Generation of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: evidence that ribosome depurination is not sufficient for cytotoxicity

Abstract

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved alpha-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5' and the 3' ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.

Ribosome Depurination Is Not Sufficient for Ricin-Mediated Cell Death in Saccharomyces cerevisiae

“…Previous studies indicated that RTA lost its depurination activity when Gly83 was deleted (18,31). A point mutation in the corresponding Gly in PAP (G75D) led to a loss of depurination in vivo (15) and affected the binding of PAP to ribosomes (14). These results suggest that ␤-strand D might be important for the interaction of RTA with the ribosome, such that a mutation in Gly83 may affect binding of RTA to ribosomes.…”

Ribosome-inactivating proteins: progress and problems

“…The retro-translocation was described when PAP was expressed by yeast cells however, this observation suggests that type 1 RIPs may also be able to follow the cellular route for misfolded proteins without being degraded by the proteasome. The same authors reported that site-directed mutagenesis of PAP expressed by yeast cells abolished cytotoxicity, although not affecting ribosome depurination, thus indicating that the inhibition of protein synthesis is not sufficient for cytotoxicity [77,78].…”

Insights into the Molecular Antiviral Mechanism of Pokeweed Protein from Phytolacca americana

“…Duggar and Armstrong [33] have observed that a protein from P. americana possessed an antiviral activity, and inhibited transmission of tobacco mosaic virus (TMV) in plants; though, not until 1978 PAP was accepted as an inhibitor of protein synthesis [25]. While the mechanism of PAP antiviral activity is somewhat unclear, recent findings, produced by the Hudak and Tumer laboratories, show that this activity is not dependent exclusively on inactivation of ribosomes [34,35]. It has been postulated that a direct interaction of PAP with viral RNA (or DNA) is an alternative antiviral mechanism in play.…”