral protein (PAP) produced by pokeweed plants is a single-chain (type I) ribosome-inactivating protein (RIP) that depurinates ribosomes at the a-sarcin/ricin loop of the large rRNA, resulting in inhibition of translation. Unlike the type II RIPs, which have an active and a binding moiety, PAP has only the active moiety. The mechanism by which toxins without a binding moiety gain access to cytosolic ribosomes is not known. We set up yeast as a simple and genetically tractable system to investigate how PAP accesses ribosomes and showed that the mature form of PAP is targeted to the cytosol from the endomembrane system in yeast. In the present study, we performed a systematic deletion analysis to identify the signal required for transport of PAP to the cytosol. We demonstrate here that processing of the C-terminal extension and sequences at the C-terminus of the mature protein are critical for its accumulation in the cytosol. Using a series of PAP mutants, we identified the C-terminal signal and demonstrated that it is distinct from the sequences required for ribosome depurination and cytotoxicity. The C-terminal motif showed sequence similarity to type II RIPs that retrotranslocate from the endoplasmic reticulum to the cytosol. These results demonstrate that a conserved sequence at the C-terminus of a type I RIP mediates its transport to the cytosol and suggest that type I and II RIPs may use a common signal to enter the cytosol.

Expression of Sambucus nigra agglutinin (SNA-I′) from elderberry bark in transgenic tobacco plants results in enhanced resistance to different insect species

“…Most RIPs specifically recognize galactosyl terminated glycoproteins on the cell surface and as such facilitate the entry of the A-chain into the cell where it can exert its enzymatic activity on ribosomes or other cellular structures. Recently Baykal and Tumer (2007) have demonstrated that a conserved sequence at the C-terminus of the pokeweed antiviral protein (type-1 RIP) mediates its transport to the cytosol suggesting that type-1 and type-2 RIPs may use a common signal to enter the cytosol.…”

The potential of toxin-based drug delivery systems for enhanced nucleic acid therapeutic delivery

“…It is less clear how the type I RIPs achieve cytosolic translocation, though there are reports of the C-terminus of PAP being necessary for cytosolic translocation and toxicity [76], when studied using a yeast expression system [76]. Here, the propensity of various PAP truncations and point mutation were examined in relation to PAP's ability to navigate ER exit and depurinate ribosomes [76]. What is not clear is how a type I RIP exits the endocytic system that by default would deliver the protein to the endolysosome and destruction.…”

“…CT, PEA, ST and PT have all been reported to translocate via the Golgi and ER en route to the cytosol, utilising a variety of retrograde transport strategies. It is less clear how the type I RIPs achieve cytosolic translocation, though there are reports of the C-terminus of PAP being necessary for cytosolic translocation and toxicity [76], when studied using a yeast expression system [76]. Here, the propensity of various PAP truncations and point mutation were examined in relation to PAP's ability to navigate ER exit and depurinate ribosomes [76].…”

“…pokeweed antiviral protein (PAP) [76] or Gelonin (Gel) [26], also have cytosolic targets. CT, PEA, ST and PT have all been reported to translocate via the Golgi and ER en route to the cytosol, utilising a variety of retrograde transport strategies.…”

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

“…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).…”