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

Katalin A. Hudak, Bijal A. Parikh, Rong Di, Marianne Baricevic, Maria Santana, Mirjana Seskar, Nilgun E. Tumer

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved α-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.

Original languageEnglish (US)
Pages (from-to)4244-4256
Number of pages13
JournalNucleic acids research
Volume32
Issue number14
DOIs
StatePublished - 2004

All Science Journal Classification (ASJC) codes

  • Genetics

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